The Roles of Psyllids, Host Plants
and Environment in the Aetiology
of Huanglongbing in Bhutan
Namgay Om
Thesis submitted in fulfilment of the requirement for the degree of
Doctor of Philosophy
School of Science and Health
Western Sydney University
January 2017
Aknowledgements
I am grateful to His Majesty the King of Bhutan for his kindness and generosity, and for
allowing me to undertake my studies.
I am sincerely grateful to the Australian Centre for International Agricultural Research
(ACIAR) for providing the John Allwright Fellowship allowing me to pursue my PhD, and
to the School of Science and Health, Western Sydney University, and the Elizabeth
Macarthur Agricultural Institute (EMAI), NSW Department of Primary Industries,
Menangle, for use of research facilities.
I would like to thank my supervisors Professors George Andrew Charles Beattie and Paul
Holford, and Dr Nerida J Donovan, for their selfless support, guidance, and patience
throughout my candidature. I am particularly grateful to Andrew for his advice in
developing my research and his assistance during sample collections. His immense
knowledge, enthusiasm, and continuous support made my PhD studies far more manageable
than I imagined. I also appreciate his endurance for travelling on the precipitous long and
winding roads of Bhutan during field work. I thank Paul for his tremendous support, and
prompt responses. Paul was most helpful in keeping me on track. His help and support in
the laboratory and result analyses, and constructive comments are very much appreciated. I
would like to thank Nerida for her guidance and support in conducting the real-time PCR,
and for financial support that made commuting between the Western Sydney University
Hawkesbury Campus and EMAI less stressful. The list of what I owe to my supervisors
does not end here. It is because of them that I enjoyed the entire period of my PhD studies. I
could not have imagined having any other mentors for my PhD studies.
My field work in Bhutan required a lot of manpower during establishment of experiments,
and for sampling and data collection. I could not have accomplished all this without the
efforts and support rendered by my colleagues at the National Plant Protection Centre
(NPPC), Department of Agriculture, Ministry of Agriculture and Forests of Bhutan. I would
like to extend my great appreciation and thanks to:
Dr Thinlay
Kinley Wangmo
Tshomo
Sangay Chophel
Karma Lham
Phuntsho Loday
Tenzin Dorji
Ata Ugyen
I thank all of them for their assistance during field work and for enduring the steep walks
along Droopchhugang-Phuensoomgang. Kinley, Tshomo, Karma and Sangay were also
involved in laboratory DNA extractions and care of plants in greenhouses at the NPPC at
Semtokha, Thimphu.
I would also like to thank the collaborative farmers in Tsirang for allowing me to use their
orchards for my field experiments.
I would also like to express my gratitude to Anna Englezou and Grant Chambers for the
lessons on handling the real-time PCR instruments, sharing their skills, and their countless
assistance during my days at EMAI.
My appreciation and thanks also go to Pat Barkley for providing references on psyllids and
huanglongbing, and useful suggestions and comments.
I would also like to acknowledge the support of Sandra Hardy, former project leader for the
ACIAR-Bhutan project. I would also like to thank all the ACIAR-Bhutan project team
members for their administrative support: ‘Citrus’ Dorjee, Lakey and Phuntsho, and Graeme
Sanderson.
Special thanks are due to Dr Susan Halbert (Division of Plant Industry, Florida Department
of Agriculture and Consumer Services, USA), and Dr Luo Xinyu, Department of
Entomology, China Agricultural University, China) for taking time to examine and identify
psyllid specimens, and Dr Zoya Yefremova (Department of Zoology, The Steinhardt
Museum of Natural History) and Dr Ekaterina Yegorenkova (Department of Geography and
Ecology, Ulyanovsk State Pedagogical University) for identifying and describing the
previously undescribed eulophid ectoparasitoid I recorded from Diaphorina communis.
I am thankful to Dr Cen Yijing, South China Agricultural University, Guangzhou,
Guangdong, China for providing samples of Cacopsylla citrisuga adults and Murraya DNA
for use in my studies.
I also owe my gratitude to Tshering Penjor (TP), Research and Development Centre,
Wengkhar, Mongar for providing the samples of the wild citrus accessions.
I would like to thank Paul Milham for providing information and support in soil nutrient
analysis and interpretation of the soil test results.
I thank Dr Jennifer Morrow, Hawkesbury Institute for the Environment (HIE) for her quick
responses to my queries and help with molecular techniques and to Dr Markus Riegler (HIE)
for his useful suggestions in interpreting molecular results.
I also thank Dr Duong Thi Nguyen for sharing her knowledge and techniques in handling
molecular analyses that saved huge amount of time.
Last but not the least, I thank my family for their all-time support. My children, Demi
Yoezer and Tenzee Kelzang Gyeltshen, for coping so well. My field work required me to
travel to Bhutan every year for at least five months for the first three years. The emotional
support, flexibility and the adaptation skills of my children added to the success of living in
two countries in a year during those field work years. My mother and my husband for their
all-time love and support.
Statement of Authentication
The work presented in this thesis is, to the best of my knowledge and belief, original
except as acknowledged in the text. I hereby declare that I have not submitted this
material, either in full or in part, for a degree at this or any other institution.
Namgay Om
January 2017
Table of Contents
List of Tables ............................................................................................................ vii
List of Figures ............................................................................................................. x
Abstract…………………………………………………………………………….xiv
Chapter 1: Introduction and literature review ........................................................... 1
1.1 Background ......................................................................................................... 1
1.2 Literature review................................................................................................. 5
1.2.1 The disease: Huanglongbing (HLB) ............................................................ 5
1.2.2 Causal agent of the Asiatic HLB ................................................................. 8
1.2.3 Impacts of huanglongbing ........................................................................... 9
1.2.4 Huanglongbing symptoms ......................................................................... 13
1.2.5 Field and laboratory confirmation of HLB infections ............................... 16
1.2.5.1 Biological indexing ............................................................................ 16
1.2.5.2 Hosts susceptible to ‘CLas’ ................................................................ 18
1.2.5.3 Light and electron microscopy ........................................................... 22
1.2.5.4 Iodine starch test and plant metabolites.............................................. 23
1.2.5.5 Serology and molecular techniques .................................................... 24
1.2.6 Psyllid fauna of Asia.................................................................................. 26
1.2.6.1 Diaphorina citri .................................................................................. 26
1.2.6.2 Other psylloids known to feed on Rutaceae ....................................... 32
1.2.7 Molecular markers used in the phylogenetic studies of plants .................. 37
1.2.8 Genes and molecular markers used in insect phylogeny ........................... 38
1.2.9 Natural enemies and biological control ..................................................... 39
1.2.10 Impacts of heat therapies on Citrus liberibacters ...................................... 43
1.2.11 Impacts of altitude, ambient temperatures and relative humidity on ‘CLas’
and its vector.............................................................................................. 44
1.3 Aims and objectives.......................................................................................... 45
Chapter 2: Phylogenetic analysis of Diaphorina communis Mathur and Diaphorina
citri Kuwayama .......................................................................................................... 47
2.1 Introduction ...................................................................................................... 47
2.2 Material and methods ....................................................................................... 48
2.2.1 Specimen collection and DNA extraction ................................................. 48
2.2.2 Amplification of Diaphorina communis DNA for phylogenetic
analyses…………………………………………………………………..48
2.2.3 Post PCR and sequencing .......................................................................... 51
i
2.2.4 Sequence assembly .................................................................................... 52
2.2.5 Phylogenetic relationships of Diaphorina communis ................................ 52
2.3 Results…. ......................................................................................................... 54
2.3.1 Phylogenetic analysis of Diaphorina communis ....................................... 54
2.4 Discussion ......................................................................................................... 54
Chapter 3: Transmission of ‘Candidatus Liberibacter asiaticus’ (α-Proteobacteria)
by Diaphorina communis Mathur (Hemiptera: Sternorrhyncha: Liviidae) ............... 57
3.1 Introduction ...................................................................................................... 57
3.2 Materials and Methods ..................................................................................... 60
3.2.1 Field site .................................................................................................... 60
3.2.2 Preparation of test plants: mandarin and curry leaf seedlings ................... 60
3.2.2.1 Mandarin seedlings ............................................................................. 60
3.2.2.2 Curry leaf seedlings ............................................................................ 61
3.2.3 Extraction of DNA of ‘CLas’ from plants ................................................. 62
3.2.4 DNA extractions from psyllids for ‘CLas’ detection ................................ 63
3.2.5 Detection of ‘CLas’ by conventional and real-time PCR .......................... 63
3.3 Preliminary Studies in 2013 ............................................................................. 65
3.3.1 Experiment 1 (2013) .................................................................................. 65
3.3.2 Experiment 2 (2013) .................................................................................. 66
3.3.3 Experiment 3 (2013) .................................................................................. 68
3.3.4 Experiment 4 (2013). ................................................................................. 69
3.3.5 Experiment 5 (2013) .................................................................................. 71
3.4 Studies in 2014 ................................................................................................. 71
3.4.1 Experiment 1 (2014) .................................................................................. 72
3.4.2 Experiment 2 (2014) .................................................................................. 83
3.4.3 Experiment 3 (2014) .................................................................................. 86
3.5 Studies in 2015 ................................................................................................. 87
3.5.1 Experiment 1 (2015) .................................................................................. 88
3.5.2 Experiment 2 (2015) ................................................................................ 100
3.6 Discussion ....................................................................................................... 104
3.6.1 Preliminary studies in 2013 ..................................................................... 104
3.6.2 Studies in 2014 and 2015......................................................................... 105
3.7 Highlights of the study ................................................................................... 108
Chapter 4: Comparison of parasitoids of Diaphorina citri Kuwayama and
Diaphorina communis Mathur (Hemiptera: Sternorrhyncha: Liviidae) .................. 109
4.1 Introduction .................................................................................................... 109
ii
4.2 Materials and methods .................................................................................... 111
4.2.1 Collection of parasitoids .......................................................................... 111
4.2.2 Molecular and phylogenetic analyses ...................................................... 111
4.2.2.1 Parasitoid DNA extraction ............................................................... 111
4.2.2.2 Amplification of DNA and sequencing ............................................ 112
4.2.2.3 Sequence analyses ............................................................................ 112
4.2.2.4 Phylogenetic relationships ................................................................ 115
4.2.3 Morphology ............................................................................................. 117
4.3 Results……. ................................................................................................... 118
4.3.1 Field observation of parasitism................................................................ 118
4.3.2 Molecular and phylogenetic analyses ...................................................... 118
4.3.2.1 COI region ........................................................................................ 118
4.3.2.2 ITS1 region ....................................................................................... 123
4.3.2.3 ITS2 region ....................................................................................... 125
4.3.3 Morphological description....................................................................... 127
4.4 Discussion ....................................................................................................... 131
4.5 Highlights of the study ................................................................................... 133
Chapter 5: Incidence of psyllids and ‘Candidatus Liberibacter asiaticus’ in
mandarin orchards at different elevations in Tsirang ............................................... 134
5.1 Introduction .................................................................................................... 134
5.2 Materials and methods .................................................................................... 137
5.2.1 Site description and experimental plots ................................................... 137
5.2.2 Plant growth assessment .......................................................................... 141
5.2.3 Soil sample analysis................................................................................. 141
5.2.4 Leaf temperature measurements .............................................................. 142
5.2.5 Incidence of psyllid infestations .............................................................. 142
5.2.6 Recording of weather data ....................................................................... 143
5.2.7 Prevalence of ‘CLas’ in plant samples at different altitudes ................... 144
5.2.7.1 Sample collection from mature trees ................................................ 144
5.2.7.2 Sample collection from young plants in the plots ............................ 145
5.2.7.3 DNA extraction and amplification ................................................... 145
5.3 Results……………………………………………………………………….146
5.3.1 Plant growth assessment and soil analysis .............................................. 146
5.3.2 Ambient temperatures, relative humidity and leaf temperatures ............. 146
5.3.3 Rainfall .................................................................................................... 147
5.3.4 Psyllid incidence ...................................................................................... 154
iii
5.3.5 Incidence of ‘CLas’ ................................................................................. 156
5.3.5.1 Samples from mature trees ............................................................... 156
5.3.5.2 ‘CLas’ detection in samples from young mandarin trees along
Droopchhugang-Phuensoomgang .............................................................. 161
5.4 Discussion ....................................................................................................... 164
5.4.1 Growth of mandarin seedlings................................................................. 164
5.4.2 Relationships between ambient temperatures, relative humidity, rainfall,
elevation, and incidence of psyllids......................................................... 164
5.4.3 Prevalence of ‘CLas’ at different elevations ........................................... 169
5.5 Conclusions .................................................................................................... 172
5.6 Highlights of the study ................................................................................... 175
Chapter 6: Preliminary investigation into the role of Cacopsylla heterogena Li in
huanglongbing .......................................................................................................... 176
6.1 Introduction .................................................................................................... 176
6.2 Materials and methods .................................................................................... 179
6.2.1 Psyllid samples for phylogenetic analyses and morphological
identification. ........................................................................................... 179
6.2.2 Psyllid DNA extraction for phylogenetic studies .................................... 179
6.2.3 Psyllid DNA amplifications and sequencing ........................................... 187
6.2.4 Phylogenetic relationships of green psyllids on citrus and Zanthoxylum
sp………………………………………………………………………..187
6.2.5 Detection of ‘CLas’ in green psyllids and their host plants .................... 188
6.2.5.1 Development of primers for the wingless gene of the green
psyllids……………………………………………………………………188
6.2.5.2 Collection of psyllid and plant samples for detection of ‘CLas’ and
detection procedures .................................................................................. 189
6.3 Results………………………………………………………………………. 192
6.3.1 Morphological descriptions of green psyllids on citrus........................... 192
6.3.2 Psyllids on Zanthoxylum sp. .................................................................... 192
6.3.3 Phylogenetic analyses .............................................................................. 195
6.3.3.1 Psyllid sequence analysis ................................................................. 195
6.3.3.2 Phylogenetic relationships of psyllids derived from the mitochondrial
COI gene………………………………………………………………….200
6.3.3.3 Phylogenetic relationships of psyllids derived from the 16S gene... 200
6.3.3.4 Psyllids phylogeny based on the ITS region .................................... 201
6.3.4 Detection of ‘CLas’ in green psyllids and host plants ............................. 205
6.3.4.1 Primer development.......................................................................... 205
6.3.4.2 Detection of ‘CLas’ in Cacopsylla heterogena and host plants ....... 205
iv
6.2.1.1 Detection of ‘CLas’ in Zanthoxylum sp. and its psyllids.................. 206
6.4 Discussion ....................................................................................................... 210
6.4.1 Phylogeny of psyllids .............................................................................. 210
6.4.2 Detection of ‘CLas’ in Cacopsylla heterogena and host plants .............. 214
6.4.3 Detection of ‘CLas’ in Zanthoxylum sp., Cacopsylla sp. and Cornopsylla
rotundiconis ............................................................................................. 215
6.5 Highlights of the study ................................................................................... 216
Chapter 7: Molecular and morphological characterisation of Murraya species and
wild citrus taxa from Bhutan and their relevance to huanglongbing ....................... 217
7.1 Introduction .................................................................................................... 217
7.2 Materials and methods .................................................................................... 224
7.2.1 Collection of Murraya accessions ........................................................... 224
7.2.1 Collection of the wild citrus .................................................................... 224
7.2.2 DNA extractions from Murraya .............................................................. 224
7.2.3 DNA extraction from the wild citrus ....................................................... 226
7.2.4 Plant genomic DNA amplification .......................................................... 226
7.2.4.1 ITS region ......................................................................................... 228
7.2.4.2 matK-5’trnK spacer .......................................................................... 228
7.2.4.3 psbM-trnDGUC spacer ........................................................................ 228
7.2.4.4 rpS4-trnT spacer ............................................................................... 228
7.2.4.5 trnCGCA-ycf6 region .......................................................................... 229
7.2.4.6 trnL-trnL-trnF region ....................................................................... 229
7.2.5 ‘CLas’ detection, gel electrophoresis and sequencing............................. 230
7.2.6 Phylogenetic nomenclature for Murraya................................................. 230
7.2.7 Phylogenetic relationships of Murraya spp. ............................................ 231
7.2.8 Morphology of Murraya.......................................................................... 241
7.2.9 Phylogenetic relationships of wild citrus................................................. 242
7.3 Results……………………………………………………………………….243
7.3.1 Sequence analysis of the Murraya accessions ......................................... 243
7.3.2 Phylogenetic relationships of Murraya ................................................... 243
7.3.2.1 trnL-trnL-trnF region ....................................................................... 243
7.3.2.2 matK-5' trnK region .......................................................................... 247
7.3.2.3 rpS4-trnT region ............................................................................... 247
7.3.2.4 ITS region ......................................................................................... 247
7.3.2.5 Phylogenetic relationships of Murraya inferred from combination of
five cpDNA and ITS region ....................................................................... 251
v
7.3.3 Morphology of Murraya accessions from Bhutan .................................. 254
7.3.4 Morphological description of wild citrus accessions .............................. 264
7.3.5 Sequence analysis of wild citrus.............................................................. 264
7.3.6 Phylogenetic relationships of wild citrus based on four cpDNA
regions…………………………………………………………………..269
7.3.7 Phylogenetic relationships of wild citrus based on the nuclear ITS
regions…………………………………………………………………..272
7.3.8 Detection of ‘CLas’ in Murraya and wild citrus ..................................... 274
7.4 Discussion ....................................................................................................... 276
7.4.1 Phylogenetic relationships and morphology of Murraya from Bhutan... 276
7.4.2 Phylogenetic relationships of wild citrus from Bhutan ........................... 281
7.4.3 Detection of ‘CLas’ in Murraya and wild citrus ..................................... 285
7.5 Highlights of the study ................................................................................... 286
Chapter 8: General Discussion .............................................................................. 287
8.1 Key findings and their significance ................................................................ 288
8.1.1 Phylogenetic relationships of Diaphorina communis and Diaphorina
citri…………………………………………………............................... 288
8.1.2 ‘Candidatus Liberibacter asiaticus’ and Diaphorina communis ............. 288
8.1.3 Comparison of parasitoids of Diaphorina citri and Diaphorina
communis………………………………………………………………. 289
8.1.4 Incidence of psyllids and ‘CLas’ at different elevations ......................... 289
8.1.5 Cacopsylla heterogena and ‘CLas’ ......................................................... 292
8.1.6 Murraya species and ‘CLas’.................................................................... 294
8.1.7 Identity of wild citrus and testing of accessions for ‘CLas’ .................... 296
8.2 Future research ............................................................................................... 296
8.2.1 Diaphorina communis and ‘CLas’ .......................................................... 296
8.2.2 ‘Candidatus Liberibacter asiaticus’ and Ct values .................................. 297
8.2.3 Incidence of ‘CLas’ and Diaphorina citri at higher elevations ............... 299
8.2.4 Phylogeny of Cacopsylla heterogena ...................................................... 300
8.2.5 Cacopsylla heterogena and ‘CLas’ ......................................................... 300
8.2.6 Morphology of Murraya species: ............................................................ 301
8.2.7 Wild citrus ............................................................................................... 301
8.3 Implications for citriculture in Bhutan ........................................................... 304
References ............................................................................................................ 306
vi
List of Tables
Table 2.1. GenBank accessions used for phylogenetic analyses of Diaphorina
communis and Diaphorina citri ................................................................................. 53
Table 3.1. ‘Candidatus Liberibacter asiaticus’-titres in Diaphorina communis adults
caged for 54 d on branches of ‘CLas’-infected mature mandarin trees ..................... 68
Table 3.2. Numbers of live Diaphorina communis adults, eggs, and nymphs on NSC
mandarin the experiment cages during the acquisition and transmission experiment in
2014………………………………………………………………………………….76
Table 3.3. Numbers of live Diaphorina communis adults, eggs, and nymphs on
2014-curry leaf seedlings in the cages during acquisition and transmission
study…………………………………………………………………………………77
Table 3.4. ‘Candidatus Liberibacter asiaticus’ titres of stumps of mature mandarin
trees (inoculum source) and NSC mandarin seedlings used in the acquisition and
transmission study by Diaphorina communis adults in 2014 (Experiment 1)…. ...... 80
Table 3.5. ‘Candidatus Liberibacter asiaticus’ titres of stumps of mature mandarin
trees (inoculum source) and 2014-curry leaf seedlings used in the acquisition and
transmission study by Diaphorina communis adults in 2014 (Experiment 1)... ........ 81
Table 3.6. Real-time PCR detection of ‘CLas’ in pooled samples of Diaphorina
communis adults representative of the population released in the cages ................... 82
Table 3.7. Real-time PCR results of the experiment to determine transmission of
'CLas' by Diaphorina communis adults from curry leaf to mandarin seedlings at
Baychhu in 2014 ........................................................................................................ 85
Table 3.8. NSC mandarin and 2015-curry leaf seedling allocations in the BugDorm
cages used in 2015 for transmission of 'CLas' from infected mandarin stumps by
Diaphorina communis. ............................................................................................... 89
Table 3.9. Ct values of samples of Baychhu mature mandarin tree stumps before
and after ATI of Experiment 1 (2015) ....................................................................... 91
Table 3.10. ‘Candidatus Liberibacter asiaticus’ titres in pooled samples of
Diaphorina communis adults and nymphs representing the samples released in the
cages on the first three occasions ............................................................................... 93
Table 3.11. ‘Candidatus Liberibacter asiaticus’ titres in Diaphorina communis
adults collected at the end of ATI for Experiment 1 (2015) ...................................... 94
Table 3.12. 'CLas' titres in samples of 2015-curry leaf seedlings collected before
and after the ATI of Experiment 1 (2015) ................................................................. 96
Table 3.13. ‘CLas’ titres in NSC mandarin seedlings before and after the ATI of the
Experiment 1 (2015) .................................................................................................. 98
Table 3.14. Number of eggs, nymphs and adults of Diaphorina communis present
on 2015-curry leaf seedlings .................................................................................... 102
Table 3.15. ‘CLas’ titres in Diaphorina communis adults, and the NSC mandarin
and 2015B-curry leaf seedlings during the experiment to determine transmission of
‘CLas’ by Diaphorina communis adults from 2015-curry leaf seedlings………….103
vii
Table 4.1. GenBank sequences used in molecular analyses of sequence data
obtained from different populations of parasitoids of Diaphorina communis and
Diaphorina citri collected in Bhutan ....................................................................... 114
Table 4.2. Specimens of parasitoids used for molecular analysis and the GenBank
accession numbers of the three genetic regions analysed ........................................ 116
Table 4.3. Sequence polymorphisms within and between populations of parasitoids
of Diaphorina citri and Diaphorina communis…………………………………… 121
Table 4.4. Comparative characters of females of Tamarixia drukyulensis sp. n. with
females of Tamarixia radiata……………………………………………………... 130
Table 4.5. Comparative characters of males of Tamarixia drukyulensis sp. n. with
males of Tamarixia radiata…………………………………………………..........130
Table 5.1. Locations of experimental sites along Droopchhugang-Phuensoomgang
in Tsirang…..………………………………………………………………………139
Table 5.2. Average numbers of leaves per terminal shoot of mandarin seedlings
planted at different elevations .................................................................................. 147
Table 5.3. Average monthly rainfall (mm) in June, July, August and September
2014–2015 at elevations ranging from 1500 m to 800 m ASL along DroopchugangPhuensoomgang in Tsirang. ..................................................................................... 153
Table 5.4. Average annual rainfall, minimum and maximum ambient temperatures,
and minimum and maximum relative humidity in 2014 at elevations ranging from
800–1500 m ASL along Droopchugang-Phuensoomgang in Tsirang……………..153
Table 5.5. Incidence of Diaphorina citri, Diaphorina communis and Cacopsylla
heterogena nymphs and adults on mandarin at different altitudes along
Droopchhugang-Phuensoomgang, Tsirang. ............................................................. 155
Table 5.6. FAM Ct values (‘CLas’) for bark samples from 10 mature mandarin trees
at each elevation along Droopchhugang-Phuensoomgang ...................................... 158
Table 5.7. qPCR FAM Ct values (‘CLas’) for roots of from 10 mature mandarin
trees at each elevation along Droopchhugang-Phuensoomgang May 2015 and March
2016………………………………………………………………………………...159
Table 5.8. qPCR ‘CLas’ FAM Ct values for mandarin tissue samples collected from
a range of altitudes in Mendrelgang Gewog, Tsirang Dzongkhag........................... 160
Table 5.9. FAM Ct values (‘CLas’) for bark samples collected from 12 young
mandarin seedlings at each elevation planted along DroopchhugangPhuensoomgang……………………………………………………………………162
Table 5.10. FAM Ct values (‘CLas’) for root samples collected from 12 young
mandarin trees planted along Droopchhugang-Phuensoomgang. ............................ 163
Table 6.1. Summary of species of psyllids feeding on Rutaceae in Asia ............... 178
Table 6.2. Host plants and locations where psyllid specimens were collected ...... 180
Table 6.3. Locations where Cacopsylla heterogena was collected in 2015 and 2016
for detection of 'CLas' .............................................................................................. 191
viii
Table 6.4.
List of GenBank accessions of psyllids used for molecular
phylogeny…………………………………………………………………………..196
Table 6.5. Sequence analysis of psyllids. ............................................................... 199
Table 6.6. Screening of Cacopsylla heterogena collected in 2015 and 2016 in
Bhutan for the presence of ‘CLas’ by real-time PCR .............................................. 207
Table 6.7. Detection of 'CLas' in host plants of Cacopsylla heterogena. .............. 208
Table 6.8. Detection of 'CLas' in Zanthoxylum sp., Cacopsylla sp. and Cornopsylla
rotundiconis ............................................................................................................ 209
Table 7.1. A list of Murraya samples collected from Bhutan and China and citrus
samples from Bhutan used for molecular analyses .................................................. 225
Table 7.2. List of primers used for the molecular analysis of Murraya and wild
Citrus specimens from Bhutan. ................................................................................ 227
Table 7.3. Proposed names for various taxa of Murraya. ...................................... 230
Table 7.4. Sequences of Murraya and Bergera accessions used for molecular
analyses in this study................................................................................................ 232
Table 7.5. GenBank accessions of citrus and hybrids used for molecular analyses of
chloroplast regions. .................................................................................................. 235
Table 7.6. GenBank accessions of citrus and hybrids used for molecular phylogeny
based on the ITS region ........................................................................................... 238
Table 7.7. Sequence analysis of the cpDNA and the ITS region of the Murraya
accessions…………………………………………………………………………..245
Table 7.8. ANOVA of the morphology of leaves taken from accessions of Murraya
elongata from Bhutan, China and Việt Nam ........................................................... 258
Table 7.9. Sequence analysis of wild citrus accessions ......................................... 268
Table 7.10. Detection of 'CLas' in the accessions of Murraya species and wild citrus
accession NCBT.9. ................................................................................................... 275
Table 8.1. A comparison of the descriptions of the flowers of Citrus cavaleriei and
Citrus latipes ............................................................................................................ 303
Table 8.2. A comparison of the descriptions of the fruit of Citrus cavaleriei and
Citrus latipes ............................................................................................................ 303
ix
List of Figures
Figure 1.1. Administrative map of Bhutan showing the twenty dzongkhags
(administrative and judicial districts).. ......................................................................... 3
Figure 1.2. Yellow shoots on orange trees in Florida. ............................................. 15
Figure 1.3. Blotchy mottle on mandarin leaves, Zn deficiency –like leaves ........... 15
Figure 1.4. Corky veins and mottling on pomelo leaves, Limethang Research
Station, Mongar, Bhutan. ........................................................................................... 15
Figure 1.5. Lop–sided fruit and aborted seeds ......................................................... 16
Figure 1.6. Diaphorina communis adult on a mandarin leaf at Kamichhu, Bhutan,
and adult of Cacopsylla citrisuga on pomelo flush at Dura Sakalgre, Garo Hills,
Meghalaya, India ........................................................................................................ 37
Figure 2.1. Adults and nymphs of Diaphorina communis on Bergera koenigii. ..... 50
Figure 2.2. Scanning electron micrographs of the abdomens of fifth instar nymphs
of Diaphorina communis and Diaphorina citri showing the differences in marginal
setae………………………………………………………………………………….51
Figure 2.3. Phylogenetic analysis of Diaphorina communis based on COI using
maximum likelihood (ML)......................................................................................... 55
Figure 2.4. Phylogenetic analysis of Diaphorina communis based on the 16S
gene………………………………………………………………………………….56
Figure 3.1. Curry leaf fruit and a seedling tray with curry leaf seedlings 30 d after
sowing……………………………………………………………………………….61
Figure 3.2. A BugDorm rearing sleeve on a branch of a ‘CLas’- infected mandarin
tree at Baychhu……………………………………………………………………...66
Figure 3.3. BugDorm rearing cages with mandarin seedlings used for rearing
nymphs of Diaphorina communis…………………………………………………...69
Figure 3.4. Average monthly minimum and maximum temperatures and relative
humidity recorded at Baychhu in 2014 and 2015…………………………………...73
Figure 3.5. A mandarin stump used as a source of ‘CLas’ for acquisition and
transmission by adult Diaphorina communis; BugDorm rearing cages placed over
mandarin stumps in the field; close up of a BugDorm with a Set A curry leaf seedling
and mandarin stump; cages while maintained under shade cloth at Baychhu until 20
October 2014, and during transmission studies from Set A 2014 curry leaf to
mandarin…………………………………………………………………………….75
Figure 3.6. Eggs of Diaphorina communis on mandarin and curry leaf seedlings. . 78
Figure 3.7. Average (n= 4) number of second generation Diaphorina communis
adults following their development on curry leaf and mandarin seedlings in the
choice and no-choice test conducted in the laboratory at NPPC in 2014. ................. 87
Figure 3.8. Average numbers of live Diaphorina communis adults in experimental
cages (Mean±SE) for six weeks after 50 adults were released into each cage on May
8 2015………………………………………………………………………………..92
x
Figure 4.1. Parasitised psyllid nymphs and thoraxic exit holes ............................. 119
Figure 4.2. Phylogenetic relationships among the parsitoids of Diaphorina
communis and Diaphorina citri collected in Bhutan based on COI……………….122
Figure 4.3. Molecular analysis of the ITS1 region of parasitoids collected from
Diaphorina citri and Diaphorina communis in Bhutan……………………………124
Figure 4.4. Phylogenetic analysis of the ITS2 region of parasitoids of Diaphorina
citri and Diaphorina communis................................................................................126
Figure 4.5. Micrographs of Tamarixia drukyulensis female and male. ................. 128
Figure 5.1. Symptoms of huanglongbing on a ‘Candidatus Liberibacter asiaticus’infected seedling mandarin tree at Richina. ............................................................. 135
Figure 5.2. Dying mature mandarin trees, stumps of dead seedling mandarin trees,
lime leaves with symptoms of huanglongbing and Diaphorina citri adults on
mandarin leaves at the Waklay orchard, Pemathang, Gosarling, Tsirang ............... 136
Figure 5.3.
Google Earth view of locations of experimental sites along
Droopchhugang-Phuensoomgang in Tsirang. View facing north from directly above
Droopchhugang-Phuensoomgang ............................................................................ 140
Figure 5.4.
Google Earth view of locations of experimental sites along
Droopchhugang-Phuensoomgang in Tsirang. .......................................................... 140
Figure 5.5. The experimental site at 783 m ASL at ‘Meme’ Tshering’s orchard at
Phuensoomgang, Gosarling, Tsirang and a plot with four mandarin seedlings at 1053
m ASL at Mandoge Subba’s orchard in Pemathang, Gosarling, Tsirang……. ....... 141
Figure 5.6. A PVC pipe containing a Tinytag data-logger and a Davis rain
gauge…………….…… ........................................................................................... 144
Figure 5.7. Results of analysis of soil samples collected from the eight experimental
sites in Tsirang in June 2015. ................................................................................... 148
Figure 5.8. Comparison of average leaf temperature and ambient temperature at the
experimental sites in Tsirang in May, June, July and August 2014......................... 149
Figure 5.9. Monthly average minimum and maximum ambient temperatures
recorded at the experiment sites along Droopchhugang-Phuensoomgang. ............. 150
Figure 5.10. Monthly average minimum and maximum relative humidity along
Droopchugang−Phuensoomgang in Tsirang. ........................................................... 151
Figure 5.11. Monthly total rainfall (mm) recorded from June 2013 to October 2015
at the sites along Droopchugang-Phuensoomgang .................................................. 152
Figure 5.12. Wart-like tubular leaf gall of an unidentified cecidomyiid observed
between 800 m and 1400 m ASL during the study, and larvae within a gall……...173
Figure 5.13. Map of peak UV indexes using a modified scale to highlight the
absolute peaks. The location of Bhutan is circled in red and indicated with an
arrow……………………………………………………………………………….174
Figure 5.14. Spider mites on abaxial leaf surfaces of Zanthoxylum sp. (1600 m
ASL) and mandarin (650 m ASL) ............................................................................ 174
xi
Figure 6.1. Sequences of primers and probes used for the amplification of the
wingless (wg) gene of the citrus green psyllids based on the primers (WGf and WGr)
designed by Li et al. (2008) and the sequences from Cacopsylla spp…. ................ 189
Figure 6.2. Cacopsylla heterogena: ovipositing female; eggs; nymphs; adult. ..... 193
Figure 6.3. Leaves and fruits of the Zanthoxylum sp. from where Cornopsylla
rotundiconis and the undescribed Cacopsylla sp. were collected. ........................... 193
Figure 6.4.
Cornopsylla rotundiconis: adult male; slender nymph with
honeydew….. ........................................................................................................... 194
Figure 6.5. Unidentified Cacopsylla sp. from Zanthoxylum .................................. 194
Figure 6.6. Phylogenetic analysis based on the COI gene of psyllids ................... 202
Figure 6.7. Phylogenetic analysis of the 16S rDNA region of psyllids ................. 203
Figure 6.8. Phylogenetic analysis based on the ITS region of psyllids.................. 204
Figure 6.9. Phylogenetic analysis of a reduced data set of the ITS region of the
Cacopsylla species using the unidentified Cacopsylla species as the
outgroup…………………………………………………………………………… 204
Figure 7.1 Nymph of Cacopsylla heterogena on wild citrus at Wengkhar; leaf
distortion caused by Cacopsylla heterogena on wild citrus at Wengkhar ............... 221
Figure 7.2. Foliage of wild citrus at Basochhu ...................................................... 222
Figure 7.3. The trnL-trnL-trnF region of Murraya showing the positions and
directions of primers ................................................................................................ 229
Figure 7.4. Phylogenetic analysis of the trnL-trnL-trnF region of Murraya ......... 246
Figure 7.5. Evolutionary history inferred based on the matK-5' trnK region of
Murraya....................................................................................................................248
Figure 7.6. Molecular phylogenetic analysis inferred based on the rps4-trnT region
of Murraya......................... ...................................................................................... 249
Figure 7.7. Phylogenetic relationships of Murraya based on the ITS region. ....... 250
Figure 7.8. Phylogenetic relationships inferred from the combination of five cpDNA
regions and the ITS region of Murraya accessions from Bhutan using ML............ 252
Figure 7.9. Phylogenetic relationships inferred from the combination of five cpDNA
regions and the ITS region of Murraya accessions from Bhutan using maximum
parsimony……………… ......................................................................................... 253
Figure 7.10. Leaves, leaflets and flowers of Bhutanese accessions of Murraya
elongata from Baychhu and leaves, leaflets and an inflorescence of Murraya
elongata from Reldri, Phuentsholing. ...................................................................... 255
Figure 7.11. Fruit and seeds of Murraya elongata from Baychhu. ........................ 256
Figure 7.12. Plot of factor coordinates for Data Set A for the first two principal
components………… .............................................................................................. 257
Figure 7.13. Plot of the coordinates for each observation (case) using the first two
factors from principal components analysis of leaves from accessions of Murraya
elongata from four locations. ................................................................................... 261
xii
Figure 7.14. Plot of factor coordinates for Data Set B for the first two principal
components.. ............................................................................................................ 262
Figure 7.15. Plot of the coordinates for each observation (case) using the first two
factors from principal components for all observations (leaves) from accessions of
Murraya elongata, Murraya paniculata, Murraya sumatrana and Murraya
heptaphylla………… ............................................................................................... 263
Figure 7.16. Accession NCBT.1 showing leaves with large winged petioles, whole
and cross-sectioned fruit, and leaf distortion caused by Cacopsylla heterogena…. 265
Figure 7.17. Accession NCBT.9 showing leaves with large winged petioles, flowers
that are mostly borne singly and Cacopsylla heterogena damage on leaves. .......... 266
Figure 7.18. Accession NCBT.3 showing leaves without large petioles, a cross
section of a fruit; side view of a fruit and the top view of a fruit ............................ 267
Figure 7.19. Phylogenetic relationships of wild citrus from Bhutan based on
combinations of four chloroplast genes (matK-5’trnK, psbM-trnDGUC, trnL-trnLtrnF, rpS4-trnT) inferred using ML ......................................................................... 270
Figure 7.20. Phylogenetic relationships of wild citrus from Bhutan based on
combinations of four chloroplast genes (matK-5’trnK, psbM-trnDGUC, trnL-trnLtrnF, rpS4-trnT) inferred using parsimony.. ............................................................ 271
Figure 7.21. Phylogenetic analysis of citrus accessions based on the ITS region
using maximum likelihood....................................................................................... 273
Figure 7.22. Fruit of Murraya elongata from Yingde, China. ............................... 279
Figure 7.23. Fruit and seeds of Murraya sumatrana from Bogor Botanic Garden,
Indonesia…….. ........................................................................................................ 279
Figure 7.24. Murraya elongata A. DC. ex Hook. Wallich Herbarium type
specimen……........................................................................................................... 280
Figure 8.1. Leaflets of Zanthoxylum sp. with mottling symptoms ......................... 294
xiii
Abstract
‘Candidatus Liberibacter asiaticus’ (‘CLas’) (α-Proteobacteria) is the pathogen
associated with the Asiatic form of huanglongbing (HLB), the most serious disease
of citrus in Asia and the Americas, in Bhutan. The principal vector of the disease,
Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Liviidae), is also present
in Bhutan. Recent detection of ‘CLas’ in the black psyllid, Diaphorina communis
Mathur (Hemiptera: Liviidae), prompted my PhD study. I examined the molecular
characteristics of Diaphorina communis and Diaphorina citri and found that the
mitochondrial COI and 16S regions can distinguish the two species, and is useful for
the identification of nymphs in the absence of adults and fifth instar nymphs.
Investigations into whether Diaphorina communis can acquire ‘CLas’ and transmit it
from ‘CLas’-infected mandarin (Citrus reticulata Blanco) to healthy seedlings of
mandarin and curry leaf (Bergera koenigii L.) and vice versa were conducted under
field conditions at Baychhu (27.2975ºN, 89.9669ºE, 784 m ASL), Wangdue
Phodrang Dzongkhag.
Several experiments were conducted starting with
preliminary experiments in 2013, and transmission experiments in 2014 and 2015.
Results showed that Diaphorina communis did not reproduce on mandarin and
preferred curry leaf to mandarin given the choice. Further, a low (~6%) proportion
of Diaphorina communis acquired ‘CLas’ after 16–47 d and 39 d of acquisition and
transmission intervals in 2014 and 2015, respectively. No transmission of ‘CLas’
from ‘CLas’-infected mandarin to healthy mandarin and curry leaf by Diaphorina
communis could be established based on periodic testing of mandarin and curry leaf
seedlings. No transmission of ‘CLas’ from curry leaf to mandarin or curry leaf could
be achieved.
Phylogenetic analyses based on ITS 1 and 2, and the COI region as well as the
morphological descriptions revealed that the individuals of the parasitoid reared from
parasitised nymphs of Diaphorina communis belonged to a new species of
xiv
Tamarixia.
The name of the new species has been proposed as Tamarixia
drukyulensis sp. n. (Hymenoptera: Eulophidae).
Assessment of the effect of altitude, ambient temperature, relative humidity and
rainfall on leaf temperatures of mandarin plants, HLB, and distribution of psyllids,
was made using eight sites ranging from 800 m to 1500 m ASL along
Droopchhugang-Phuensoomgang in Tsirang. My investigations revealed that, in
contrast to my hypotheses, leaf temperatures were lower than ambient temperature at
all elevations and did not influence the low incidences of ‘CLas’ and Diaphorina
citri at higher elevations; mandarin seedlings grew better at 800–1000 m ASL than
seedlings planted at 1100 and 1500 m ASL in spite of higher incidences of ‘CLas’
and Diaphorina citri at 800–1100 m ASL; ambient temperatures and relative
humidity at all elevations were within the range of conditions favourable for
development and survival of Diaphorina citri. UV radiation could have a role in the
distribution of psyllids in addition to the interaction of rainfall and ambient
temperatures at elevations ≥ 1200 m ASL; Diaphorina communis were observed on
Bergera koenigii at 800, 900 and 1000 m but not on mandarin seedlings in the
experimental plots.
Cacopsylla heterogena Li (Hemiptera: Sternorrhyncha:
Psyllidae) was found on the mandarin seedlings from 1300 m to 1500 m ASL in the
experimental plots in Tsirang. It was also recorded on citrus above and below this
range in other locations.
The molecular phylogenetic analyses using COI, 16S and ITS regions revealed that
green psyllids on citrus belonged to one species, Cacopsylla heterogena.
Two
species of psyllids occurring on Zanthoxylum sp. were identified as Cornopsylla
rotundiconis Lou, Li, Li & Cai and an unidentified Cacopsylla sp. (Hemiptera:
Sternorrhyncha: Psyllidae). However, the molecular phylogenetic analyses could not
resolve the separation of Cacopsylla heterogena from Cacopsylla citrisuga (Yang &
Li), a species occurring in China. Real-time PCR analyses did not detect ‘CLas’ in
Cornopsylla rotundiconis and the unidentified Cacopsylla sp. Only a small portion
(~2%) of adults of individuals of Cacopsylla heterogena were ‘CLas’ positive
despite most citrus plants from the same the locations where the insect was collected
xv
being positive for ‘CLas’; no nymphs were ‘CLas’ positive. The insects on the
Zanthoxylum sp. were regarded as not harbouring ‘CLas’.
The Murraya plants occurring in Baychhu, Wangdue Phodrang and in Reldri along
Rinchending-Pasakha at Phuentsholing (26.8400°N, 89.4045°E, 400 m ASL) were
identified as Murraya elongata A. DC. ex Hook. f. based on molecular analyses
using five cpDNA (matK-5’trnK spacer, psbM-trnDGUC spacer; rpS4-trnT spacer,
trnCGCA-ycf6 region trnL-trnL-trnF region) and the ITS region.
Analyses of
morphological features of leaves also separated it from Murraya paniculata (L.), and
Murraya heptaphylla Spanoghe but not from Murraya sumatrana Roxburgh.
Morphological differentiation between Murraya elongata and Murraya sumatrana
was, therefore, based on the shapes of fruit and seeds. Adults of both Diaphorina
communis and Diaphorina citri were observed on Murraya elongata in Baychhu but
the eggs and nymphs occurring on these plants were confirmed as those of
Diaphorina citri. No confirmation of the presence of ‘CLas’ in Murraya elongata
could be established.
The identity of a wild citrus species observed at near Wengkhar in Mongar and at
Basochhu in Wangdue Phodrang could not be resolved based on four cpDNA and
ITS, and morphological descriptions.
Although, leaf damage and nymphs of
Cacopsylla heterogena were observed on these wild citrus, leaf samples tested for
‘CLas’ did not indicate any presence of the pathogen.
The study provides information on the role of other citrus-feeding psyllids and host
plants that could be involved in the transmission of HLB in Asia. It has implications
for management of HLB and citrus production in Bhutan and other countries with
similar situations. Based on my results, it should be feasible to grow citrus above
1200 m ASL if suitable varieties can be found and if powdery mildew is controlled.
Citrus production below 1200 m should also be feasible if all citrus below 1200 m in
Bhutan is progressively removed and replanted at high densities with certified
pathogen-free trees under strict quarantine.
xvi
Chapter 1: Introduction and literature review
__________________________________________________________________
1.1
Background
Citrus is one of the most important agricultural commodities to the economy of
Bhutan (Figure 1.1). It is an important cash crop for many small farmers and is the
leading export commodity of Bhutan (RNR Statistical Coordination Section, Policy
and Planning Division 2015). The majority of citrus cultivars grown in Bhutan and
elsewhere in the region are mandarins (Ladaniya 2008). However, the industry, as
elsewhere throughout most of the world, faces major challenges, particularly
huanglongbing (HLB) (Ahlawat & Baranwal 2003; Doe Doe et al. 2003; Donovan et
al. 2012a; Bové 2014).
Huanglongbing is the most serious disease of citrus (da Graça 1991; 2008; Bové
2006; Gottwald 2010). It is caused by three putative species of phloem-limited αProteobacteria that have not been cultured (Halbert & Manjunath 2004; Bové 2006;
Manjunath et al. 2008; Gottwald 2010):
‘Candidatus Liberibacter asiaticus’ (‘CLas’) which causes an Asiatic form of HLB;
‘Candidatus L. africanus’ (‘CLaf’) which causes an African form of the disease; and
‘Candidatus L. americanus’ (‘CLam’) which has only been recorded from Brazil.
The incidence of ‘CLam’ is declining as the incidence of ‘CLas’ increases (Lopes et
al. 2008; 2010; 2013). ‘Candidatus Liberibacter asiaticus’ is more tolerant of warm
to hot ambient temperatures than ‘CLaf’ and ‘CLam’ (Bové 2006; Gottwald 2010;
Lopes 2010; 2013).
‘Candidatus Liberibacter asiaticus’ has been recorded in Asia (Aubert 1992; Halbert
& Manjunath 2004; Bové 2006), where it is assumed to have originated (Halbert &
Manjunath 2004; Bové 2006; Beattie et al. 2012), New Guinea (Weinert et al. 2004),
Ethiopia (De Bac et al. 2010), and the Americas, including Cuba in the Caribbean,
where it has spread rapidly since 2004 (Halbert & Manjunath 2004; Bové 2006; Luis
et al. 2009; Lopes et al. 2008 & 2010; Manjunath et al. 2008 & 2010; Martínez et al.
1
2009). ‘Candidatus Liberibacter africanus’ occurs in Africa and the south east of the
Arabian Peninsula, and the Mascarene island (Bové 2006; da Graça 2008; Aubert
2009; Phahladira et al. 2012). ‘Candidatus Liberibacter americanus’ has only been
recorded in Brazil (Bové 2006; Lopes et al. 2010).
The Asiatic citrus psyllid, Diaphorina citri Kuwayama, can transmit each of the
three pathogens, and the African citrus psyllid (Trioza erytreae (del Guerico)
(Triozidae) can transmit the Asiatic and African pathogens (Aubert 1987; Halbert &
Manjunath 2004, Bové 2006; Yamamoto et al. 2006; Gasparoto et al. 2010).
‘Candidatus Liberibacter asiaticus’ has recently been recorded in the black psyllid,
Diaphorina communis Mathur, collected from HLB-symptomatic mandarin (Citrus
reticulata Blanco) in Bhutan (Donovan et al. 2012a; 2012b) and in the pomelo
psyllid, Cacopsylla citrisuga (Yang & Li), collected from symptomatic and ‘CLas’positive lemon (Citrus × limon L.) trees in China (Cen et al. 2012a). Transmission
by Cacopsylla citrisuga has also been reported (Cen et al. 2012b).
The symptoms of HLB (cited as citrus greening) were first recorded in southern
Bhutan in Phuentsholing during surveys between 1972–1990 (Lama & Amtya 1991;
Lama & Amatya 1993). In the mid-1990s, orchards in West-Central Region showed
symptoms attributed to HLB, and the presence of the disease was confirmed in 2002
(Ahlawat & Baranwal 2003; Doe Doe et al. 2003) though earlier records from
Bangladesh, India and Nepal suggest that both the disease and the psyllid may have
been present in southern Bhutan since the mid-1960s (Catling 1968a; Catling et al.
1978; Lama & Amtya 1991; Lama & Amatya 1993; Ohtsu et al. 1998). Currently,
many citrus orchards in the West-Central and the Southern Regions of Bhutan are
affected by HLB (pers. obs.). This has led to reduced yield and premature death of
many trees which is more apparent in the low altitude areas e.g., in Tsirang
Dzongkhag (pers. obs.).
The recent detection of ‘CLas’ in adult Diaphorina communis in Bhutan (Donovan et
al. 2012a) has intensified interest in the impact of HLB in Bhutan and the Indian
subcontinent.
2
Figure 1.1. Administrative map of Bhutan showing the twenty dzongkhags (administrative and
judicial districts). The dzongkhags are further divided into 205 gewogs which are administrative units
below a dzongkhag). The gewogs in turn are divided into chiwogs which are comprised of groups of
villages. (Image: http://www.nationsonline.org/oneworld/map/bhutan_map.htm with modifications).
The only information on this psyllid is contained in the records of Mathur (1935,
1975) in which it was first recorded as Diaphorina sp. n. (Mathur 1935) with
Bergera koenigii L. (as Murraya koenigii (L.) Spreng.; curry leaf) as the host plant.
Despite these observations, transmission of ‘CLas’ by Diaphorina communis and the
role of curry leaf in the epidemiology of the disease have not been determined. With
the exception of the recent report of transmission of ‘CLas’ by Cacopsylla citrisuga
(Cen et al. 2012b), all transmission studies on HLB pathogens have focused on
Diaphorina citri and Trioza erytreae (da Graça 1991; Bové 2006; Beattie & Barkley
2009; Lopes et al. 2010).
Moreover, curry leaf was not known to be a host of ‘CLas’ or the other HLB
pathogens prior to commencement of this study. Further, HLB-symptoms are less
common at higher altitude than at lower altitudes. Based on the above knowledge,
3
the scope of my study was initially designed to compare the molecular
characterisation of Diaphorina communis to that of Diaphorina citri (Chapter 2) and
to investigate whether Diaphorina communis could acquire and transmit ‘CLas’ from
infected mandarin to healthy mandarin and curry leaf and vice versa (Chapter 3),
identify natural enemies of Diaphorina communis (Chapter 4), and to evaluate the
effect of altitude, ambient temperature and relative humidity on leaf temperatures of
mandarin plants, HLB, and distribution of Diaphorina communis and Diaphorina
citri (Chapter 5). The rest of my study (Chapters 6 & 7) stemmed from observations
made during the initial year of my study. These observations indicated (a) that
Diaphorina communis does not develop on mandarin, secondly, (b) the presence of a
previously unrecorded green psyllid species in Bhutan, initially on mandarin and
subsequently on lime, lemon, orange and a wild citrus taxon, and (c) the presence of
a wild species of Murraya not previously recorded in Bhutan. Therefore, in Chapter
6, I focused on morphological and molecular identification of the green psyllid
species, and the presence or absence of ‘CLas’ in it and its host plants; in Chapter 7, I
focused on the molecular and morphological characterisation of a wild species of
Murraya and its status as host of ‘CLas’.
The outcomes of my studies have
significant importance for management of HLB in Bhutan and more widely on the
Indian subcontinent where Diaphorina citri and Diaphorina communis occur. The
research also has implications for understanding the role of other citrus-feeding
psyllids in transmission of HLB and other citrus diseases in Asia. Moreover, my
results will influence world-wide quarantine practices for psyllids and HLB.
4
1.2
Literature review
1.2.1 The disease: Huanglongbing (HLB)
Although huanglongbing is the official common name of the disease (Moreno et al.
1996; van Vuuren 1996; Bové 2006), it is also known by several other names. These
include likubin (decline) in Taiwan, citrus dieback in India, citrus leaf mottle yellows
in the Philippines, citrus vein-phloem degeneration (CVPD) in Indonesia, greening
or blotchy-mottle disease in South Africa, and greening disease in Florida and Brazil
(da Graça 1991; Bové 2006).
Huanglongbing literally means ‘yellow dragon
disease’ in Mandarin, but the intended meaning when this name was first used by
farmers in the Chaozhou region of Guangdong in the early 1900s was ‘yellow shoot
disease’ (Zhao 1981; Bové 2006). Huanglongbing was unanimously adopted by
citrus virologists as the official name of the disease at the 13th Conference of the
International Organisation of Citrus Virologists in Fuzhou, Fujian, China, in 1995
(van Vuuren 1996; Moreno et al 1996; Bové 2006) in honour of Prof Lin Kung
Hsiang who was the first to demonstrate transmissibility of the disease (Lin 1956a).
The following literature review mainly focuses on the Asiatic form of the disease.
It is widely assumed (Lin 1956a; 1956b; Zhao 1981; da Graça 1991; Bové 2006) that
the first report of HLB symptoms in English was by Reinking (1919) in his
descriptions of maladies of citrus in China and that the disease originated in China.
However, Reinking (1919) did not describe the disease. He described die-back
resulting from overcrowding of orange trees with intercrops, leaf mottling leaf was
attributed to nutrient deficiency, and most yellowing of leaves was attributed to poor
growing conditions.
Lin (1956a; 1956b) claimed, based on Reinking (1919),
interviews with farmers, and his studies on graft transmission that the disease was
present in southern China in the late 1800s. However, Diaphorina citri was not
recorded in Guangdong until 1934 (Hoffman 1936) and in Fujian in 1947 (Huang
1953; Beattie et al. 2012), despite it being described from specimens collected in
Taiwan in 1907 (Kuwayama 1907). Huanglongbing could not have been widespread
in southern China in the absence of Diaphorina citri and, according to Lin (1956a;
1956b), the disease did not reach ‘devastating proportions’ until the mid 1940s, a
5
decade after Diaphorina citri was recorded in Guangdong. Moreover, HLB was not
recorded in Fujian until 1947 (Huang 1953; Beattie et al. 2012) or in Taiwan (as
likubin) until 1951 (Su 2000). Beattie et al. (2008; 2009) hypothesised that ‘CLas’
may have been introduced to Guangdong with plants imported from India or
Indochina in the late 1920s and early 1930s, the latter coinciding with first record of
Diaphorina citri in Guangdong.
Aubert (1992) and Beattie et al. (2009) also
speculated that the disease spread to Taiwan, the Philippines, Indonesia, Thailand
and Malaysia through movement of plant material from Guangdong.
Beattie et al. (2009) noted that Husain and Nath (1927) were the first to
unknowingly, but unambiguously, describe symptoms now associated with HLB
when they described feeding damage by Diaphorina citri in Sargodha, Lyallpur,
Gujranwala and other parts of Punjab region of pre-partition India. They noted that
the insect probably ‘injected some poison’ into the trees causing premature death of
trees and making the fruit small and insipid’ in taste. Prior to this, Citrus trees in
India were observed to exhibit sudden wilting, reduced vigour and growth, and death
of twigs, branches and trees as early as the mid 1700s (Capoor 1963; Fraser & Singh
1966; Raychaudhuri et al. 1972; Raychaudhuri 1991). The causes of these symptoms
were attributed to many factors ranging from water logging and nutritional
deficiencies to fungal and viral infections including, Citrus tristeza virus (Capoor
1963).
The cause of symptoms associated with the disease, as distinct from
symptoms caused by other maladies, was subsequently recognised in the late 1960s
to be HLB (Fraser 1966; Fraser et al. 1966; Varma et al. 1993). By 1967, the disease
was then reported from different Northeastern States of India including Arunachal
Pradesh, Assam, Bihar, Manipur, Meghalaya, Sikkim, and West Bengal (Fraser
1966; Nariani & Raychaudhuri 1968; Bhagabati 1993; Varma et al. 1993). Many of
these states share land borders with Bhutan. In Bhutan, mandarin trees with HLBlike symptoms were observed in the mid 1990s in Punakha in the West Central
Region (Doe Doe et al. 2003). The presence of ‘CLas’ in this region of Bhutan was
confirmed by 2002 (Ahlawat & Baranwal 2003; Doe Doe et al. 2003).
6
Wallace (1978), in reviewing leaf-mottle yellows disease in the Philippines,
concluded that the ‘mottle leaf’ studied by Lee (1921) was caused by Zn deficiency,
and the severity of leaf mottle on mandarin and Valencia orange scions on pomelo
rootstock was due to the susceptibility of the stock to CTV. Reinking (1921), in his
report on ‘Citrus diseases of the Philippines, Southern China, Indo-China and Siam’,
did not mention symptoms similar to those of HLB. The first valid records of HLB
in the Philippines stem from reports by Martinez & Wallace (1967a; 1967b) and
Catling (1968b).
Numerous trees became unproductive by 1957 (Martinez &
Wallace 1967a; 1967b), and the disease reached epidemic proportions that drastically
reduced the total area of citrus production by 1960s (Altamirano et al. 1976).
Martinez & Wallace (1967a) demonstrated CTV and seedling yellow complex
diseases were present in all citrus trees with leaf mottling. This was supported by
transmission tests using the black citrus aphid, Toxoptera citricida (Kirkaldy), but it
was not clear whether the symptoms were caused by a different strain of the virus
that was not present in other countries facing similar tree decline problem. Their
attempts to transmit citrus leaf mottle yellows with Diaphorina citri were initially
inconclusive (Martinez & Wallace 1967a), but they subsequently (Martinez &
Wallace 1967b; 1969) demonstrated that it was the vector of this disease.
In Indonesia, Tirtawidjaja et al. (1965) who sought to determine the causes of citrus
decline, observed that West Indian lime (Citrus x aurantiifolia (Christm.) Swing.)
seedlings inoculated with scions from chlorotic trees showed collapsed phloem and
starch accumulation that was generally accompanied by vein clearing. However,
seedlings that showed vein clearing did not always show collapsed phloem and
starch accumulation. Graft inoculation of ‘djeruk Siem’ mandarin (Citrus reticulata
Blanco) and rough lemon (Citrus × taitensis Risso (= Citrus jambhiri Lushington))
showed that collapsed phloem and starch accumulation were induced only when
grafted with scions from trees showing these symptoms. Unlike on West Indian
lime, ‘djeruk Siem’ mandarin and rough lemon did not show collapsed phloem and
starch accumulation or chlorosis when inoculated with scions from chlorotic trees.
Further, transmission with Toxoptera citricida (cited as Aphis tavaresi del Guercio)
and other insects, including Diaphorina citri, revealed that transmission with
7
Diaphorina citri resulted in starch accumulation and phloem necrosis. Tirtawidjaja
et al. (1965) concluded that CVPD was caused by a distinct virus(es), not Citrus
tristeza virus or other known viral diseases at the time. They hypothesised that the
‘virus’ could be the same as the pathogen causing ‘likubin’ in Taiwan (Matsumoto et
al. 1961). Surveys between 1960 and 1980 revealed that CVPD was widespread in
Java and Sumatra (Tirtawidjaja 1980). The disease and the vector have since spread
eastward through the Indonesian archipelago to Timor and to New Guinea (Weinert
et al. 2004; Davis et al. 2005).
1.2.2 Causal agent of the Asiatic HLB
The Asiatic form HLB is caused by a phloem-restricted bacterium ‘Candidatus
Liberibacter asiaticus’ (α-Protebacteria) (Jagoueix et al. 1994). It, and its congeneric
putative species, ‘CLaf’’ and ‘CLam’, have not been cultured artificially, despite
recent claims (Schaad et al. 2009; Sechler et al. 2009), and Koch’s postulates have
not been fulfilled (Bové 2006; Gottwald 2010). The Asiatic form is more heat
tolerant than ‘CLaf’ and ‘CLam’, and can withstand ambient temperatures above
30ºC (Bové 2006; Lopes et al. 2010 & 2013; Gasparoto et al. 2012; Hoffman et al.
2013).
As noted above, the transmissible nature of the casual agent of the disease in Asia
was demonstrated in 1956 in China by Professor KH Lin (Lin Kung Hsiang) through
his work on graft-inoculation (Bové 2006). However, the pathogen was considered
to be a virus (Tirtawidjaja et al. 1965; Martinez & Wallace 1967a; 1967b; Zhao
1981) before Laflèche & Bové (1970) suggested it may be a mycoplasma-like
organism, based on its presence in the phloem of infected tissue and its nonculturable nature. However, mycoplasmas do not have a cell wall (van Iterson &
Ruys 1960), and subsequent studies revealed that the liberibacters possess a threelayered envelop consisting of an inner and outer membrane enclosing a thin layer of
peptidoglycan (Moll & Martin 1974; Garnier et al. 1984a) typical of Gram-negative
bacteria (Beveridge 1981). Jagoueix et al. (1994) cloned and sequenced the 16S
ribosomal DNA (16S rDNA) of the Asiatic and African HLB organism and
compared them with 16S rDNA sequences from GenBank and established that the
8
liberibacters belong to the α-Proteobacteria. A putative genus name ‘Liberobacter’
was proposed (Jagoueix et al. 1994). Following the Rules of International Code of
Nomenclature of Bacteria, the spelling of the putative genus was changed to
Liberibacter because the connecting vowel preceding ‘bacter’, of masculine gender
and Latin origin, should be ‘i’ not ‘o’ (Garnier et al. 2000).
1.2.3 Impacts of huanglongbing
Huanglongbing is the most severe disease of citrus: there is no known cure for the
disease on a field scale (Bové 2006; Gottwald 2010). Infection leads to reduced
yields, poor fruit quality, premature tree death and higher than normal production
costs (da Graça 1991; Roistacher 1996; Bové 2006; Gottwald 2010; Lopes et al
2009).
The first unambiguous impact of the disease was reported by Husain & Nath (1927)
in the Punjab region of pre-partitioned India in their description of damage caused by
Diaphorina citri. Husain & Nath (1927) noted that psyllid infestations led to the
death of fresh leaves and buds, sparse foliage on branches and a slow drying up of
trees, reduced yield and ‘insipid’ fruits which are all now associated with damage
caused by HLB. They commented that the psyllid appeared to inject ‘some poison’
into the trees, and that the impact on the citrus industry in the region was serious and
of greatest importance to the citrus industry.
However, for several decades,
quantitative data were lacking to support the impact of the ‘citrus decline problem’ in
India (which was attributed to CTV by some authors) until the Government of India
requested Australian Government for a specialist to evaluate the causes of the citrus
problem (Fraser & Singh 1966). Fraser (1966) noted that the problem was becoming
a substantial threat causing much concern in many districts of India and suggested
that citrus greening may in part be the cause. Raychaudhri (1991) reported that the
citrus dieback problem affected profitable citrus cultivation so much that growers
contemplated to replacing citrus with other crops.
Huanglongbing is the major limiting factor for citrus production in many parts of
Asia and Africa (Aubert et al. 1985; da Graça 1991; Aubert 1992; Buitendag & von
9
Broembsen 1993; Bové 2006; Gottwald 2010).
Records of Jiaogan mandarin
production over 40 years in Shantou in China showed sporadic reductions of
production area and yield following each epidemic of HLB caused by ‘CLas’
(Aubert 1990). An epidemiological study over 8 years in Réunion showed 65%
infection by ‘CLas’ of pathogen-free trees planted seven years earlier. The trees
subsequently became non-productive (Aubert et al. 1996). Huanglongbing infection
at the nursery level accelerates deterioration of a citrus orchard, and orchards
established with asymptomatic plants from infected areas can result in 100%
infection of trees within three years of planting (Aubert 1990).
Martinez & Wallace (1967b) reported that 60–90% of citrus trees in certain orchards
of the Philippines were infected by leaf mottle disease with many trees showing
progressing symptoms of declining in 1960. A survey of 83 orchards in Lipa,
Philippines revealed that citrus production decreased from 11,680 t in 1955–1960 to
6,300 t in 1964. The production decline was estimated to to have reached 1,000 t by
1968 (Martinez et al. 1971). The Philippine citrus production area of 25,000 ha in
1960s was reduced to 40% by 1990s (Aubert 1992).
From 1960 to 1970, three million trees were lost in Indonesia due to the disease
(Gottwald et al. 2007). A survey in Java in 1984 revealed that orchards that appeared
healthy 3–4 years earlier were all infected by HLB (Aubert et al. 1985), an indication
of rapid development and spread of the disease. Efforts initiated in 1970 to control
the disease through injection of an antibiotic, tetracycline, in a nation-wide program
met with total failure (Bové et al. 2000). An attempt to re-establish the citrus
industry in Bali following the removal of 3.6 million infected trees between 1984 and
1987 and subsequent planting of pathogen-free trees also ended in failure. By 1995,
76% of trees replanted between 1991 and 1993 expressed symptoms of the disease.
Efforts to re-establish the industry were subsequently impeded as a consequence of
an economic crisis in Indonesia in 1998 (Bové et al. 2000).
FAO production
statistics show peaks and troughs in production related to plantings of new trees
followed by HLB epidemics, with major peaks and troughs occurring every 8–10
10
years and declines in cropping area falling by up to 82% over two years (from
132,195 ha in 1996 to 96,000 in 1998).
In Thailand, where citrus is an important cash crop, Roistacher (1996) reported that
HLB destroyed almost 10–15% of tangerine trees each year. He noted that the
disease was exceptionally destructive in the northern regions and cited an economic
analysis by Grenzebach (1994) that showed a 12-years cycle of production in which
most trees declined to a point of non-profitability after eight years, only four years
after they became profitable. Akarapisan (2012) reported that HLB was primarily
responsible for a 41% reduction in annual tangerine yields between 2006 and 2009,
from 871,644 t to 514,678 t, and that the area planted to tangerines had fallen by 43.6
% over the same period, from 67,922 ha to 38,295 ha. Cyclical impacts of the
disease are also evident on FAO production statistics for Thailand. Similar impacts
appear to be evident in production statistics for Malaysia and Việt Nam.
Severe impacts of the disease are now being recorded in the Americas where ‘CLas’
was first recorded in Brazil in South America in 2004 and in Florida in North
America in 2005 (Gottwald 2010; Lopes et al. 2010; 2013; Bassanezi et al. 2011).
Where HLB is present, there is a rapid spread of the disease and symptom
development in the orchard (Bassanezi et al. 2009). Within 4 years of the pathogen
being detected in Brazil, nearly three million trees were removed in an effort to
eradicate the disease (Bassanezi et al. 2009).
Bassanezi & Bassanezi (2008)
calculated the incidence and severity of the disease and their impacts on yield in the
absence of disease control in relation to tree age. Their calculations showed that the
disease incidence would reach 50% in 0–2-years trees two years after the initial
appearance of disease symptoms, whereas in trees older than 10 years, it would take
10 years to get to the same level of incidence. Similarly, the disease severity in
younger trees (0–2 years) would reach 100% in almost two years compared with the
duration of 12 years for older trees. This showed that disease incidence and severity
progression is relatively faster in younger trees than older trees and would result in
massive yield reduction of these trees 2–4 years after the appearance of the first
symptoms (Bassanezi & Bassanezi 2008). Bassanezi et al. (2011) assessed the
11
severity of HLB symptoms and impacts and yields of Brazilian sweet orange
cultivars. They performed a single tree approach for estimating yield losses for 949
trees. For each assessed tree, symptomatic and asymptomatic fruits were counted
and weighed separately. For disease severity, each tree was divided into an upper
and lower hemisphere, and each hemisphere was further divided into four quadrants.
The 8 quadrants were then assessed on 0–5 scale corresponding to 0–100% of the
canopy section area with HLB symptoms.
The mean of the eight sections
represented the disease severity of the tree. Results showed poor correlation between
the number of symptomatic fruits per tree and disease severity for all cultivars
although symptomatic fruits weighed less than the asymptomatic fruits. Bassanezi et
al. (2011) noted that the poor correlation between symptomatic fruits and disease
severity is an indication that either fewer fruits are set on symptomatic branches or
that more symptomatic fruits have prematurely abscise from the trees as the disease
severity increased. They (Bassanezi et al. 2011) used a negative exponential model
to describe the relationship between relative yield and HLB severity. Relative yield
was calculated by dividing yield from symptomatic tree by the mean yield from
asymptomatic trees. The results showed a relative yield of 14–19% for trees with
100% of canopy with symptoms using this model (Bassanezi et al. 2011).
In the presence of HLB, the cost of production is higher. This results from having to
grow seedlings under protection, conduct surveys to detect HLB symptoms and the
vector, remove infected trees, and apply insecticides (Lopes et al. 2008; Morris et al
2008; Hodges & Spreen 2012).
With the discovery of HLB in Florida and
implementation of new State and Federal laws to prevent spread of the disease, the
additional cost of growing a citrus plant was estimated to be of US $8–10/plant
(Morris et al. 2008; Spann et al. 2008). Hodges & Spreen (2012) reported that the
estimated total revenue of the Florida orange growers was $10.9 billion without HLB
compared to $9.2 billion with HLB thus causing a 16% reduction during five years
from 2006/07 to 2010/11. The economic impact analysis of orange processing with
and without HLB showed that without HLB, the total revenues impact during the
five year period was $28,816 compared to $24,275 with HLB. Without HLB, the
labour income impact was $11.1 billion compared to $9.3 billion with HLB. Overall,
HLB has adverse impacts on the gross domestic product, income, and significant
12
employment impacts on the agriculture sector (Hodges & Spreen 2012). At present,
the overall citrus production in Florida has been reduced by more than 70% and the
production of oranges for the 2017 season is forcast to be 67 milllion boxes
compared to the 240 million boxes prior to HLB (Halbert, pers. comm.).
1.2.4 Huanglongbing symptoms
The symptoms caused by HLB are not specific, but some are very characteristic of
HLB (Bové 2006). Lin (1956b) described ‘yellow shoots’ (Figure 1.2), leaf drop and
rotting of rootlets as characteristic symptoms of the disease. Infected trees exhibiting
early symptoms are conspicuous because of yellowing (chlorosis) of leaves on
infected branches with medium to advanced symptoms stand out from leaves on
healthy branches or branches with leaves exhibiting mild symptoms (McClean &
Schwarz 1970; Aubert 2008; Bové 2006). As the disease progresses, whole trees,
particularly those with severe symptoms, may appear yellow (Bové 2006). Initial
leaf symptoms are characterised by leaves with patchy yellow and green or pale
green and yellow mottling. This is referred to as ‘blotchy mottle’ (Figure 1.3A).
Veins of such leaves can become yellow (McClean & Schwarz 1970). Blotchy
mottling is, by definition, not symmetrical on opposing leaf surfaces (adaxial or
abaxial) separated by leaf midribs. These symptoms differ from those of zinc (Zn)
deficiencies that are characteristically presented as symmetrical chlorosis on both
sides of midribs (Bové 2006). Moreover, Zn-deficient leaves (Figure 1.3B) exhibit
interveinal chlorosis whereas with botchy-mottled leaves also exhibit yellowing of
veins (Schneider 1968). Schneider (1968) described botchy mottle as a primary
symptom.
Leaves exhibit secondary symptoms as disease severity progresses.
Chlorosis becomes more evenly distributed and leaves become small, leathery and
upright. Veins become corky (Figure 1.4). These symptoms are mostly due to
accumulation of starch in the parenchyma cells as movement of sugars ceases due to
hyperplasia and necrosis that leads to vein phloem collapse (Salibe & Cortez 1966;
Matsumoto et al. 1961; Tirtawidjaja et al. 1965; Su & Huang 1990; Schneider 1966;
McClean & Schwarz 1970; Achor et al. 2008; Etxeberria et al. 2009; Gonzalez et al.
2012). Ultimately, leaves with severe symptoms abscise, leaving sparse foliage
13
(McClean & Schwarz 1970). Mineral deficiencies symptoms that appear on the
severely symptomatic leaves with secondary symptoms resemble Zn, Fe, Mn, Ca, S
and B deficiencies among which Zn-like deficiency symptoms are the most common
(Schneider 1968; McClean & Schwarz 1970).
Fruits of infected trees exhibit ‘colour inversion’ manifested as abnormal colouring
from the stylar to the penduncular ends. These symptoms are more apparent in
temperate regions where cool conditions in autumn and winter lead to chlorophyll
depletion in the rinds in contrast to fruit grown in the tropics that remain green
(Hodgson 1967). Fruit also become lop-sided (Figure 1.5A), a symptom that is more
apparent when fruit are cut open parallel to the fruit axis (Bové 2006). Schneider
(1968) explained that lop-sided fruit ripen on one side as the other side remains
immature with the external surface of the latter remaining green, the symptom
leading to use of the common name, ‘greening’, for the disease in South Africa.
Infected fruits may also have aborted seeds (Figure 1.5B) which appear as brown and
shriveled seeds when fruit are cut open (Bové 2006); the fruit, themselves, become
dry and insipid (Schneider 1968; McClean & Schwarz 1970, da Graça 1991; Bové
2006). These latter symptoms were among those attributed by Husain & Nath (1927)
to Diaphorina citri injecting ‘some poison’ into trees.
These symptoms may also be induced by other causes. Yellow shoot can result from
damage on the trunks by phytophthora gummosis (Bové 2006), water-logging, winter
yellows (Broadbent & Fraser 1979) and, as noted above, mineral deficiencies. Citrus
tristeza virus infections and B deficiency can lead to corky veins (Aubert 2008).
Moreover, temperature, light, host susceptibility, and virulence of the putative
‘Candidatus Liberibacter’ species and strain can affect symptom expression (da
Graça 1991; Bové 2006; Folimonova et al. 2009).
14
Figure 1.2. Yellow shoots on orange trees in Florida (GAC Beattie).
Figure 1.3. Blotchy mottle on mandarin leaves (A), Zn deficiency –like leaves (B) (Photos: N. Om).
Figure 1.4. Corky veins and mottling on pomelo leaves, Limethang Research Station, Mongar,
Bhutan (Photos: GAC Beattie).
15
Figure 1.5. Lop–sided fruit (left), aborted seeds (right) (Photos: Pat Barkley).
1.2.5 Field and laboratory confirmation of HLB infections
Several techniques can be used to confirm the presence of HLB pathogens in
symptomatic tissues.
These techniques include biological indexing (Lin 1956a;
Tirtawidjaja 1972; Tatineni et al. 2008), electron and light microscopy (Schwarz,
1965, Laflèche & Bové 1970; Manthey 2008), an iodine-starch test (Eng 2007) and
serological and molecular methods (Villechanoux et al. 1992; Garnier & Bové 1996;
Hocquellet et al. 1999; Li et al. 2006; 2007a; 2009).
The accuracy of these
procedures is often affected by the uneven distribution of the pathogens within the
tree and non-specific symptom exhibition (Bové 2006; Manjunath et al. 2008; Li et
al. 2009).
Selection of tissues for testing is generally based on sampling of
symptomatic leaves. As noted above, Koch’s postulates have not been proven for the
HLB liberibacters and the pathogens have not been cultured (Jagoueix et al. 1994;
Bové 2006; Li et al. 2009).
1.2.5.1 Biological indexing
Biological indexing was initially used to demonstrate that symptoms of a malady
now known to be caused by ‘CLas’ were caused by a graft-transmissible plant
pathogen (Lin 1956a). Lin (1956a) based his tests on mandarin trees with yellow
shoots. Tirtawidjaja (1972) reported 45% disease transmission using infected tips of
twigs grafted onto Cleopatra mandarin compared to 40% and 7% transmission with
leaf segments with midvein attached, and leaf segments alone, respectively. On
16
rough lemon (Citrus × taitensis Risso), transmission was 26% using bark with buds
compared to 2% transmission with bark alone (Tirtawidjaja 1972). This showed that
the pathogen was unevenly distributed in the plants. Uneven distribution of the
pathogen was also noted by others who concluded the pathogen to be present only in
symptomatic branches (Martinez et al. 1971; Huang 1979; Gonzales & Viñas 1981).
Teixeira et al. (2008) observed that samples negative by two PCR techniques, i.e.,
with no or very few liberibacters, were located near blotchy mottle leaf samples i.e.,
samples with high amounts of liberibacters, reflecting, locally, an uneven distribution
of liberibacters. da Graça (1991) cited references to the use of sweet oranges,
Orlando tangelo (Citrus × aurantium), Key, Kagzi or Indian acid lime (Citrus ×
aurantiifolia (Christm.) Swingle) and mandarins as indicator plants.
Although,
biological indexing was the earliest method used to demonstrate the grafttransmissibility of the disease (Lin 1956a), its use is limited by the uneven
distribution of the pathogen within a tree, and the long latent period before the
development of symptoms.
Recently, it has been noted that ‘CLas’ is not confined to symptomatic portions of
HLB-infected trees (Gottwald et al. 2008; Tatineni et al. 2008; Li et al. 2009). Using
real–time PCR, Gottwald et al. (2008) analysed two entire trees and detected ‘CLas’
not just in the symptomatic branches but in the scions, rootstock, and in almost 76%
and 26% of the main branches and roots, respectively, using leaf veins, petiole tissue
and cambium samples. Tatineni et al. (2008) examined different parts of ‘CLas’infected trees and detected the pathogen in all floral parts, except in the endosperm
and embryo. To determine systemic infection of HLB, Tatineni et al. (2008) also
tested fully expanded young to mature leaves collected from a branch further away
from the site of graft inoculation and detected the pathogen in all samples except one.
The bacterium was also detected in the roots of infected trees grown in a greenhouse.
All samples were tested using conventional and real–time PCR (Tatineni et al. 2008).
Li et al. (2009) performed quantitative PCR to quantify the distribution of ‘CLas’
genomes in six species of citrus. Root samples were taken from one symptomatic,
field-grown tree each of Persian lime (Citrus × latifolia (Yu. Tanaka) Tanaka,
Palestine sweet lime (‘Citrus limettioides Tan.’), and lemon (Citrus × limon (L.)
17
Burm. f.), or three trees each of sweet orange (Citrus × aurantium L),
Mexican/Tahitian lime (Citrus aurantiifolia) and kaffir lime (Citrus hystrix DC).
They (Li et al. 2009) assayed samples from midribs, petioles, leaf blades, green stem
bark, mature bark and roots. No significant difference was observed in the pathogen
genome level based on the tissue type (midrib/petiole, leaf blade, bark) from the
same tree, but differences were observed among tissues samples from different trees.
The pathogen genome level ranged from 1 × 1010 to 6 × 1010 copies per gram of root
tissue in all greenhouse plants and in one field-grown tree. The pathogen was also
consistently detected in symptomatic fruits. Higher titres of the pathogen in fruit
were detected in locular membranes and septa than in the peduncle, pericarp, or
central axis. Greenhouse trees, that were systematically sampled, showed higher
genome levels in midribs than in the bark taken from lower portions of the tree. The
pathogen was detected in all samples of leaf blades, midribs, stem barks and roots (Li
et al. 2009). Sagaram et al. (2008) found a wide variation in concentration of ‘CLas’
among tissues with the highest concentration in fruit peduncles.
These studies
indicate that the pathogen can be present in infected trees almost systematically but
may not be present at a detectable level (Gottwald et al. 2008). The PCR method
used detects all living and dead DNA (Li et al. 2009). Therefore, detection of
pathogen based on quantitative PCR from any plant part does not guarantee detection
of only viable bacteria. Thus, variable results may be obtained when used for
biological indexing.
1.2.5.2 Hosts susceptible to ‘CLas’
Several species and hybrids of Rutaceae and some non-rutaceous plants are known
hosts of ‘CLas’. Different hosts react differently to the pathogen. Beattie & Barkley
(2009) compiled an extensive list of hosts of different forms of HLB. Species and
hybrids of Aurantioideae within the Aurantieae recorded as expressing symptoms or
known to be susceptible are:
Aegle marmelos (L.) Corr. (Ramadugu et al. 2016);
Aeglopsis chevalieri Swingle (Ramadugu et al. 2016);
18
Atalantia buxifolia (Poir.) Oliv. (Deng et al. 2008): (Severinia buxifolia (Poir.)
Tenore (Martinez & Wallace 1969; Su et al. 1995; Koizumi et al. 1996; Hung
et al. 2000, 2001; Hu 2012; Hu et al. 2014);
Balsamocitrus daweii Stapf (Koizumi et al. 1996; Ramadugu et al. 2016);
Citropsis gilletiana Swing. & M. Kell. Jour. (Hu 2012);
Citrus amblycarpa (Hassk.) Ochse (Tirtawidjaja 1981; Ramadugu et al. 2016);
Citrus australasica F. Mueller: Microcitrus australasica (F. J. Muell.) Swingle
(Koizumi et al. 1996; Ramadugu et al. 2016);
Citrus cavaleriei H. Léveillé ex Cavalerie: Citrus ichangensis Swingle
(Miyakawa & Zhao 1990);
Citrus glauca (Lindl.) Burkill hybrid: Eremocitrus glauca (Lindl.) Swing.
(Ramadugu et al. 2016);
Citrus hystrix DC (Miyakawa & Zhao 1990);
Citrus japonica Thunb.: Fortunella sp., Fortunella hindsii and Fortunella
japonica (Miyakawa & Zhao 1990; Ramadugu et al. 2016);
Citrus maxima (Burm.) Merr.: Citrus grandis (L.) Osbeck (Nariani 1981;
Miyakawa & Zhao 1990; Ramadugu et al. 2016);
Citrus medica L. (Su & Matsumoto 1972; Nariani 1981);
Citrus halimii B.C. Stone (Ramadugu et al. 2016);
Citrus latipes (Swing.) Tan. (Ramadugu et al. 2016);
Citrus reticulata Blanco: Citrus reticulata (Tirtawidjaja et al. 1965;
Tirtawidjaja 1981); Citrus depressa Hayata; Citrus keraji hort. ex Tan.; Citrus
oto hort. ex. Yu. Tanaka and Citrus unshiu (Mack.) Marc (Miyakawa & Zhao
1990); Citrus leiocarpa hort. ex Tanaka (Ramadugu et al. 2016); Citrus sunki
hort. ex Tanaka (Su & Matsumoto 1972; Miyakawa & Zhao 1990; Ramadugu
et al. 2016);
19
Citrus trifoliata L.: Poncirus trifoliata (L.) Raf. (Martinez & Wallace 1969;
Miyakawa 1980; Nariani 1981; Miyakawa & Zhao 1990; Koizumi et al. 1996;
Ramadugu et al. 2016); trifoliate and hybrids (Gonzales et al. 1972); ×
Citroncirus sp. (Ramadugu et al. 2016);
Citrus × aurantiifolia (Christm.) Swingle: Citrus aurantifolia (Christm.)
Swingle (Schwarz et al. 1973; Tirtawidjaja 1981; Miyakawa & Zhao 1990);
Citrus × davaoensis (Wester) Yu. Tanaka) (Ramadugu et al. 2016);
Citrus × aurantium L.: Citrus hassaku hort. ex Tanaka, Citrus natsudaidai
Hayata, Citrus nobilis Lour., Citrus sinensis (L.) Osbeck, Citrus paradisi
Macfad. (Fraser & Singh 1969; Su & Matsumoto 1972; Nariani 1981;
Miyakawa & Zhao 1990; Koizumi et al. 1993; 1994; 1996; Pelz–Stelinski et al.
2010; Fan et al. 2012); Citrus intermedia hort. ex. Tanaka, Citrus taiwanica
Tan. & Shimada, Citrus neo-aurantium Tanaka, Citrus nobilis Lour., Citrus
lycopersiciformis hort. ex. Tan., Citrus maderaspatna, Citropsis daweana
Swing. & M. Kell (Ramadugu et al. (2016);
Citrus × insitorum Mabb.: (× Citroncirus webberi J. Ingram & H. E. Moore
(Martinez & Wallace 1969, Nariani 1981, Miyakawa & Zhao 1990);
Citrus × junos Siebold ex Tanaka (Miyakawa & Zhao 1990);
Citrus × limon (L.) Osbeck: Citrus limon (L.) Burm. f., Citrus limonia Osbeck,
Citrus meyeri Tan. (Tirtawidjaja 1981; Gonzales et al. 1972; Miyakawa &
Zhao 1990); Citrus limettioides Tan.; Citrus excelsa Wester (Ramadugu et al.
2016);
Citrus longispina Wester (winged lime) (Ramadugu et al. 2016);
Citrus × volkameriana Osbeck: Citrus volkameriana Osbeck (Ramadugu et al.
2016);
Citrus × macrophylla Wester: Citrus macrophylla (Ramadugu et al. 2016);
Citrus × microcarpa Bunge (Su & Matsumoto 1972); × Citrofortunella
microcarpa (Hu 2012);
20
Citrus × taitensis Risso: Citrus jambhiri Lush. (Tirtawidjaja 1981; Koizumi et
al. 1993; 1994; Ramadugu et al. 2016);
Citrus × webberi Wester: Citrus webberi Wester (Ramadugu et al. 2016);
Limonia acidissima L. (Koizumi et al. 1996; Su et al. 1995; Hung et al. 2000);
× Microcitronella sp.: Citrus australasica × calamondin (Ramadugu et al.
2016);
Murraya paniculata (L.) Jack: Tirtawidjaja et al. (1981); Aubert et al. (1985);
Li & Ke (2002); Damsteegt et al. (2010); Walter et al. (2012); Ramadugu et al.
(2016); Murraya exotica L. (Lopes et al. 2010)
Pamburus missionis (Wight) Swingle: Atalantia missionis Oliver (Tirtawidjaja
1981);
Triphasia trifolia (Burm. f.) P. Wilson (Koizumi et al. 1996).
A few species of Aurantioideae within the Clauseneae are recorded as hosts or
possible hosts of ‘CLas’:
Bergera koenigii L. (Ramadugu et al. 2016);
Clausena indica Oliv. (Clausena indica (Dalz.) Oliver (Tirtawidjaja 1981); and
Clausena lansium (Lour.) Skeels (Tirtawidjaja 1981, Koizumi et al. 1996, Ding
et al. 2005).
Some species in the Rutoideae recorded as being host of ‘CLas’ are:
Casimiroa edulis La Llave & Lex. (Ramadugu et al. 2016);
Choisya ternata Kunth ‘Sundance’ (Hu 2012);
Choisya ternata Kunth × Choisya dumosa (Torr.) A. Gray: Choisya aztec
‘Pearl’ (Hu 2012);
Esenbeckia runyonii C.V.Morton (Hu 2012);
Zanthoxylum ailanthoides Siebold & Zucc. (Ramadugu et al. 2016);
21
Zanthoxylum fagara (L.) Sarg (Hu 2012).
However, HLB infections in Casimiroa and Zanthoxylum species may be transient
(Halbert, pers. com.)
Some non–rutaceous species recorded as hosts of ‘CLas’ are:
Catharanthus roseus (L.) G. Don (= Vinca rosea L.) (Gentianales:
Apocynaceae) (Tirtawidjaja 1981; Garnier & Bové 1983; Garnier et al. 1984b;
Zhou et al. 2008);
Cleome rutidosperma (Capparales: Capparaceae) (Brown et al. 2011);
Cuscuta sp. (Solanales: Convolvulaceae) (Tirtawidjaja 1981);
Cuscuta australis R. br. (Solanales: Convolvulaceae) (Su & Huang 1990);
Cuscuta campestris (Ghosh et al. 1977; Garnier & Bové 1983), and Cuscuta
pentagona (Duan et al. 2008, Zhou et al. 2008);
Nicotiana tobacum L. (Solanales: Solanaceae) cited as Nicotiana xanthii
(Garnier & Bove 1993)
Pisonia aculeate (Caryophyllales: Nyctaginaceae) (Brown et al. 2011);
Pithecellobium lucidum Benth (Fabales: Fabaceae) (Fan et al. 2011);
Solanum lycopersicum L. (Solanales: Solanaceae) (cited as Lycopersicon
esculentum cvs. Manapal and FL47) (Duan et al. 2008); and
Trichostigma octandrum (Caryophyllales: Phytolaccaceae) (Brown et al.
2011).
1.2.5.3 Light and electron microscopy
Schwarz (1965) reported the presence of a compound that exhibits a bright-violet
blue florescence under ultraviolet light when extracts of fruit tissues infected with
‘Ca. L. africanus’ were separated using paper chromatography. The fluorescent
substance was identified as gentisoyl glucose (Hooker 1993). Significant amounts
occur in fruits of trees with leaves exhibiting severe HLB symptoms than in fruit of
22
trees less symptomatic foliage (Hooker 1993).
Hooker (1993) concluded that
gentisoyl glucose was not a specific test for HLB since increased levels of gentosoyl
glucose were also found associated with diseases other than HLB (e.g., CTV).
Phloem hyperplasia and necrosis caused by ‘CLas’, and starch accumulation
resulting from degeneration of the phloem, were observed under light microscopy by
Matsumoto et al. (1961) and, subsequentlyby others including Tirtawidjaja et al.
(1965), Schneider (1966, 1968), Aubert (2008) and Su & Huang (1990).
‘Candidatus Liberibacter africanus’ was first observed by electron microscopy in
sweet orange phloem in 1970 (Laflèche & Bové 1970).
The technique was
subsequently used by Catling et al. (1978), Bové & Garnier (1984) and Wu & Zhang
(1985) to observe ‘CLas’, but it was not suitable for differentiating between ‘CLas’
and ‘CLaf’.
Nevertheless, it remained the only technique for detection of the
pathogens until 1992 (Garnier & Bové 1996).
1.2.5.4 Iodine starch test and plant metabolites
Iodine starch test is based on Schneider (1968), who observed that starch
accumulated in parenchyma cells of leaves infected with ‘CLaf’. Eng et al. (2007)
described different iodine formulations and types of abrasive paper used in iodine
starch test. Iodine formulations of 1.2% and 1% gave 90% positive results using
leaves from PCR-positive honey mandarin trees.
Increased levels of host-plant metabolites associated with HLB symptomatic leaves
were reported by Manthey (2008) who detected high levels of hydroxycinnamates
and flavonoids in HLB-infected leaves using reversed–phase high pressure liquid
chromatography–mass spectrometry (HPLC–MS). HLPC–MS also detected higher
concentrations of limonin glucoside in symptomatic leaves than in healthy leaves
(Manthey 2008).
23
1.2.5.5 Serology and molecular techniques
Bové (2006) noted that 13 monoclonal antibodies specific for the HLB pathogens
were produced between 1987 and 1993, the number limited by the fact that the
bacteria were not available in culture. However, the technique could not accurately
separate ‘CLaf’ and strains of ‘CLas’ and was superseded by the use of molecular
techniques (Garnier & Bové 1996).
Villechanoux et al. (1992) were the first to use molecular techniques to detect the
pathogens. They obtained two DNA probes, In-2.6 and AS-1.7, through differential
hybridisation of DNA from infected and healthy periwinkle plants. The probes could
detect strains from India, China, and Southeast Asia. Jagoueix et al. (1994) designed
primers based on the sequence of these probes that amplify portions of the 16S
rDNA of the Asian and African HLB strains: OI1 and OI2C from Asiatic strain and
OI3c from the African strain. Jagoueix et al. (1996) used the same primers to
amplify rDNA and showed that the pair OI1/OI2c amplified both ‘CLaf’ and ‘CLas’
while the pair O2c/OA1 amplified ‘CLaf’. Thus, in places where both ‘CLaf’ and
‘CLas’ were suspected, use of the OI2c/OI1/OA1 primers were recommended
(Jagouiex et al. 1996; Bové 2006). Moreover, digestion of the amplified DNA with
the restriction enzyme, XbaI, allowed distinction between ‘CLaf’ and ‘CLas’,
producing two fragments of 520 bp and 640 bp for ‘CLas’, and three fragments of
520 bp, 506 bp and 130 bp for ‘CLaf’ (Jagouiex et al. 1996). Subsequently, another
PCR method based on ribosomal protein genes of the beta operon using primers A2
and J5 was developed for distinguishing ‘CLaf’ from ‘CLas’ (Hocquellet et al. 1999;
Bové 2006).
A third putative HLB liberibacter species, ‘Candidatus Liberibacter americanus’
(‘CLam), was found in São Paulo, Brazil, in 2004 when attempts to detect ‘CLas’ in
strongly symptomatic sweet orange plants using the primers OI1/OI2c/OA1 yielded
negative results in contrast to the positive controls of ‘CLas’ and ‘CLaf’ (Teixeira et
al. 2005b). This led to a new pair of primers, GB1 and GB3, for detection of
‘CLam’.
24
More recent PCR methods include:
nested PCR, where a second primer set is added to amplify the product of the
first PCR, was used to analyze lemon and wampee samples regardless of
HLB symptoms and was found to be 104 more sensitive than single step PCR
(Ding et al. 2005).
Multiplex PCR can detect all three forms of HLB
pathogens in a single test using all the different primer sets together (Bové
2006; Li et al. 2006);
loop mediated isothermal amplification (LAMP) was developed to facilitate
rapid detection of the HLB pathogens in laboratories without a thermocycler
(Okuda et al. 2005) and has been adapted and improved over the years to detect
‘CLas’ in both plant and psyllid samples (Rigano et al. 2014; Manjunath et al.
2015); and
real-time PCR using 16S rDNA-based TaqMan primer-probes developed to
provide rapid, sensitive and robust tests for ‘CLaf’, ‘CLam’ and ‘CLas’ (Li et
al. 2006; 2007a). Comparison of results of the real-time PCR test designed by
Li et al. (2006) used for detecting ‘CLas’ and assessments based on visual
inspection of symptoms in Florida revealed that the incidence of infection was
two-fold greater than determined by the visual inspections due to the test being
able to detect the pathogen in asymptomatic tissues (Irey et al. 2006). Li et al.
(2007a) compared conventional PCR, LAMP and TaqMan real-time PCR to
assess their sensitivity, accuracy and specificity in detecting HLB pathogens.
The results showed that TaqMan real-time PCR was 10–100 times more
sensitive than conventional PCR or LAMP (Li et al. 2007a). Lin et al. (2010)
developed a system comprising nested PCR and TaqMan PCR that can be
performed sequentially in the same tube. This dual system involving two
primer pairs and two different annealing temperatures proved to be a very
sensitive and quantitative system to detect ‘CLas’.
PCR has also been used to detect liberibacters in psyllids. Hung et al. (2004) used
conventional PCR to detect ‘CLas’ in third, fourth and fifth instar, and adult
Diaphorina citri, including single specimens.
25
Teixeira et al. (2005a) used
conventional PCR with GB1/GB3 primer pair to detect ‘CLam’ in Diaphorina citri
in Brazil. TaqMan based real-time PCR developed by Li et al. (2006) was adapted
by Manjunath et al. (2008) to detect ‘CLas’ in Diaphorina citri in Florida. Psyllids
collected from asymptomatic trees and locations where HLB was not confirmed
tested positive for ‘CLas’ (Manjunath et al. 2008). This suggested that testing of
psyllids may be a rapid means of determining the presence of HLB pathogens in
regions where presence of the disease is suspected in the absence of symptomatic
trees. Real-time and conventional PCRs were also used by Lee et al. (2015) to test
both plant and psyllid samples for HLB in the experiment to determine the latent
period for HLB. They (Lee et al. 2015) were able to show that HLB-free plants
became infectious in less than 15 days of feeding by pathogen-bearing Diaphorina
citri adults, as shown by HLB-positive second-generation adults and plant parts
where the second-generation psyllid nymphs developed. Conventional and real-time
PCR were used by Donovan et al. (2012a) to detect ‘CLas’ in adult Diaphorina
communis collected in Bhutan.
Real-time method of Li et al. (2006) and
conventional PCR using primers A2/J5 (Hocquellet et al. 1991) were applied for
detection of ‘CLas’ in the current study.
1.2.6
Psyllid fauna of Asia
1.2.6.1 Diaphorina citri
Diaphorina citri was described by Kuwayama (1907) from males and females
collected on citrus in Taiwan. It was described as Euphalerus citri (Kuwayama) by
Crawford (1912), and this name was briefly used in literature (Lal 1917; 1918;
Fletcher 1917; 1919, Crawford 1913; 1917; 1919).
Crawford (1912) based his
descriptions on one female from Adra (now in) West Bengal, India, previously
described by JT Jenkins in 1909 (Husain & Nath 1927), and 17 specimens including
both sexes, collected by George Compere from the Philippines. Crawford (1912)
commented that Compere found the same insect in India on citrus trees in
considerable numbers during a visit to India (in 1907). Crawford (1924) transferred
the species to Diaphorina. Crawford (1919) and Kuwayama (1931) considered
Diaphorina citri to be widely distributed and common throughout the Eastern
26
Hemisphere—in Taiwan, the Philippines, Malaysia, Indonesia (Java, Ambon,
Moluccas) and southern China. Clausen (1933) noted its presence in Myanmar and
Sri Lanka. Lal (1917; 1918), Fletcher (1917; 1919), Husain & Nath (1927), Pruthi &
Batra (1938) and Pruthi & Mani (1945) mentioned it as pest in pre-partition India. It
was recorded in Brazil about 1940, but not elsewhere in the Americas until 1998,
when it was found in Florida (Halbert & Manjunath 2004). It has since spread
rapidly in the Americas and continues to do so (Halbert & Manjunath 2004; Halbert
& Núñez 2004; Halbert et al. 2010; Hall et al. 2013). It also occurs in Réunion and
Arabia (Halbert & Manjunath 2004) and was recorded in New Guinea in 2002
(Weinart et al. 2004) and Pacific islands of Hawaii (Conant et al. 2006), Guam (Poe
& Shea 2007), American Samoa (www.aphis.usda.gov) and Samoa (Richard Davis,
pers. comm., 2012). It can transmit ‘CLaf’, ‘CLam’ and ‘CLas’ (Aubert 1987;
Halbert & Manjunath 2004; Bové 2006; Lopes et al. 2010; 2013). In Bhutan,
Diaphorina citri was recorded in Phuentsholing, a town in the south on the border
with India, during surveys between 1972 and 1990 (Lama & Amtya 1991; Lama &
Amatya 1993), but the psyllid is now found in 14 of the 16 citrus growing districts
(NPPC report; pers. obs.).
The presence of Diaphorina citri in southern China, as reported by Crawford (1919),
was based on the collection of six specimens collected in Macau by Frederick Muir
in November 1906. Mention of its presence in southern China by Clausen (1933)
was most probably based on these specimens. It is not clear from the literature
whether its distribution in southern China extended from Macau into Guangdong or
elsewhere in mainland China, or whether specimens collected by Muir were from
established populations. The host plant was not recorded (Crawford 1919). Huang
(1953), citing Hoffmann (1936), stated that the psyllid was first recorded in
Guangzhou in 1934. Hoffmann (1936) reported receiving adult specimen collected
from lemon (Citrus × limon (L.) Osbeck), wong p’ei (wampee, huangpi: Clausena
lansium (Lour.) Skeels), Kam (Citrus × aurantium L. as Citrus nobilis Lour.), Kat
(Citrus × aurantium as Citrus nobilis Lour. var. delicosa (Ten.) Swingle)), sweet
orange (Citrus × aurantium as Citrus sinensis Osbeck)), and pomelo (Citrus maxima
(Burm.) Merr. as Citrus grandis Osbeck). By spring of 1935, both nymphs and
27
adults of Diaphorina citri were recorded feeding on different citrus plants in
orchards at Lingnan University (now Zhongshan University) and nearby orchards on
Honam Island, Guangzhou (Hoffmann 1936).
A. Description of adult Diaphorina citri and sex ratio
Husain & Nath (1927) cited Crawford’s (1912) description of the adult Diaphorina
citri as: body length of 2.4 mm; generally brown coloured; variable abdomen colour
from usual greyish brown to bluish or orange depending on the contents of the
abdomen; orange abdomen colour related to gravid females or sexual maturity;
antennae 10 segmented with black tips, and shorter than the head; forewings broadest
at half of the apex and subhyaline. Halbert & Manjunath (2004) reported that
Diaphorina citri has very clear wing pattern that allows for easy differentiation from
other species. In contrast to Crawford (1912) and Husian & Nath (1927), Wenninger
& Hall (2008) reported that females with green or bluish abdomens can be gravid
and even males can have orange abdomens, and that abdomen colour has no relation
to sexual maturity.
The ratio of males to females is approximately 1:1. In field samples, Catling (1968b,
1970) recorded 44.8% males, and Aubert & Quilici (1988) 49% and 54.5% in
separate samples. Males have abdomen tips bent upwards in contrast to the straight
end of the females (Husain & Nath 1927). Adults are weak flyers and usually found
feeding on the lower side of the leaves forming an angle of 30–45 °C to the leaf
surface (Husain & Nath 1927; Hall et al. 2013).
B. Life cycle and seasonal fecundity
Husain & Nath (1927) reported that adults mate shortly after eclosion. Pande (1971)
observed that mating took place 12–60 h after eclosion.
The fecundity of
Diaphorina citri females is high. Husain & Nath (1927) recorded up to 807 eggs per
female, Pande (1971) 180–520, Huang (1990) up to 1,900 with averages ranging
from 630–1230, Liu & Tsai (2000) a maximum fecundity of 748 eggs at 28°C on
orange jasmine, and Tsai & Liu (2000), means of 858, 626, 572 and 612, and
28
maxima of 1378, 830, 818 and 994, respectively, on grapefruit, orange jasmine,
rough lemon and sour orange seedlings. The eggs are about 0.3 mm long, almondshaped with a thick base that narrows towards the distal end with a curve. Newly
laid eggs are pale and then turn yellow and subsequently orange just before hatching
(Husain & Nath 1927). They may be laid on tender shoots in such large numbers
that the shoots are rendered orange. Just before hatching, two red spots are visible
that develop into the nymph’s eyes (Husain & Nath 1927). Incubation periods for
eggs are influenced by ambient temperatures. Husain & Nath (1927) reported that
egg hatch took 4–6 d in summer and 22 d in winter in the Punjab. Pande (1971)
reported 4–6 d from late spring to mid autumn, and 8–18 d from late autumn to late
winter in nearby Rajasthan (Pande 1971).
Tsai & Liu (2000) observed that
development took around 4 d at 25°C. Pre-oviposition period and fecundity are
affected by light intensity and duration (Yang et al. 2006). They (Yang et al. 2006),
citing Chinese literature, noted that light intensity below 11000 1x and light duration
less than 18 h per day increased the number of eggs laid and pre-oviposition period.
There are five nymphal instars (Husain & Nath (1927; Yang 1984). Husain & Nath
(1927) reported a total duration of 11–25 d in the Punjab, with each instar lasting
about 3 d in summer and 4 d in winter, the exception for first instar that lasted 11 or
12 d in winter.
Catling (1970) reported development taking 11–15 d in the
Philippines. Yang et al. (2006) noted that nymphal development, especially for
females, depended on obtained nutrients from phloem.
Husain & Nath (1927) reported a total life span of 15–47 d in the Punjab, and Pande
(1971) 25–30 d from early spring to mid autumn, and 35–75 d from late autumn to
late winter in Rajasthan. A similar trend of development was observed by Xu et al.
(1988a) with an average period of nymphal development of 31.3 and 10.3 d in spring
and summer, respectively, in Fujian.
Females were observed to live longer with decreasing temperature when observed
within a range of 15–33°C, average longevity of females was recorded 88 d at 15°C
while individual female longevity was 117 d at 15°C, and 51 d at 30°C under
29
artificial conditions (Liu & Tsai 2000). Similar observation was made by Pande
(1971) who recorded average longevity as 40 and 45 d for males and females,
respectively.
Husain & Nath (1927) noted that all stages of Diaphorina citri occur throughout the
year in the Punjab, with slower development in winter. Most activity was observed
when host plants produce new flush growth, particularly after monsoonal rain
(Husain & Nath 1927). Husain & Nath (1927) recorded nine annual generations
under experimental conditions, with considerable overlap between generations
(Husain & Nath 1927). Pande (1971) reported up to 10 overlapping generations in
Rajisthan. Literature reviewed by Yang et al. (2006) indicated that, depending on
climate, 6–11 generations may occur in regions of China where the psyllid occurs,
and that overwintering stages comprise mostly adults. Diapause does not occur
(Husain & Nath 1927).
In China, due to warmer winters in recent years, Diaphorina citri is now able to
survive inland and in northern of regions where it did not occur previously (Yang et
al. 2006). Yang et al. (2006) also noted that local weather conditions and host
phenology are key factors in determining the active life stages during winter. All life
cycle stages are active in the presence of young flush growth and favourable warm
winter temperatures in Taiwan (Yang et al. 2006). In the absence of young flush
growth in winter, only adults are present with restricted movement in Guangzhou in
southern China.
C. Acquisition and transmission of liberibacters by Diaphorina citri
Huanglongbing is mainly spread in the field by Diaphorina citri (Halbert &
Manjunath 2004).
Early reports on acquisition and transmission of ‘CLas’ by
Diaphorina citri include studies by Tirtawidjaja et al. (1965), Celino et al. (1966),
Salibe & Cortez (1966), Capoor et al. (1967; 1974), Martin & Wallace (1967),
Raychaudhuri et al. (1972), Huang et al. (1984; 1990) and Xu et al. (1985; 1988b;
1988c).
30
Following observations on the graft transmissibility of the disease by Salibe &
Cortez (1966) in the Philippines, Celino et al. (1966) conducted transmission tests
with adult Diaphorina citri collected from citrus orchards with mottling symptoms.
The adults were allowed to feed on healthy sweet orange seedlings for two weeks or
more. They (Celino et al. 1966) established that Diaphorina citri transmitted the
disease. The results were confirmed using buds from symptomatic orange seedlings
resulting from psyllid feeding to graft inoculate onto healthy seedlings of ‘szinkom’
and ‘ladu’ mandarins. All test plants showed symptoms within 90–150 d (Celino et
al. 1966).
Capoor et al. (1974) found that 68% of plants became infected when grown singly
with a single psyllid that had fed on an infected plant for 24 h.
A minimum
acquisition period of 15 min was achieved with psyllids in groups of five when
starved for 2 h before feeding. This resulted in 90% infection of the test plants. A
low percentage of infection was achieved with feeding for 15 min, but 100%
infection was achieved after feeding for 1 h.
Capoor et al. (1974) reported a
minimum incubation period of 8–12 d before transmission could occur. Xu et al.
(1988b) reported that the incubation period could vary from 1–2 d or from 23–25 d
depending on the psyllid. Pelz-Stelinski et al. (2010) reported 4–10% transmission
with single psyllid compared to 88% infection with groups of 100 or more psyllids.
Capoor et al. (1974) showed that psyllids remain infective throughout their lives after
acquiring the pathogen. They (Capoor et al. 1974) also reported that fourth and fifth
instars were able to acquire and transmit the pathogen in contrast to the younger
instars, and higher numbers of nymphs feeding resulted in higher percent of infection
as shown by 100% infection with 25 nymphs feeding on a test plant compared to
15% infection with a single nymph. Xu et al. (1988b; 1988c) reported that fourth
and fifth instars could acquire and transmit the pathogen and that adults emerging
from such nymphs were infective. Huang et al. (1984) obtained low transmission
rates by adult Diaphorina citri with acquisition feeding and infection feeding periods
of one month each. They reported that successful transmission was obtained with
test psyllids that acquired the pathogen as nymphs.
31
Inoue et al. (2009) were the first to use PCR to confirm transmission of the disease
by Diaphorina citri. Inoue et al. 2009 monitored the concentration of ‘CLas’ in fifth
instar nymph and adult Diaphorina citri. They observed that the bacterial titre in the
adults did not increase significantly, and the psyllids failed to transmit the pathogen
on test plants compared to fifth instar nymphs. Titres increased significantly by 25-,
360- and 130-fold from the first acquisition day to 10, 15 and 20 d with acquisition
by fifth instars leading to 67% transmission by emerging adults. They suggested that
bacterium multiplies in the nymph, and the adults eclosing from infected nymphs are
able to transmit the pathogen more effectively than adults that acquired the pathogen
after eclosion. Pelz-Stelinski et al. (2010) reported similar results. Inoue et al.
(2009) noted that the adult psyllids did not transmit the bacterium persistently after a
24 h acquisition period as suggested by Capoor et al. (1974), and attributed the
different outcomes to different techniques and strains of the pathogen used in the two
studies. Capoor et al. (1974) used indicator plants, whereas Inoue et al. (2009) used
both conventional and quantitative PCR to detect infection.
Huang et al. (1990) reported high transmission rates during the spring in Taiwan, the
peak period for production of new flush by citrus trees. Chavan (2004) also reported
that, in the Indian state of Maharashtra, maximum infection in nymphs (66.6%) and
adults (45–50%) occurred in late winter and early spring.
Transovarial transmission of ‘CLaf’ in Trioza erytreae was reported by van den Berg
et al. (1991–1992).
Hansen et al. (2008) reported transovarial transmission of
‘Candidatus Liberibacter solanacearum’ (syn. ‘Candidatus L. psyllaurous’), the
cause of Zepra chip disease of potato, by Bactericera cockerelli (Šulc).
Pelz-
Stelinski (2010) reported 2–6% transovarial transmission of ‘CLas’ by Diaphorina
citri in contrast to Capoor et al. (1974), Xu et al. (1988b) and Hung et al. (2004)
reported no transovarial transmission of the pathogen by the psyllid.
1.2.6.2 Other psylloids known to feed on Rutaceae
Several psyllid species other than Diaphorina citri and Trioza erytreae are known to
feed on Rutaceae, including species and hybrids of Citrus. Only limited studies have
32
been undertaken to determine if they can transmit liberibacters. Species (some
possibly conspecific: Kandasamy 1986; Burckhardt 1994; Inoue et al. 2006; Inoue
2010; Cen et al. 2012a) in Asia include:
Cacopsylla (Psylla) citricola (Yang & Li);
Cacopsylla (Psylla) citrisuga (Yang & Li);
Cacopsylla (Psylla) evodiae (Miyataki);
Cacopsylla heterogena Li; (Li 2011 cited by Cen et al. 2012a); Chapter 6 of
the current study;
Cacopsylla (Psylla) murrayi (Mathur);
Cacopsylla toddaliae (Yang);
Diaphorina communis Mathur;
Diaphorina murrayi Kandasamy; and
Trioza citroimpura Yang & Li.
Burckhardt (1994) commented that Cacopsylla citricola and Cacopsylla citrisuga
may be identical to Cacopsylla murrayi.
Diaphorina communis was first collected on curry leaf at Dehra Dun (700 mASL),
Uttar Pradesh, northern India, in 1932 (Mathur 1935), but it was not described until
four decades later (Mathur 1975). Diaphorina mathuri Loginova is a synonym of
Diaphorina communis (Hodkinson 1986). Other hosts include orange jasmine and
citrus (Mathur 1975). It was reported as major pest of curry leaf in the Jammu region
of India by Tara & Sharma (2010). It was recently recorded as host of ‘CLas’ in
Bhutan (Donovan et al. 2012a; Chapter 3 of the current study), where it has been
recorded on citrus and more commonly, on curry leaf at elevations ranging from
~200 to 1223 m ASL.
Mathur (1975) provided keys and complete descriptions to distinguish nine species
of Diaphorina, including Diaphorina citri and Diaphorina communis, from each
other. Key distinguishing features separating adult Diaphorina communis from other
33
Diaphorina species are that they are black with more densely maculated wings. The
shape of the head, genal cones and genitalia are also distinguishing features.
The following description of Diaphorina communis adults is based on Mathur (1935,
1975). The wings and body are generally black with greyish-brown tinge. The body
length of males (2.1 mm) is shorter than that of females (2.4 mm). The head is
smaller than the thorax, and 0.6 mm wide, including the eyes. Antennae are 10segmented, 0.52 mm long and pale-yellow with few setae. The thorax is arched and
sparsely pubescent. The pronotum is flat, convexly round when viewed dorsally.
The legs are coarsely pubescent. The tibiae are longer than femora. Hind tibiae lack
a basal spur but have seven tooth-like spines at the apex. The forewings are large
and more than two times longer than wide, widest subapically, roundish at the apex
and narrow at the base. The first marginal cell is almost equal in length and width,
but shorter and wider than the second. Each of the four cells has one spot, two spots
present between Cu1 and Cu2 and 1 spot near the clavus; veins are lined with two
rows of tiny setae. The hind wings have 7 or 8 simple, and 4–6 hooked setae on the
coastal margin. The abdomen is long and has sparse hairs. Male genitalia are
smaller than the abdomen, and the anal valve is pyriform in shape and about 0.38
mm long. Female genitalia are smaller than the abdomen and the ovipositor is
pointed. Mathur (1975) also described the five nymphal instars. The nymphs cause
little damage to curry leaf plants if present in small numbers, but when they are
numerous, the leaves wilt and fall off gradually, and young growth can be killed
completely and tender branches deformed (Mathur 1935).
The female lays her oval (0.26 × 0.14 mm), smooth and light yellowish eggs singly
scattered about in leaf axils on the ventral surface of leaflets and tender branches
(Mathur 1935). Mathur (1935) recorded a female laying 600 eggs over 35 d in
summer, and egg hatch in 3–5 d in summer, 4–6 d in autumn and 9–14 d in late
winter. Full grown fifth instars are about 1.62 mm long and 1.45 mm wide, dark
grey with a green tinge and black antennae. The abdominal margin of fifth instar is
lined with 45–50 lanceolate setae (Mathur 1975).
34
Mathur (1935) observed that all instars could walk at a steady pace and exude large
quantities of honeydew in the form of translucent filaments. He (Mathur 1935) also
observed:
that nymphal development varied from 11–37 d in summer, 66–79 d in winter,
and 33–40 d in spring;
that the longevity of males and females in summer was 59 and 45 d,
respectively;
that female longevity was longer in the cold season than in summer;
that all developmental stages were present on the host plant from mid spring to
mid autumn;
nine annual generations, with considerable over-lapping; and
life cycles of 14–41 d in summer, 70–83 d in winter, and 47–54 d in spring.
Cacopsylla citricola and Cacopsylla citrisuga were collected from pomelo (Citrus
maxima (Burm.) Merr.) and citron (Citrus medica L.) trees at elevations ranging
from 750 to 1650 mASL in western Yunnan (Yang & Li 1984). The latter (Figure
1.6) was collected from a pomelo tree by GAC Beattie at Dura Sakalgre (25.4983°N,
90.2932°E, 1190 m ASL) in the Garo Hills of Meghalaya, India, on 25 October
2009. These records suggest that Cacopsylla citricola occurs on Citrus at 700–1800
m ASL in humid subtropical regions of Laos, Myanmar, Yunnan in China, and
Meghalaya, Nagaland, Manipur and Mizoram in northeast India (Beattie et al. 2012).
‘Candidatus Liberibacter asiaticus’ was recently detected in Cacopsylla citrisuga
nymphs collected 1200 mASL from ‘Eureka’ lemon (Citrus × limon L.) trees in
Yunnan (Cen et al. 2012a) and subsequently reported to transmit the pathogen (Cen
et al. 2012b).
Inoue et al. (2006) recorded Cacopsylla evodiae feeding on orange jasmine in Japan,
and noted that it occurs on Tetradium glabrifolium (Champ. ex Benth.) T. G. Hartley
(cited as Euodia meliifolia (Hance) Benth.) [Rutoideae] and feeds on ‘Zanthoxylum
beecheyanum var. alatum (Nakai) Hara’ [Rutoideae]. Inoue (2010) listed Toddalia
asiatica (L.) Lamarck [Rutoideae] as another host, and noted that Cacopsylla evodiae
35
is probably most closely related to Cacopsylla murrayi and similar to Cacosylla
toddaliae (Yang), which feeds on Toddalia asiatica in both Taiwan and Japan, and
Cacopsylla fagarae (Fang & Yang), which feeds on Zanthoxylum cuspidata Champ.
(cited as Fagara cuspidata (Champ.) Engl.) in Taiwan (Beattie et al. 2012).
Cacopsylla murrayi has been recorded on curry leaf, citron, acid lime (Citrus × limon
(L.) Osbeck, cited as Citrus acida Korel) and sour orange (Citrus × aurantium L.) in
northern India (Mathur 1975; Lahiri & Biswas 1979; 1980). Osman & Lim (1992)
recorded it feeding on curry leaf plants in Malaysia, and observed it occasionally on
citrus plants. Comments by Lahiri & Biswas (1980) suggest that it may have been
introduced to Meghalaya. They (Lahiri & Biswas 1980) reported that it inflicted
great loss to citrus cultivation: foliage of infested plants became deformed, resulting
in a depleted photosynthetic activity over and above the loss caused by intake of
plant sap by the nymphs (Beattie et al. 2012).
Cacopsylla toddaliae occurs on Toddalia asiatica (L.) Lam. [Rutoideae] in Japan and
Taiwan (Inoue 2010).
Diaphorina murrayi was recorded on curry leaf by Kandasamy (1986) in Chennai
(Madras) in Tamil Nadu, Southern India.
Prior to my study, Cacopsylla heterogena was known to occur on citrus in China (Li
2011, Cen et al. 2012a).
Adult males (42) and females (24) of Trioza citroimpura were collected and
described from mandarin trees 540 mASL in southern Yunnan (Yang & Li 1984),
near the Laos and Myanmar. Prior to this record, Trioza erytreae was the only
species of Trioza known to feed and develop on Rutaceae (Hollis 1984; Beattie et al.
2012).
36
Figure 1.6. Diaphorina communis adult on a mandarin leaf at Kamichhu, Bhutan, May 2009 (left);
adult of Cacopsylla citrisuga on pomelo flush at Dura Sakalgre, Garo Hills, Meghalaya, India (right)
(GAC Beattie).
1.2.7
Molecular markers used in the phylogenetic studies of plants
Molecular techniques such as DNA sequencing are fast, convenient and produce
large distinct data sets and have become a widely-used tool for phylogenetic studies
(Johnson & Soltis 1994). In plants, chloroplast DNA (cpDNA) has been widely used
to infer phylogenetic relationships at different levels, because it has a stable genetic
structure, is haploid and generally uniparentally inherited, there is no (or very rare)
recombination, and universal primers can be used to amplify target sequences
(Palmer et al. 1988; Taberlet et al. 1991; Gielly & Taberlet 1994; Small et al. 2004;
Dong et al. 2012). It comprises of protein coding regions, and introns and intergenic
spacers that do not encode proteins usually referred to as non-coding regions (Shaw
et al. 2007).
The non-coding regions evolve more rapidly and contain greater
variation than the coding regions (Shaw et al. 2005; 2007; 2014) that is applicable for
lower taxonomic levels (Small et al. 2004; 2005). Many of the non-coding regions
have been used in the molecular phylogeny of a wide range of plants including those
belonging to the Rutaceae (Nocolasi et al. 2000; Morton et al. 2003; Bayer et al.
2009; Morton 2009 & 2015; Lu et al. 2011; Nguyen 2011; French et al. 2016).
However, because cpDNA is uniparentaly inherited, it reveals information of only
one parent, and limits the use of cpDNA in plants of hybrid and polyploid origins
(Small et al. 2004). This limitation is compensated by use of the nuclear ribosomal
DNA (rDNA).
37
Although rDNA is ‘biparentally inherited, the process of concerted evolution, array
expansion/contraction and the presence of paralogous sequences can make isolation
of both parental copies difficult’ (Small et al. 2004). The interal transcribed spacer
(ITS) of 18S-26S nuclear rDNA, which includes the 5.8S subunit, an evolutionarily
highly conserved sequence, and two spacers known as ITS-1 and ITS-2 is commonly
used for plant phylogenetics (Baldwin 1992; Baldwin et al 1995; Álvarez &Wendel
2003) and has been used in Rutaceae (Morton 2009 & 2015; Othman et al. 2010;
Nguyen 2011; French et al. 2016).
Other nuclear genes employed in molecular phylogeny of plants includes the lowcopy nuclear genes: Adh (alcohol dehydrogenase), G3PDH (glyceraldehyde 3phosphate dehydrogenase), GBSSI (granule-bound starch synthase), MADS-box
genes (e.g. pistil/ala, apetalal, apetala3, leafY), PHY (phytochrome) and PGI
(phospho-glucose isomerase (Small et al. 2004), hyb (beta-carotene hydroxylase) and
mdh (malate dehydrogenase) (Schwartz 2011; Schwartz et al. 2015). Sang et al.
(2002) commented that although low-copy nuclear genes in plants are a ‘rich source
of phylogenetic information’ and have the potential to improve phylogenetic
reconstruction and resolve close interspecific relationships (especially where
universal markers such as cpDNA and rDNA are unable to do so), they remain
underutilised because of complicaitons related to evolutionary dynamics of nuclear
genes, and lack of universisal markers or primers.
1.2.8
Genes and molecular markers used in insect phylogeny
In insects, the most commonly studied regions are those within the mitochondrial
DNA (mtDNA) and the nuclear ribosomal DNA (rDNA) (Zhang & Hewitt 1997;
Caterino et al. 2000; Cameroon 2014). The frequently used mtDNA genes and
regions are COI and COII (cytochrome oxidase I & II), and 16S rDNA, followed by
12S rDNA, COIII, ND5 (NADH dehydrogenase 5), cytb (cytochrome b) with ND1,
ND2, and ND4 being less commonly used (Hwang & Kim 1999; Caterino et al.
2000; Mandal et al. 2014). For the nuclear rDNA, the 18S rRNA is used for higher
taxonomic levels, and popular regions for lower levels are the ITS and 28S regions;
among the nuclear protein-coding genes, EF-1α (elongation factor 1-alpha) PEPCK
38
(phosphoenolpyruvate carboxykinase), DDC (dopa-decarboxylase), wingless, white
gene among others have been used (Caterino et al. 2000; Danforth et al. 2005; Simon
et al. 2010).
1.2.9
Natural enemies and biological control
There are two primary parasitoids of Diaphorina citri, the ectoparasitic eulophid,
Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae), and the endoparasitic
encyrtid, Diaphorina aligarhensis (Shafee, Alam and Agarwal) (Hymenoptera:
Encyrtidae). A number of hyperparasitoids are associated with them in Southeast
Asia, with species composition varying across the region (Waterhouse 1998; Halbert
& Manjunath 2004). Both primary parasitoids were described from India, Tamarixia
radiata as Tetrastichus radiatus by Waterston (1922) and Diaphorina aligarhensis
by Shafee et al. (1975), and both species have been introduced, or spread naturally,
to regions to where Diaphorina citri has spread (Beattie et al. 2012).
Tamarixia radiata adults are 0.92–1.04 mm long with a black body, and hyaline
wings with pale veins. They have a short generation time and high reproduction
under laboratory conditions with total development period of 11.4 d. Females lay 1–
2 eggs under Diaphorina citri nymphs, and pupation takes place in the mummified
nymphs (Mann & Stelinski 2010). Adults of Tamarixia radiata emerge from the
thorax of the nymph leaving an exit hole (Aubert 11987).
Diaphorencyrtus aligarhensis adults are ~1–1.5 mm in length with yellow legs and
antennae, black head and thorax. Sexual dimorphism is exhibited by differences in
the morphology of antennae and the abdomen: females have smooth, clubbed
antennae while males have hairy and slightly longer antennae that are not clubbed
(Rohrig 2011). The total life cycle takes about 16–18 d under laboratory condition at
25°C (Rohrig 2011). One to two weeks-old females produced an average of 6.6
offspring under laboratory conditions (Skelly & Hoy 2004).
nd
aligarhensis parasitises 2
Diaphorencyrtus
th
to 4 instar nymphs of Diaphorina citri by ovipositing,
usually, a single egg into the host. Diaphorina citri nymphs continue to feed and
grow after parasitism until they are killed by the developing parasitoid. The mature
39
parasitoid leaves the mummified body of the Diaphorina citri nymph through its
abdomen (Rohrig 2011).
The introduction of both parasitoids to Réunion led to successful biological control
of their host (Aubert et al. 1996). Chien et al. (1989) introduced Tamarixia radiata
to Taiwan from Réunion in 1983–1986 to control Diaphorina citri; its establishment
was confirmed by March 1986. Although it was well established by 1987, parasitism
was variable (Chien et al. 1989). Chien et al. (1989; 1991) considered Diaphorina
aligarhensis to be indigenous to Taiwan, and to be quite active in host searching.
However, its rare occurrence during January to May results in resurgence of
Diaphorina citri in spring (Chien et al. 1989).
Chien et al. (1991), and Chein & Chu (1996) reported that the efficacy of Tamarixia
radiata and Diaphorina aligarhensis as parasitoids of Diaphorina citri was
influenced by environmental stability. Chien & Chu (1996) reported that populations
of Diaphorina citri were kept at 0 to 0.7 adults per 100 mm of branch under a stable
environment, whereas they reached 0.1 to 2.5 adults in an area where host plants
were pruned every 3–4 months and insecticides were sprayed.
Gavarra et al. (1990) reported that Diaphorina aligharensis was the more important
of the two species in orchards in Luzon and Mindanao in the Philippines where
average parasitism over one year ranged from 8.5–31%, and 10–70%, respectively.
Over the same interval, parasitism by Tamarixia radiata ranged from 0.3–67% and
6.7% to 22.2%, respectively.
Average percent parasitism by Diaphorina
aligarhensis and Tamarixia radiata in Luzon (n = 2977 nymphs) was 16.3% and
6.2%, respectively. Average percent parasitism by the two parasitoids in Mindanao
(n = 620) was 36% and 10.8%, respectively. Leong (2006) reported that parasitism
by Tamarixia radiata and Diaphorina aligharensis in Sarawak ranged from 4.4–
22.3% and 2.6–35.3%, respectively. Peak parasitism by the two species occurred in
November and December and coincided with peak abundance of Diaphorina citri.
In Malaysia, parasitism suspected to be from Tamarixia radiata ranging from 20% in
January to 36% in May on fourth and fifth instars of Diaphorina citri on Murraya
40
paniculata were observed during field surveys (Osman & Lim 1992). Introduction
of Tamarixia radiata to Guadeloupe successfully reduced Diaphorina citri
populations (Étienne et al. 2001).
In Mauritius, biological control of Diaphorina citri using Tamarixia radiata was not
successful in contrast to the control of Trioza erytreae by its parasitoid, Tamarixia
dryi (Toowara 1998). Toowara (1998) noted that the success of control of Trioza
erytreae was because its parasioid was able to survive on another host when citrus
had no flush growth and population of Trioza erytreae was low but Tamarixia
radiata did not have an anternaive host while its host, Diaphorina citri, flourished on
Murraya in the absence of citrus flush. Biological control with Tamarixia radiata
and Diaphorina aligarhensis was initiated in the United States of America but
Diaphorina aligarhensis failed to establish (Hall 2008). More recently, Tamarixia
radiata has been released in California (Hoddle 2012). However, the parasitism
rates of Tamarixia radiata in Florida are lower than in Réunion and other locations
(Hall & Nguyen 2010). Environmental factors, competition with other organisms,
genetic traits, chemical and nutrient sprays in the orchards could all affect the
efficiency of Tamarixia radiata (Hall & Nguyen 2010), and these factors probably
affect biological control by other natural enemies.
Species of ladybirds (Coleoptera: Coccinellidae), hoverflies (Diptera: Syrphidae),
lacewings (Neuroptera: Hemerobiidae, Chrysopidae), bugs (Hemiptera: Reduviidae)
and spiders (Aranae: Anyhpaenidae, Clubionidae, Salticidae, Oxyopidae) prey on the
adults and nymphs (Waterhouse 1998; Michaud 2002).
Aubert (1987) reported that the Citrus sooty mould fungus, Capnodium citri Berk. &
Desm. (Polychaeton citri (Persoon) Léveillé) (Capnodiales: Capnodiaceae)
(Reynolds 1999; Chomnunti et al. 2011), caused fungal epizootics affecting
artificially reared Trioza erytreae and Diaphorina citri nymphs when the relative
humidity was at saturation point.
Aubert (1987) also cited Grech & Samways
(1985), who reported that Cladosporium sp. nr. oxysporum Berk. & M.A. Curtis
(Capnodiales: Davidiellaceae) caused death and hyphal growth on Trioza erytreae
41
nymphs. Samways & Gretch (1986) reported that this species caused death and
hyphal growth on Trioza ertyreae nymphs, and that it had a considerable initial
impact on populations of the psyllid in the field. Hall (2008) cited Aubert’s (1987)
report on sooty mould fungus and Cladosporium Link. However, Aubert’s report for
the sooty mould fungus was erroneous due to misidentification (Bernard Aubert,
pers. comm. with Andrew Beattie, 28 January 2013).
Hirsutella citriformis Speare (Hypocreales: Ophiocordycipitaceae) and another
fungus, Isaria fumosorosea Wize (syn. Paecilomyces fumosoroseus (Wize) A.H.S.
Br. & G. Sm) (Eurotiales: Trichocomaceae) were found infecting adult Diaphorina
citri in citrus orchards in Indonesia (Subandiyah et al. 2000). Étienne et al. (2001)
recorded low levels of Hirsutella citriformis infection of nymphs and adults of
Diaphorina citri in the humid areas of Guadeloupe. In a recent two year study in
Florida, Hall et al. (2012) found high numbers of adult Diaphorina citri infected with
Hirsutella citriformis during the humid summer of the first year but not during the
second year. Hall et al. (2012) attributed low incidence of dead psyllids to low
psyllid populations and application of copper and mineral oil sprays in the orchards
during the second year.
Exposure of uninfected adult psyllids to Hirsutella
citriformis infected Diaphorina citri resulted in the death of the former psyllids
within 7–9 d (Subandiyah et al. 2000; Meyer et al. 2007; Hall et al. 2012). In
contrast, Isaria fumosorosea killed psyllids within 3 d (Meyer et al. 2007). This
difference in the rapidity of parasitism is probably due to the mechanism involved.
Hirsutella citriformis penetrates into the haemolymph of the psyllid and grows until
the host dies, whereas Isaria fumosorosea does not appear to enter the haemolymph
but rather releases toxins to kill the psyllids then utilises the dead insect for
development (Meyer et al. 2007; 2008).
Species of entomopathogens associated with Diaphorina citri in China include
Acrostalagmus
aphidum
Oudem
(Hypocreales:
Hypocreaceae),
Isaria
(Paecilomyces) javanicus (Friederichs & Bally) Samson & Hywel-Jones (Eurotiales:
Trichocomaceae), Lecanicillium lecanii (Zimm.) Zare & W. Gams (Hypocreales:
incertae sedis) (syn Cephalosporium lecanii Zimm. and Verticillium lecanii (Zimm.)
42
Viégas),
and
Beauveria
(Balsamo)
bassiana
Vuillemin
(Hypocreales:
Clavicipitaceae) (Yang et al. 2006). Hall (2008) cited Isaria fumosorosea, Hirsutella
citriformis, Lecanicillium lecanii, Beauveria bassiana, Cladosporium sp. nr.
Oxysporum Berk. & M.A. Curtis, and Capnodium citri Berk. & Desm as
entomopathogens of Diaphorina citri.
Notwithstanding the effectiveness of these natural enemies and impacts of orchard
chemicals on their effectiveness, biological control has limited scope in orchards
where HLB is present (Halbert & Manjunath 2004). Reliance on it is limited to
suppression of Diaphorina citri populations on Citrus and other hosts in home
gardens, parks and recreational areas.
1.2.10 Impacts of heat therapies on Citrus liberibacters
‘Candidatus Liberibacter asiaticus’ is more heat tolerant than the other two citrus
liberibacters, ‘CLaf’ and ‘CLam’ (Bové 2006; da Graça 2008; Lopes et al. 2010;
Lopes et al. 2013). Several studies have been undertaken on the impacts of heat
therapies on the pathogens. Early work by Lin & Lo (1965) showed that ‘CLas’infected trees treated with vapour saturated hot air at 48, 49, and 50 °C for 31, 35, or
40 min recovered after treatment and remained healthy for 28 months. Martinez et
al. (1971) demonstrated that 70% of the budsticks from infected tree treated in a hot
water bath at 45°C for 5 h survived the treatment and when budded onto healthy
seedlings, 35.7% of the budded trees did not show HLB symptoms. They (Martinez
et al. 1971) also treated infected seedlings with dry heat at 45°C for 5 h. Buds from
treated seedlings when budded onto test plants, some showed symptoms while some
did not. Similar tests with a hot water bath and with moist air were also conducted
by Raychaudhuri et al. (1974). Hot water treatment of buds at 50°C for 30 min
yielded viable buds and none of the plants produced HLB symptoms (6 of 6).
Similarly, buds treated with hot, moist air at 45°C for 6 h, and 47°C for 4 h resulted
in 100% viable buds but with only 5 of 8 plants in both treatment with no HLB
symptoms. Plants placed in a hot water bath at 55°C for 15 min resulted in only 2
out of 6 plants remaining HLB-free, while treatment with hot moist air at 51°C for 1
h did not yield any HLB-free plants (0/8 plants) though plants produced viable buds
43
in boht instances (Raychaudhuri et al. 1974). In Taiwan, separate studies conducted
by Su & Chang (1976) and Huang (1978) demonstrated recovery of infected
seedlings. Su & Chang (1976) observed recovery of seedlings of Ponkan orange on
Rangpur lime treated with hot air at 40°C in a growth chamber for 10 d whereas
reducing the days to three or seven did not suppress infection. Diseased seedlings
held at 30 and 40°C for 8 and 16 h cycle resulted in notable recovery of the plants
with healthy normal shoots (Su & Chang 1976). Huang (1978) demonstrated that
diseased buds grafted on Rangpur lime treated at 40°C during the light and 30°C
during the dark for 4 weeks or longer showed no HLB symptoms after two years.
Cheema et al. (1982) obtained a low incidence (20%) of HLB when infected buds
were treated at 47°C for 2 h and then budded onto grapefruit seedlings.
Lopes et al. (2013) observed low ‘CLas’ titres in plants maintained at 24–38°C
compared to plants held at 12 to 24°C or 18 to 30°C. Hoffman et al. (2013) used a
16 h light period at 40°C and an 8 h dark period at 30°C for four months to heat-treat
dormant budwood of grapefruit, sweet orange, lemon and pomelo. Their results
showed that only 12.5% (3 of 24) of treated budwood was ‘CLas’ positive after 12
months of treatment compared to 52% (11 of 21) for untreated budwood. In a
separate experiment, 2.5 years old grapefruit plants were exposed to 42°C for 19 h
and 30°C for 5 h, and 45°C for 16 h and 30°C for 8 h for a period of 2, 4, and 6 d
with intermittent cooler periods to reduce heat damage to the trees (Hoffman et al.
2013). All 27 surviving test plants had undetectable level of the pathogen 60 d after
treatment (Hoffman 2013).
1.2.11 Impacts of altitude, ambient temperatures and relative
humidity on ‘CLas’ and its vector
Aubert et al. (1985; 1988), and Yang et al. (2006) reported that the incidence of
Diaphorina citri on its Citrus hosts declined with increasing altitude above sea level.
Aubert (1987) observed that Diaphorina citri was more sensitive to high rainfall and
humidity.
Beattie et al. (2012) suggested that high saturation deficits favour
Diaphorina citri. This suggestion was based on observations in Pakistan where
44
ambient temperature recorded were ≥ 45°C at 160 to > 600 m ASL, and in Bhutan
where ambient temperatures of 36°C can occur at 1300 to 1500 m ASL. Beattie et
al. (2012) also noted that, in contrast to observations made by Liu & Tsai (2000),
Diaphorina citri can thrive at ambient temperatures above 33°C if relative humidity
is not high. In Bhutan, Diaphorina citri and HLB are common in orchards near
Punakha (27.5805°N, 89.8653°E, 1200 m ASL) where the saturation deficits exceed
0.55 kPa (Beattie et al. 2012).
Beattie et al. (2012) assumed that, under such
conditions, the psyllid can survive due to the impacts of evaporative cooling on leaf
temperatures and psyllid body temperatures.
Hoffman et al. (1975) observed
increased survival of the psyllid, Acizzia russellae Webb & Moran (Hemiptera:
Psylliade) on the host plant (Acacia karoo Hayne, Fabales: Fabaceae) at 43°C and
46°C and saturation deficits at 30–40 mm Hg (= 5.33 kPa) compared to lower or
higher saturation deficits under laboratory conditions. They (Hoffman et al. 1975)
hypothesised evaporative cooling by the host plant or the psyllid at these ranges
caused the increased survival whereas death due to less evaporative cooling and
thermal condition, and dessication occurred at low and very high saturation deficits
respectively.
Preliminary experiments with potted trees conducted in China on the effect of
saturation deficits and high leaf temperatures on ‘CLas’ showed low titres of the
pathogen in plants kept in clear polycarbonate cylinders compared to plants kept at
ambient temperature and humidity during September and October (Beattie et al.
2012). Leaf temperatures were also higher in the cylinder caged plants than the
control plants.
1.3 Aims and objectives
The overall aim of the study was to determine the roles of vectors, environment and
potential host species in the aetiology of huanglongbing in Bhutan. The specific
objectives of the research were to determine:
the
phylogenetic
relationships
between
Diaphorina citri;
45
Diaphorina
communis
and
whether Diaphorina communis can acquire ‘Candidatus Liberibacter
asiaticus’ from mandarin and transmit it from mandarin to mandarin and from
mandarin to curry leaf or vice versa, and to study the host preferences of
Diaphorina communis;
the molecular and morphological characteristics of the parasitoid of
Diaphorina communis, and to determine the phylogenetic relationships
between the parasitoids collected from Diaphorina communis and Diaphorina
citri;
the effect of altitude, ambient temperature and relative humidity on leaf
temperature, and determine the incidences and types of psyllids, and
prevalence of ‘CLas’ at different elevations;
the morphological and molecular characteristics of the green psyllids found
on mandarin and wild citrus, and other psyllids found on an unidentified
Zanthoxylum sp., and to determine the presence of ‘CLas’ within the green
psyllids and their host plants; and,
the phylogenetic and morphological relationships of species of Murraya
occurring in Bhutan with those proposed by Nguyen (2011), to determine the
phylogenetic relationship of the wild citrus species found in Bhutan in
relation to other Citrus species and hybrids, and whether the Murraya species
and wild citrus found in Bhutan can be hosts for ‘CLas’.
46
Chapter 2: Phylogenetic analysis of Diaphorina
communis Mathur and Diaphorina citri Kuwayama
___________________________________________________________________
2.1
Introduction
Diaphorina communis was first recorded by Mathur (1935; 1975) on curry leaf
(Bergera koenigii L., cited as Murraya koenigii Spreng.). Mathur (1935) noted that:
females could lay 600 eggs in 35 d; nymphal development varied from 11–37 d in
summer, 66–79 d in winter and 33–40 d in spring; and males lived for 45 d in midsummer and females for 59 d.
Morphologically, adult Diaphorina communis are mostly black (Mathur 1975) and
are easily distinguished from adults of Diaphorina citri Kuwayama, which are
mostly brown (Figure 2.1). However, the nymphs of the two species are similar with
key distinguishing features detectable only in fifth instar nymphs. The fifth instar
nymphs of Diaphorina communis are dark grey with a green tinge, black abdomen
and antennae, and a margin lined with 45–50 lanceolate setae (Mathur 1935, 1975).
In contrast, the fifth instar nymphs of Diaphorina citri are light yellow with an
orange tinge, dark antennae, and 53–60 lanceolate setae on the margin of their
abdomen (Husain & Nath 1927) (Figure 2.2).
Molecular data can supplement
morphological characters particularly when challenged with identification of
nymphs. Thus, this chapter focuses on molecular differences between Diaphorina
communis and Diaphorina citri using the mitochondrial COI and 16S regions.
47
2.2
Material and methods
2.2.1 Specimen collection and DNA extraction
Adult specimens of Diaphorina communis were collected by tapping branches of
Bergera koenigii L. (curry leaf) onto a white enamel tray, and aspirating the adults
falling on the tray into a glass vial.
Adults were collected from four sites, at
Basochhu (27.3646°N, 89.9124°E, 1036 m ASL) and Basochuu 2 (27.4136°N,
89.9036°E, 1223 m ASL) in Wangdue Phodrang Dzongkhag (district), Sunkosh
(Dagana-Tsirang Highway Junction (27.02789°N, 090.07511°E, 391 m ASL), and at
Reldri (Rinchending-Pasakha Highway, 26.8400ºN, 89.4045ºE, 400 m ASL) in
Chukhha Dzongkhag.
Extraction of DNA from psyllids and PCR analysis were performed in the Molecular
Laboratory of the School of Science and Health (SSH), Western Sydney University,
Hawkesbury Campus. Psyllid genomic DNA was extracted from specimens stored
in 100% ethanol. Each specimen was placed in a 1.5 mL tube and washed with 100
µL of 2% bleach plus Triton X-100 for 1 min with gentle vortexing and rinsed three
times with Milli Q water.
Then, DNA was extracted by crushing the washed
specimen with a sterile plastic pestle in a new 1.5 mL tube using the ISOLATE II
Genomic DNA Kit (Bioline). The pre-lysis incubation was performed overnight at
56ºC and 300 rpm in a shaking incubator. During the DNA elution step, DNA was
eluted in 80 µL of elution buffer and stored at −20°C until further use.
2.2.2 Amplification of Diaphorina communis DNA for phylogenetic
analyses
The COI region was amplified by conventional PCR (cPCR) using primers DCITRI
COI-L (5′-AGG AGG TGG AGA CCC AAT CT) and DCITRI COI-R (5′- TCA
ATT GGG GGA GAG TTT TG) (Boykin et al. 2012). Reactions were performed
using 2 µL of template DNA in a 20 µL reaction volume following the GoTaq®
Flexi DNA polymerase (Promega) protocol with final concentrations of 2.5 mM
MgCl2, 0.24 mM dNTP mix (Bioline), 0.4 µM of each primer and 1.25 U of GoTaq
48
Flexi DNA polymerase. The cycling parameters used by Boykin et al. (2012) were
applied: initial denaturation at 94°C for 2 min followed by 35 cycles of 30 sec
denaturation at 94°C, 30 sec annealing at 53°C, 1 min extension at 72°C followed a
final step of extension of 72°C for 10 min.
49
Figure 2.1. Adults (top) and nymphs (bottom) of Diaphorina communis on Bergera koenigii (Photos:
N. Om & GAC Beattie).
50
Figure 2.2. Scanning electron micrographs of the abdomens of fifth instar nymphs of Diaphorina
communis (A) and Diaphorina citri (B) showing the differences in marginal setae.
The 16S region was amplified using the same reaction mixtures as for the COI but
using the primers, LR13943F (5’-CAC CTG TTTA TCA AAA ACA T-3’) and
LR13392R (5’-CGT CGA TTT GAA CTC AAA TC-3’) (Costa et al. 2003). PCR
conditions for the 16S gene were adapted from Costa et al. (2003) with an initial
denaturation at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 1
min, annealing at 42°C for 90 sec, extension at 64°C for 90 sec followed by a final
extension at 64°C for 5 min.
2.2.3 Post PCR and sequencing
The PCR products were subjected to electrophoresis in 1% agarose containing
GelRed (0.1 L/mL, Biotium). Gel images were visualized under a UV
transilluminator and acquired with a GelDoc (BIORAD). Successful amplifications
were selected for sequencing. Prior to sequencing, PCR products were cleaned with
a mixture of thermosensitive alkaline phosphatase (TSAP (1U/µL, Promega) and
Exonuclease I (20 u/µL, New England Biolab). The TSAP mixture was prepared by
adding 2.5 µL of Exonuclease I, and 25 µL of TSAP to 172.5 µL of molecular
biology grade water. The TSAP mixture was added to PCR products at the rate of 2
µL TSAP mixture per 20 µL PCR product and the mixture incubated at 37°C for 30
51
min followed by a deactivation step at 94°C for 5 min. The cleaned PCR products
were sent for sequencing at Macrogen Inc. (Seoul, Korea).
Sequencing was
performed in both directions with the same primer sets as used for PCR.
2.2.4 Sequence assembly
Contiguous sequences of DNA obtained from this study were assembled with DNA
Baser Sequence Assembler software (Version 4, Heracle BioSoft) or with Geneious
(Version 8, Kearse et al. 2012). Sequences of other accessions were obtained from
GenBank. The sequences were aligned using Muscle as enabled in MEGA (Version
6, Tamura et al. 2013). Each alignment was cross checked by eye to verify base
calling and for the presence of indels.
2.2.5 Phylogenetic relationships of Diaphorina communis
Phylogenetic relationships among the Diaphorina communis accessions from Bhutan
and those of Diaphorina citri were determined based on the two mitochondrial DNA
regions. Aligned sequence datasets were subjected to phylogenetic analyses by
maximum likelihood (ML) using MEGA 6. For COI, the ML analysis was based on
the Tamura 3-parameter model (Tamura 1992) with a discrete Gamma distribution (6
categories (+G, parameter = 1.0753)) and, for 16S, the Tamura-Nei model (Tamura
& Nei 1993) with a discrete Gamma distribution (6 categories (+G, parameter =
0.4622)). For both analyses, initial trees for the heuristic search were obtained
automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of
pairwise distances estimated using the maximum composite likelihood approach, and
then selecting the topology with the superior log likelihood value. Each analysis was
replicated with 1000 bootstraps. GenBank accessions used for the two analyses are
shown in Table 2.1.
52
Table 2.1. GenBank accessions used for phylogenetic analyses in this study.
Region
Accession number
Reference
COI
AY971889
Liu et al. (2006)
FJ190289
Boykin et al. (2012)
FJ190375
Boykin et al. (2012)
FJ190377
Boykin et al. (2012)
KC509561
Lashkari et al. (2014)
KF702297
Chaitanya et al. (unpublished)
KF702299
Chaitanya et al. (unpublished)
KF702304
Chaitanya et al. (unpublished)
KU647697
Wu et al. (2016)
AB721011
Katoh et al. (2013)
16S
53
2.3
Results
2.3.1 Phylogenetic analysis of Diaphorina communis
BLAST searches using the COI sequences obtained in this study found that my
Diaphorina communis sequences shared 94% identity with Diaphorina citri; for the
16S region, the Diaphorina communis sequences showed 97–98% sequence identity
to Diaphorina citri.
The phylogenetic relationship of Diaphorina communis with Diaphorina citri based
on the COI region is shown in Figure 2.3. The tree was rooted with Bactericera
cockerelli (Šulc) (Hemiptera: Triozidae).
Accessions of Diaphorina communis
formed a separate clade from Diaphorina citri with strong bootstrap support (99%).
Variations within the group were observed but were not location specific. The
phylogenetic analysis of Diaphorina communis based on the 16S region is given in
Figure 2.4. The tree is rooted with one of the accessions of Cacopsylla heterogena
Li obtained in this study (see Chapter 6). Two distinct clades with high bootstrap
support are obtained, but no variation within the groups was found.
2.4
Discussion
The phylogenetic relationships between Diaphorina communis and Diaphorina citri
were determined based on COI and 16S regions. In both the analyses, two distinct
groupswere formed with strong bootstrap support, with one comprising accessions of
Diaphorina communis and the other of Diaphorina citri. Variation within the groups
was observed in the COI region but the variation was not related to locations from
which specimens were collected. The results indicate that differences in COI and
16S regions may prove useful for differentiating between early instar Diaphorina
communis and Diaphorina citri nymphs when fifth instar nymphs and adults are not
present on host plants.
54
Figure 2.3. Phylogenetic analysis of Diaphorina communis based on COI using maximum likelihood
and the Tamura 3-parameter model (Tamura 1992) with a discrete gamma distribution (6 categories
(+G, parameter = 1.0753)) to model evolutionary rate differences among sites. Name of the collection
sites are given next to the accession numbers. GenBank accession numbers are given in parentheses.
55
Figure 2.4. Phylogenetic analysis of Diaphorina communis based on the 16S gene using maximum
likelihood and the Tamura-Nei model (Tamura & Nei 1993) with a discrete gamma distribution (6
categories (+G, parameter = 0.4622)) to model evolutionary rate differences among sites. The names
of the collection sites are given next to the accession numbers. Genbank accession numbers are given
in parentheses. The tree is rooted with Cacopsylla heterogena (Chapter 6).
56
Chapter 3: Transmission of ‘Candidatus Liberibacter
asiaticus’ (α-Proteobacteria) by Diaphorina
communis Mathur (Hemiptera: Sternorrhyncha:
Liviidae)
____________________________________________________________________
3.1
Introduction
Many species of psyllids are important pests of agricultural crops (Burckhardt 1985,
1994). Their feeding may cause necroses, galling, and malformations of their host
plants (Wallis 1955; Markkula & Laurema 1971; Burckhardt 1994). Certain species
are also vectors of serious plant pathogens, including bacteria, phytoplasmas, and
viruses (Munyaneza 2010). The Asiatic citrus psyllid, Diaphorina citri, is the main
vector of ‘Candidatus Liberibacter asiaticus’ (‘CLas’), the pathogen primarily
responsible for devastating impacts on citrus production in Asia, and most recently in
the Americas (Bové 2006; Lopes et al. 2013; Hall et al. 2013). Diaphorina citri is
also the vector of the less important ‘Candidatus Liberibacter americanus’ (‘CLam’)
in Brazil and, under experimental conditions, has transmitted ‘Candidatus
Liberibacter
africanus’
(‘CLaf’),
the
pathogen
naturally
associated
with
huanglongbing in sub-Saharan Africa (Aubert 1987; Halbert & Manjunath 2004;
Bové 2006; Chiyaka et al. 2012; Wang & Trivedi 2013). ‘Candidatus Liberibacter
asiaticus’ has also been detected in adults of the black psyllid, Diaphorina communis
Mathur, a species collected in Bhutan from ‘CLas’-infected mandarin trees in May
2009 (Donovan et al. 2012a; 2012b). Beside these two species, ‘CLas’ has also been
recorded in Cacopsylla citrisuga (Yang & Li) collected from ‘CLas’-infected lemon
trees in Yunnan, China (Cen et al. 2012a), and from Cacopsylla heterogena Li
collected from infected mandarin trees in Bhutan (Chapter 6). Cacospylla citrisuga
has also been reported to transmit ‘CLas’ under laboratory conditions (Cen et al.
2012b).
As mentioned in earlier chapters Diaphorina communis was first recorded by Mathur
(1935; 1975) on curry leaf.
Mathur (1935) noted that wilting and defoliation
occurred when numerous nymphs attacked a plant. Mathur (1975) described the
57
insect in greater detail and commented that it was commonly found on curry leaf and
‘Murraya paniculata (L.) Jack’, but rarely on Citrus spp. near Dehra Dun,
Uttarakhand, in north-west India. Decades later, Diaphorina communis was reported
as a serious pest of Bergera koenigii (cited as Murraya koenigii) in the state of
Jammu and Kashmir in India (Tara & Sharma 2010a; 2010b). In Bhutan, it was first
identified in 2007 (Yamamoto 2007; Bové 2014) but could have been present much
earlier. It is mainly found on curry leaf and occasionally observed on mandarin.
Huanglongbing was first recorded, as citrus greening, in south-western Bhutan in the
Phuentsholing region in surveys for citrus greening between 1972 and 1990 (Lama &
Amtya 1991; Lama & Amatya 1993). Citrus orchards in the west-central region of
Bhutan were observed with severe dieback symptoms in the early 1990s. This was
attributed to HLB. However, the presence of ‘CLas’ was only confirmed in 2002,
mainly in the orchards along the Punakha-Wangdue Valley through which the
Punatsangchhu-Sunkosh River flows (Ahlawat & Baranwal 2003; Doe Doe et al.
2003; Bové 2014). Since then, the disease has been detected in other parts of the
country, and it is now the most serious impediment to citrus production nationally
(Donovan et al. 2016; pers. obs.). Diaphorina citri was also first recorded in Bhutan
in the Phuentsholing region in surveys for citrus greening between 1972 and 1990
(Lama & Amtya 1991; Lama & Amatya 1993). Records from Nepal and Sikkim
suggest that both the disease and the psyllid may have been present in southern
Bhutan since the mid-1960s (Knorr et al. 1970; Catling 1978; Lama & Amtya 1991,
1992; Ohtsu et al. 1997).
Diaphorina communis was shown to acquire ‘CLas’ (Donovan et al. 2012a) but the
potential of this psyllid to transmit ‘CLas’ has not been determined. Moreover, the
status of curry leaf as a host of ‘CLas’ has not been unambiguously determined. At
the start of this study, assessments of Bergera spp. as potential hosts of ‘CLas’ was
limited to studies by Hung et al. (2000), Damsteegt et al. (2010) and Ramadugu et al.
(2016). Hung et al. (2000) attempted graft transmission with Bergera euchrestifolia
(Hayata) (cited as Murraya euchrestifolia and mistakenly referred to as curry leaf)
from ‘CLas’-infected sweet orange plants in Taiwan, but they did not observe the
58
symptoms commonly observed in Citrus species and hybrids. Moreover, they did
not detect ‘CLas’ by dot hybridisation. Here, it should be noted that curry leaf
(Bergera koenigii) does not occur in Taiwan (Huang et al. 1997) and the distribution
of Bergera euchrestifolia is restricted to Taiwan, Hainan, Yunnan, Guangxi, Guizhou
and Guangdong (Swingle & Reece 1967; Zhang et al. 2008). Toorawa (1998) noted
that ‘CLas’ was never detected in Murraya paniculata and Bergera koenigii (cited as
Murraya koenigii) by both electron microscopy and PCR. Damsteegt et al. (2010)
did not detect the pathogen in Bergera koenigii following transmission experiments
with Diaphorina citri. Ramadugu et al. (2016) reported a single detection with a
‘CLas’, based on a Ct value of 36 by real-time PCR, during a six-year evaluation
program with a range of hosts of the pathogen and Diaphorina citri. However,
symptoms were not reported, and DNA of the pathogen was not sequenced.
Therefore, the overall aim of this chapter was to evaluate whether Diaphorina
communis can transmit ‘CLas’ and whether curry leaf is a host of the pathogen. The
evaluations involved several small experiments, particularly preliminary experiments
in 2013, and field experiments in 2014 and 2015. For simplicity, general methods
relevant to all experiments are described in the Materials and Method section of the
chapter. Materials and methods for each experiment is followed by the results of
each experiment.
The specific objectives of this chapter were to:
1. determine whether Diaphorina communis can acquire ‘Candidatus
Liberibacter asiaticus’ from mandarin and transmit it from mandarin to
mandarin and from mandarin to curry leaf or vice versa; and
2. study the host preferences of Diaphorina communis.
59
3.2
Materials and Methods
3.2.1 Field site
For this study, potted plants infected with ‘CLas’ and pathogen-free populations of
Diaphorina communis were not available.
Therefore, the experiments were
conducted under field conditions in order to assess transmission of ‘CLas’ by
Diaphorina communis from naturally-infected mandarin trees to healthy mandarin
and curry leaf seedlings.
A mandarin orchard at Baychhu (27.2975ºN, 89.9669ºE, 784 m ASL), Wangdue
Phodrang Dzongkhag, was selected.
It was chosen because it was one of the
mandarin orchards where ‘CLas’ was first confirmed as being present in Bhutan in
2002 (Doe Doe et al. 2003) and because of its proximity, ~75 km (3–4 hours by car)
to the National Plant Protection Centre (NPPC), Semtokha, Thimphu (27.4411°N,
89.6682°E, 2301 m ASL). It originally comprised ~500 mandarin trees planted in
the mid-1980s.
When the study commenced, many trees had been recently
skeletonised or cut down (but not poisoned).
Those that remained had severe
dieback, mostly related to ‘CLas’. Both Diaphorina citri and Diaphorina communis
were present in the orchard. Temperature and relative humidity in the orchard were
recorded using Tinytags (PLUS 2-TGP-4500, Omni Instruments Australia Pty Ltd)
set to log records every 30 min.
3.2.2 Preparation of test plants: mandarin and curry leaf seedlings
3.2.2.1
Mandarin seedlings
One hundred and thirty, 15-month-old mandarin seedlings were obtained from the
nursery of the National Seed Centre (NSC) at Bhur, Gelephu (26.9075°N,
90.4293°E, 381 m ASL) in April 2013. These seedlings are referred to as the April
2013 consignment. They were used for all the experiments conducted in 2013 and
2014. Another consignment of 100 seedlings was obtained from the NSC in April
2014. These seedlings were used for the transmission experiment in 2015. All
seedlings were maintained in the greenhouse at the NPPC in Thimphu until they
60
were transported to Baychhu. The plants were maintained in 80–100 mm diameter,
170 mm deep, black, plastic tubes (polytubes). The potting mix was changed prior to
transporting to the field. For the plants remaining in a greenhouse, the potting mix
was changed once a year. The potting mix comprised a 1:1 mixture of autoclaved
garden soil:forest top soil. The seedlings were pruned every 45–60 d to maintain
their height at approximately 300–450 mm.
Seedlings were watered daily and
fertilized with 1 g of urea every 30–45 d. Occasional 0.25% (v/v) sprays of an nC24
agricultural mineral oil (SK EnSpray 99) were applied to suppress infestations of soft
scales and spider mites.
3.2.2.2
Curry leaf seedlings
Mature curry leaf fruit that had turned dark red were collected on 14 June 2013 from
Baychhu and sown within 24 h. Curry leaf usually grows in sandy soils in Bhutan.
Therefore, a potting mix was prepared by mixing river sand and forest top soil at a
ratio of 1:1. The seedlings were grown in the potting mix in 38-cell, black, plastic
seedling trays with 58 mm diameter and 65 mm deep cells (Figure 3.1). Each curry
leaf seed was squeezed from the fruit and sown directly into the seedling tray.
A
B
Figure 3.1. Curry leaf fruit (A) and a seedling tray with curry leaf seedlings 30 d after sowing (B).
61
The transplanting of curry leaf seedlings into polytubes (dimensions as above)
commenced one month after sowing and continued for another two and half months.
All seedlings were transplanted into black polytubes with a slightly modified version
of the potting mix used for seeds. The potting mix used for transplanting comprised
a 1:2:4 mixture of autoclaved garden soil:river sand:forest top soil, because the 1:1
river sand to forest soil mix was too porous. The seedlings were then kept in the
same greenhouse at NPPC as the mandarin seedlings with similar management
operations, except that the curry leaf seedlings did not require daily watering. Curry
leaf seedlings raised in the greenhouse were used for experiments in 2014 and 2015.
3.2.3 Extraction of DNA of ‘CLas’ from plants
Most DNA extractions from plants were performed in the Plant Pathology
Laboratory of NPPC in Bhutan unless specified. Samples included both leaves and
bark. Samples were washed in tap water and blot dried with tissue paper. For
detection of ‘CLas’ from leaf samples, only mid ribs were used by excising the mid
ribs with disposable razor blades. Half a gram (0.5 g) of each sample was placed in
an extraction pouch with 3.5 mL of CTAB buffer (2% CTAB, 0.2% β–mercapto
ethanol, 2% PVP10, 1 M Tris HCl, 0.5 M EDTA, 1.4 M NaCl, pH 8) and ground
with a homogenizer (HOMEX 6, Bioreba) at 300 rpm. The extracts were transferred
into 2 mL tubes and incubated in water bath at 65ºC for 30 min, shaking the tubes at
intervals. The extracts were then centrifuged for 5 min at 8000 × g and 20°C. Then,
1 mL of the supernatant was transferred into another 2 mL tube, and 1 mL of
chloroform:isomyl alcohol (24:1) was added and mixed well by inverting the tube.
The tubes were centrifuged at 16,100 × g and 20ºC for 5 min. The supernatants were
transferred to 1.5 mL tubes, and cold isopropanol was added (540 µL of isopropanol
for 900 µL of supernatant) and incubated at −20ºC overnight. After incubation, the
tubes were then centrifuged for 20 min at 16100 × g and 4°C to collect the pellets.
The pellets were washed twice with 70% ethanol and centrifuged at 16,100 × g for 5
min after each wash. The washed pellets were air- dried for 30 min and dissolved in
100 µL 1 × TE buffer, and stored at –20ºC in NPPC until transportation to Western
Sydney University and stored at −20ºC again until further use.
62
3.2.4 DNA extractions from psyllids for ‘CLas’ detection
Extractions of DNA from psyllids were performed in the Molecular Laboratory of
the School of Science and Health (SSH), Western Sydney University (WSU),
Hawkesbury Campus, and the Plant Pathology laboratory at NSW Department of
Primary Industries Elizabeth Macarthur Agricultural Institute (EMAI), Menangle,
NSW. Psyllids stored in 100% ethanol were placed in 1.5 mL tubes and the tubes
left open for 5 min in a laminar hood to remove the ethanol from the samples. DNA
was extracted using the Sigma REDExtract-N-Amp™ Plant PCR Kit (Sigma)
following the manufacturer’s protocol except for modifications of the volumes of
extraction and dilution buffers. For extractions from single adults 10 µL of each
buffer was used, for extractions from 2–3 adults (i.e., insects combined to form a
composite sample) 15 µL of each buffer was used, and 25 µL of each buffer was
used for extractions from five adults. For nymphs, 10 µL and 15 µL of each buffer
was used for extractions from 2–3 and five nymphs, respectively.
All psyllid
samples were crushed after adding the extraction buffer using sterile, 20–200 µL
pipette tips made blunt by flaming and pressing on the lid of the sample tube. The
blunt pipette tips were more efficient at crushing the psyllids than plastic pestles.
The crude DNA extracts were stored at −20ºC till further use.
Psyllid samples for phylogenetic studies were extracted from single adults as
described in Chapter 2 using an ISOLATE II Genomic DNA Kit (BIOLINE) except
the psyllids were not crushed (non-destructive DNA extraction).
3.2.5 Detection of ‘CLas’ by conventional and real-time PCR
All work on detection of ‘CLas’ using real-time PCR (qPCR) was performed at
EMAI. Detection of ‘CLas’ was performed by multiplexing the TaqMan probe
(HLBp) and primer set (HLBas and HLBr) targeting the 16S rDNA of the bacterium
in combination with either the probe and primers for the plant cytochrome oxidase
(COX) gene (Li et al. 2006) or the insect nuclear gene, wingless (wg) (Manjunath et
al. 2008), as internal controls. The TaqMan probe, HLBp, was labelled at its 5’terminal with 6-carboxy-fluorescein (FAM) reporter dye, and the labelling of probes
63
at the 5’- terminals for wg (DCP) and COX (COXp) were both replaced with CAL
Fluor 560 Orange (CAL) instead of hexachlorofluorescein (HEX) or tetrachloro-6carboxy-fluorescein (TET) reporter dyes as used by Manjunath et al. (2009) and Li et
al. (2006), respectively. All probes were labelled at the 3’-terminals with Black Hole
Quencher (BHQ)-1.
Real-time PCR reactions were performed using 0.1 mL, four–strip tubes with caps in
a Rotor Gene Q cycler (Qiagen) containing final concentration of 1 × PCR buffer,
6.0 mM MgCl2, 0.24 mM dNTPs (Bioline), 240 nM of each primer
(Invitrogen/Sigma), 240 nM of each probe (Biosearch), and 1U Taq immolase
(Bioline). To each reaction, 1 µL of template DNA was added to prepare a final
reaction volume of 12.5 µL. Cycling parameters previously optimized by EMAI
were used: pre–incubation at 50°C for 2 min followed by 95°C for 10 min and 40
amplification cycles of 95°C for 30 sec and 58°C for 40 sec. For all assays, positive
and non-template controls were included.
For plant samples obtained in 2015
onwards, an extraction control (healthy control) made from midribs of peach or apple
was included. Samples of citrus plants and Diaphorina citri that were known to be
‘CLas’-positive were used as positive controls for plant and psyllid assays,
respectively. Samples were interpreted as ‘CLas’ positive or not-detected based on
threshold cycle (Ct) values. The threshold cycle is the number of cycles required for
the fluorescent signal within a reaction to cross the threshold, and the Ct value of a
particular sample is the point at which sufficient amplicons have accumulated
(Adams 2006). Interpretation of results was based on the guidelines set by the
United States Department of Agriculture (USDA-APHIS, 2012). For plant assays,
samples were considered ‘CLas’-positive if the FAM Ct values were in the range of
0.00 < FAM Ct ≤ 36, and not detected if the FAM Ct was zero. If the samples
produced FAM Ct values in the range of 36 < FAM Ct ≤ 40 then the results for the
samples were deemed to be inconclusive. For psyllid assays, samples were positive
if they produced FAM Ct values in the range of 0.00 < FAM Ct ≤ 32, and ‘CLas’
was not detected if the FAM Ct is equal to zero, and the result is inconclusive if the
FAM Ct values were in the range of 32< FAM Ct ≤ 40. Representative samples
showing a FAM Ct value > 0.00 were then selected and subjected to conventional
64
PCR (cPCR) using primers A2/J5 (Hocquellet et al. 1999).
Reactions were
performed using 2 µL of template DNA in a 25 µL reaction volume following the
GoTaq® Flexi DNA polymerase (Promega) protocol with final concentrations of 2.5
mM MgCl2, 0.24 mM dNTP mix (Bioline), 0.4 µM of each primer and 1.25 unit of
GoTaq Flexi DNA polymerase.
Touch-down PCR was used with an initial
denaturation at 92°C for 2 min, followed by 45 cycles of denaturation at 92°C for 45
s, and annealing at 69–65°C (decrease 1°C/cycle for 5 cycles) for 45 sec, extension at
72°C for 1 min followed by a final extension step at 72°C for 10 min. Conventional
PCR assays were conducted at WSU. Post PCR and sequencing were performed as
described in Chapter 2, Section 2.2.3.
3.3
Preliminary Studies in 2013
Five preliminary experiments were conducted at Baychhu. They were undertaken to
evaluate survival and development of Diaphorina communis on Citrus reticulata,
and the acquisition and transmission of ‘CLas’ by psyllid nymphs and adults. The
mandarin trees were about 20 years old and severely affected by the disease. Curry
leaf plants used in the experiments were obtained from within the orchard. They are
referred to as Baychhu curry leaf plants.
3.3.1 Experiment 1 (2013)
The objective was to determine survival of Diaphorina communis nymphs
transferred from Baychhu curry leaf plants to soft, fully-expanded, mandarin leaves
and the acquisition of ‘CLas’ by the nymphs from the mandarin leaves.
The
experiment was based on the assumption that ‘CLas’ was not present in the curry leaf
plants, and that Diaphorina communis nymphs caged on ‘CLas’-infected mandarin
leaves would acquire the pathogen.
Materials and Methods. Six mandarin trees were selected on 2 April 2013. One
branch on each of the six trees was caged within a BugDorm rearing sleeve (300 ×
700 mm) (Australian Entomological Supplies Pty. Ltd.) (Figure 3.2). Each sleeve
was labelled. Young Diaphorina communis nymphs were collected by brushing
65
them from infested, immature, Baychhu curry leaf shoots onto white enamel trays
with a camel hair brush. Nymphs that fell onto the trays were sorted according to
their size. The smallest nymphs (< 0.5 mm) were placed in a glass Petri dish. The
nymphs were counted by placing the Petri dish over a ruled notebook page and using
a tally counter. Nymphs were then transferred onto the mandarin leaves. Batches of
106 to 130 nymphs were transferred to each branch.
Results. All nymphs died within 7 d.
Figure 3.2. A BugDorm rearing sleeve on a branch of a ‘CLas’- infected mandarin tree at Baychhu.
3.3.2 Experiment 2 (2013)
The objective was to determine survival of Diaphorina communis adults on fullyexpanded immature leaves on ‘CLas’-infected mandarin trees.
Materials and Methods.
‘Candidatus Liberibacter asiaticus’-infected, mature
mandarin trees used for the previous experiment were also used in this experiment.
66
Adults were collected from Baychhu curry leaf plants by tapping infested branches
of the plants over a white enamel tray and collecting adults that fell into the tray with
an aspirator or a glass vial. The adults were placed in batches of 20 or 25 in the
vials, and groups of 105 adults were released into BugDorm rearing sleeves, one on
each of six trees, on 4 April 2013. The presence of eggs and nymphs on immature
flush growth was observed after seven days. Surviving adults were collected after 54
d and stored in 100% ethanol to determine if they had acquired ‘CLas’.
Results. No psyllid eggs or nymphs were observed on the mandarin leaves. The
average number of live adults in the cages at after 54 d was 32. ‘CLas’ titres in
adults sampled on day 54 are presented in Table 3.1. Fifty-three samples were
tested, 29 prepared from single adults and 24 samples each comprising 5 adults.
Twenty of the single adult samples produced FAM Ct values ranging from 20.58 to
39.57. Of these 20 samples, only three had FAM Ct values in the range of 0.00 <
FAM Ct ≤ 32, values regarded as ‘CLas’-positive.
For the 24 samples, each
comprising five adults, 21 samples produced FAM Ct values > 0.00 with a range of
21.53–38, out of which only 11 samples showed FAM Ct values in the range of 0.00
< FAM Ct ≤ 32. Eighteen samples with FAM Ct values > 0.00 were selected for
cPCR. Five samples with FAM Ct values ranging from 21.53 to 25.56 showed
amplification, and four samples were successfully sequenced.
BLAST searches
using sequences obtained in this study showed 100% identity and 99% coverage with
‘CLas’ isolates from Bhutan (JF346109), India (KT164840–KT16484045), Indonesia
(LCO90231) and China (CP010804).
67
Table 3.1. ‘Candidatus Liberibacter asiaticus’-titres in Diaphorina communis adults caged for 54 d
on branches of ‘CLas’-infected mature mandarin trees.
Parameters
Results
Number of samples based on single adults
29
FAM1 Ct range of samples based on single adults
20.58–39.572
Number of single adult with FAM1 Ct values > 0.00
20
Number of single adult with FAM1 Ct values in the range of 0.00 < FAM Ct
3
≤ 32
Number of samples based on five adults
24
FAM Ct range of samples based on five adults
21.53–38.112
Number of adult composite samples with FAM1 Ct values > 0.00
21
Number of single adult samples with FAM1 Ct values in the range of 0.00 <
11
FAM Ct ≤ 32
1
‘CLas’ DNA.
Eighteen samples with FAM Ct values 21.53–36.39 were selected for cPCR. Five samples with
FAM Ct values between 21.53 & 25.56 produced amplicons of which four were successfully
sequenced.
2
3.3.3 Experiment 3 (2013)
The objective was to determine possible transmission of ‘CLas’ from Baychhu curry
leaf plants to mandarin seedlings by Diaphorina communis nymphs transferred from
the curry leaf plants to the mandarin seedlings. The experiment was based on the
assumption that ‘CLas’ may have been present in the Baychhu curry leaf plants and
that late instar Diaphorina communis nymphs developing on the plants would
acquire the pathogen, and that adults eclosing from these nymphs would transmit the
pathogen to healthy mandarin seedlings.
Materials and Methods. Nine mandarin seedlings from the April 2013 consignment
from the NSC were planted in 250 mm diameter plastic pots containing potting mix
(1:1 sterilised garden soil and forest top soil). Nine 475 × 475 × 475 mm BugDorm
insect rearing cages (Australian Entomological Supplies Pty. Ltd.) were placed on a
flat area in the orchard (Figure 3.3). One mandarin seedling was placed in each cage.
Small stones were placed in the corners of the cages to prevent them from being
68
dislodged by wind. Twenty-five 4−5th instar Diaphorina communis nymphs were
transferred from curry leaf plants, as above, to each of the six mandarin seedlings.
Three of the mandarin seedlings were retained as controls. In order to confirm that
all adults that eclosed were Diaphorina communis some nymphs from the curry leaf
plants were held for five days with young curry leaf shoots in plastic containers with
perforated lids.
Results. All nymphs that were transferred to the mandarin seedlings died within 7 d.
All adults that eclosed from nymphs maintained on curry leaf shoots for five days in
the plastic containers were Diaphorina communis.
Figure 3.3. BugDorm rearing cages with mandarin seedlings used for rearing nymphs of Diaphorina
communis.
3.3.4 Experiment 4 (2013).
The objective was to determine possible transmission of ‘CLas’ from Diaphorina
communis-infested Baychhu curry leaf plants to healthy mandarin seedlings. As for
Experiment 3 above, this experiment was based on the assumption that ‘CLas’ may
have been present in the Baychhu curry leaf plants and that late instar Diaphorina
communis nymphs developing on the plants would acquire the pathogen, and that
adults eclosing from these nymphs would transmit the pathogen to healthy mandarin
seedlings.
69
Materials and Methods. In this instance, the Diaphorina communis-infested curry
leaf plants were caged with healthy mandarin seedlings in May and again in June
2013. In May, nine curry leaf plants with abundant eggs and nymphs were selected
from within the orchard. These plants were watered thoroughly and left overnight
for easy uprooting on the following day and then planted in 180 mm diameter plastic
pots. Soil collected from the sites where curry leaf plants were uprooted was used
for planting curry leaf in the pots. Eighteen mandarin seedlings were planted in 180
mm diameter plastic pots containing a potting mix consisting of sterilised garden soil
and forest top soil (1:1). The mandarin seedlings were pruned before being placed in
the cages. All mandarin seedlings were kept in BugDorm cages during preparation
of the experiment. Each curry leaf plant was examined carefully, and all adult
Diaphorina communis present were removed before each curry leaf plant was placed
in a cage with one mandarin seedling. Nine mandarin seedlings were placed singly
in nine other cages. All cages were kept in the orchard for 34 d. Emergence of
Diaphorina communis adults in the cages was recorded every 3–6 d for 17 d. The
mandarin seedlings were pruned 17 d after planting in order to maintain their height
within the cages. After 34 d, all mandarin plants were transported to the NPPC and
where they were kept in a rearing room at room temperature for general observations
and PCR analysis after one year.
In June 2013, for logistical reasons related to moving plants to and from Baychhu to
the NPPC, fewer plants were used and, in this instance, four mandarin seedlings were
caged with curry leaf plants infested with Diaphorina communis eggs and nymphs
and four were kept as controls.
Results. In May, adults emerged in all except one cage five days after the Baychhu
curry leaf plants were placed in the cages. The lowest and highest numbers of adults
that emerged at this time were two and six, respectively. Five days after the first
record of adults, all cages contained adults, and the highest number of adults
emerging was 23 and the lowest three. Twelve days after the first observation, the
highest number of adults increased to over 50 while the lowest was two. However,
after 34 d, except for one cage, adults in all cages had died. In June, one week after
70
the Baychhu curry leaf plants were placed in the cages, four and two adults emerged
in cages 1 and 4, respectively, and no adults were present in the other cages. One to
two adults emerged in other cages but died ~28 d after first being observed. Plant
samples, used in both May and June 2013, were tested after one year, showed FAM
Ct values between 33.81 and 38.08 but selected samples subjected to cPCR did not
produce any amplicons.
3.3.5 Experiment 5 (2013)
The objective was to determine development of Diaphorina communis on mandarin
flush, in this instance, from budbreak on ‘CLas’-infected mature mandarin trees.
Materials and Methods. Development was determined by: (a) caging 30 adults in
BugDorm sleeve cages for 14 d in each of five cages with new buds and developing
flush on skeletonised branches of ‘CLas’-infected mandarin trees 28 d after mature
leaves were removed; and (b) likewise in two cages for 14 d on new buds 30 d after
branches were pruned.
Results. With the exception of two adults in one cage, all adults died within 11 d of
their release. These adults were collected and preserved in 100% ethanol. One
nymph was observed. It died within seven days of being observed.
3.4
Studies in 2014
The preliminary studies in 2013 indicated that Diaphorina communis does not
develop on Citrus reticulata.
Therefore, emphasis shifted to evaluating adult
acquisition and transmission of ‘CLas’ from new-growth on stumps of the naturallyinfected, mature mandarin trees at Baychhu in 2014.
Greenhouse-raised curry leaf and mandarin seedlings were used for the experiments
on transmission of ‘CLas’ from mandarin.
The greenhouse-raised mandarin
seedlings are referred as NSC mandarin, and the greenhouse raised curry leaf
seedlings are referred as 2014-curry leaf seedlings for the studies in 2014.
71
3.4.1 Experiment 1 (2014)
The objective was to determine possible acquisition and transmission from flush
growth on stumps of ‘CLas’-infected mature mandarin trees to ‘CLas’-free mandarin
and curry leaf seedlings.
Materials and Methods. This experiment commenced in the orchard at Baychhu on
8 May 2014. Average weather data for 2014 are presented in Figure 3.4.
Twenty-two stumps of mature, ‘CLas’-infected trees cut down in early 2013 were
selected in March 2014. In order to initiate flush growth, shoots present on the
stumps were pruned to a height of 250–300 mm above ground level 45 d prior to the
commencement of the experiment. Pruned tissues were collected from the stumps in
order to confirm that ‘CLas’ was present.
New flush growth on each stump was inspected for Diaphorina citri and Diaphorina
communis eggs and nymphs prior to each stump being enclosed within a BugDorm
cage modified by cutting a ~ 150 mm diameter hole in its base so as to enable it to be
placed over a stump. Soil around the base of each stump was levelled beforehand
and stones were placed around the cages in order to prevent them being dislodged by
wind. Diaphorina communis adults were collected from wild curry leaf plants at
Basochhu (27.3646°N, 89.9124°E, 1036 m ASL), ~10 km from Baychhu, on 7 May
2014. Branches of the plants were tapped onto a white, enamel tray and adults
falling on the tray were placed in batches of 30 in glass tubes and released overnight
onto greenhouse-raised curry leaf seedlings in BugDorm cages not being used in the
experiment. The psyllids were then transferred on 8 May 2014 in batches of 30 to
glass vials. Fifty-five adults were then released into each of the 22 BugDorm cages
enclosing the mandarin tree stumps and immature flush growth on the stumps. Five
psyllids from each vial were used to test for the presence of ‘CLas’.
Single, 10-month-old 2014-curry leaf seedlings from the NPPC were placed in each
cage on 19 May 2014. They were pruned to heights of approximately 150 mm in
72
order to encourage new growth that was 2–5 mm long when the seedlings were
placed in the cages.
Figure 3.4. Average monthly minimum and maximum temperatures and relative humidity recorded
at Baychhu in 2014 and 2015.
Single NSC mandarin seedlings from April 2013 consignment were placed in each
cage on the same day. These seedlings were 28-months-old and pruned to heights of
approximately 120 mm to encourage new growth that was 2–5 mm long when the
seedlings were placed in the cages. The plants were allocated randomly to the
BugDorm cages. Further batches of 60–65 adults were released into each cage four
and 30 d after the 2014-curry leaf and NSC mandarin seedlings were placed in the
cages.
Adult psyllids were maintained in the BugDorm cages until 5 July 2014. Thus,
Diaphorina communis adults were provided with opportunities to acquire ‘CLas’
from shoots on infected stumps over intervals up to 58 d and to transmit the pathogen
73
to the 2014-curry leaf and NSC mandarin seedlings over an acquisition and
transmission interval (ATI), depending on survival after release, of 16–47 d.
Numbers of eggs, nymphs and adults on the plants were recorded one and two weeks
after the second release of adults on 23 May 2014. Adults within each cage were
counted visually through a clear plastic panel at the front of each cage and by
examining the plants and internal cage surfaces through the sleeve opening of each
cage. Eggs and nymphs were counted using a 10× hand lens by removing the plants
temporarily from the cages after shaking adults off the plants inside the cages. At the
end of the ATI (5 July 2014), surviving Diaphorina communis adults were collected
from each BugDorm cage and stored in 100% ethanol for determining the presence
or absence of ‘CLas’ by PCR. Five adults from each cage were tested individually
unless specified.
The NSC mandarin seedlings were placed in groups of three or four in large, 475 ×
475 × 930 mm BudDorm rearing cages under shade cloth within the orchard (Figure
3.5) for 107 d before they were transported to NPPC. The 2014-curry leaf seedlings,
all bearing eggs and nymphs of Diaphorina communis, were maintained separately in
other cages under shade cloth for use in the following experiment. The midribs of
symptomatic leaves were sampled from mandarin stump-shoots on 5 July 2014 to
further confirm the presence or absence of ‘CLas’. Periodic sampling for detection
of ‘CLas’ in midribs of leaflets from the 2014-curry leaf seedlings and leaves of the
NSC mandarin seedlings commenced 44 d after the ATI, and continued at intervals
ranging from 72−95 d for 541 d (~18 months) to test for ‘CLas’.
Results. Numbers of adults surviving, and numbers of eggs and nymphs on mandarin
and 2014-curry leaf seedlings, during the acquisition and transmission experiment
after adults were released twice into each of the 22 cages are summarised in Tables
3.2 and 3.3. All adults caged with 2014-curry leaf seedlings in the 11 cages died
within 11 d of the first release of 50 adults into each cage. All adults in eight of the
11 cages with mandarin seedlings also died within 11 d of being released. One week
after the subsequent release of 60 adults into each cage, an average of seven adults
74
per cage were alive in cages with NSC mandarin seedlings and an average of nine
adults were present in the cages containing 2014-curry leaf seedlings. At this point,
the cages containing NSC mandarin seedlings had an average of 1 egg per cage
compared to the cages housing 2014-curry leaf seedlings that contained 71 eggs per
cage (Figure 3.6); no nymphs were recorded at this point. Two weeks after the
second release of adults, the cages containing 2014-curry leaf seedlings had higher
average numbers of adults (~6), eggs (65), and nymphs (46) compared to the cages
with the NSC mandarin seedlings (adults = 2; eggs = 0; nymphs= 0). The average
numbers of immature shoots of similar age on the NSC mandarin and 2014-curry leaf
seedlings were 8 and 9, respectively at 14 d after the second release of psyllids.
Figure 3.5. A mandarin stump used as a source of ‘CLas’ for acquisition and transmission by adult
Diaphorina communis (A); BugDorm rearing cages placed over mandarin stumps in the field (B);
close up of a BugDorm with a Set A curry leaf seedling and mandarin stump (C); cages while
maintained under shade cloth at Baychhu until 20 October 2014, and during transmission studies from
Set A 2014 curry leaf to mandarin (D).
75
Table 3.2. Numbers of live Diaphorina communis adults, eggs, and nymphs on NSC mandarin in the experiment cages during the acquisition and transmission experiment in
2014.
Seedling and
stump/cage
number
Live adults 11 d after 50
adults were released per
cage on 8 May 2014
Live adults, eggs and nymphs 7 and 14 d after 60 adults were released per cage on 23
May 2014
At 7 d (30 May 2014)
Number of
shoots on 6 June
2014
At 14 d (6 June 2014)
Adults
Eggs
Nymphs
Adults
Eggs
Nymphs
Mandarin B1
1
1
0
0
0
0
0
3
Mandarin B2
2
2
0
0
0
0
0
8
Mandarin B9
0
0
0
0
0
0
0
5
Mandarin B10
0
1
0
0
1
0
0
12
Mandarin B11
0
1
2
0
0
0
0
8
Mandarin B12
0
7
4
0
1
2
0
14
Mandarin B13
0
8
0
0
1
0
0
9
Mandarin B14
0
28
0
0
14
0
0
8
Mandarin B16
10
23
0
0
7
0
0
10
Mandarin B18
0
8
0
0
0
0
0
9
Mandarin B19
0
1
0
0
0
0
0
6
7.27
0.55
0
2.18
0.18
0
8.36
Average
76
Table 3.3. Numbers of live Diaphorina communis adults, eggs, and nymphs on 2014-curry leaf seedlings in the cages during acquisition and
transmission study by Diaphorina communis adults in 2014 (Experiment 1).
Seedling and
stump/cage
number
Live adults 11 d after 50
adults were released per
cage on 8 May 2014
Live adults, eggs and nymphs 7 and 14 d after 60 adults were released per cage on 23
May 2014
At 7 d (30 May 2014)
Number of
shoots on 6 June
2014
At 14 d (6 June 2014)
Adults
Eggs
Nymphs
Adults
Eggs
Nymphs
Curry leaf B24
0
8
86
0
3
34
35
10
Curry leaf B3
0
5
30
0
2
33
20
2
Curry leaf B6
0
7
1
0
2
85
0
10
Curry leaf B7
0
7
100
0
0
7
135
14
Curry leaf B8
0
9
80
2
6
153
30
8
Curry leaf B15
0
8
68
0
7
113
32
11
Curry leaf B17
0
23
30
0
15
12
3
5
Curry leaf B20
0
4
11
0
0
0
23
11
Curry leaf B21
0
3
120
0
0
0
88
13
Curry leaf B22
0
10
70
0
9
123
60
9
Curry leaf B23
0
23
186
0
19
155
77
5
9.73
71.09
0.18
5.73
65
45.73
8.91
Average
77
Figure 3.6. Eggs of Diaphorina communis on mandarin (top) and curry leaf (bottom) seedlings
(Photos: N. Om).
78
‘Candidatus Liberibacter asiaticus’ titres in tissues sampled from the mandarin
stumps prior to the commencement of the experiment and at the end of the ATI, and
the results of periodic testing of test plants (2014-curry leaf and NSC mandarin
seedlings) are presented in Tables 3.4 and 3.5. Titres at the beginning and the end of
ATI produced FAM Ct values ranging from 14.18 to 25.70 and 15.66 to 22.77,
respectively. Four stumps had died by the end of the ATI. Seven of the 11 NSC
mandarin seedlings produced FAM Ct values ranging from 34.97 to 39.39 when
tested 44 d after the ATI compared to four 2014-curry leaf seedlings. Except for one
NSC mandarin seedling (FAM Ct = 39.2), none of the NSC mandarin seedlings and
2014-curry leaf seedlings sampled 125 to 305 d after ATI produced FAM Ct values >
0.00. One of the NSC mandarin seedlings died 305 d after the ATI and two had
growth unsuitable for sampling. At 377 and 458 d after ATI, three NSC mandarin
and two 2014-curry leaf seedlings produced FAM Ct values between 35.09−37.79
and 37.81−38.38, respectively. All seedlings produced FAM Ct values equal to zero
when tested at 541 d. The positive controls showed FAM Ct values ranging from
23.21–34.60, and the internal controls (CAL) showed Ct values ranging from 13.36
to 20.47. Samples with FAM Ct values between 34.62−36.69 did not yield any
amplicons when subjected to cPCR with primers A2/J5.
‘Candidatus Liberibacter asiaticus’ titres in psyllids at the beginning of the
experiment, when adults were released into the cages, and at the end of the ATI are
shown in Table 3.6. All psyllid samples at the time of release into the cages showed
FAM Ct values equal to zero, except for one sample that showed a FAM Ct value of
38.9.
At the end of the ATI, only seven out of the 22 cages contained live
Diaphorina communis adults.
Only two adult samples from these cages tested
positive by qPCR with the individuals having FAM Ct values of 30.17 and 31.06.
79
Table 3.4. ‘Candidatus Liberibacter asiaticus’ titres in stumps of mature mandarin trees (inoculum source) and NSC mandarin seedlings used in the acquisition and
transmission study by Diaphorina communis adults in 2014 (Experiment 1).
Seedling
stump/cage
number1
and
FAM Ct values of
stumps2
Before2a
After 2b
FAM Ct values of test plants (days after the ATI))
44 d
125 d
210 d
305 d
377 d
458 d
39.29
0.00
0.00
0.00
35.09
0.00
541 d
Mandarin B1
20.28
21.00
Mandarin B2
16.89
dead
0.00
0.00
0.00
0.00
0.00
37.76
0.00
Mandarin B9
18.85
dead
39.39
0.00
NS
0.00
0.00
0.00
0.00
Mandarin B10
16.05
dead
39.38
0.00
0.00
0.00
0.00
0.00
0.00
Mandarin B11
20.62
17.83
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Mandarin B12
15.75
19.21
0.00
0.00
0.00
0.00
0.00
37.79
0.00
Mandarin B13
20.04
17.67
0.00
0.00
0.00
dead
dead
dead
dead
Mandarin B14
19.43
18.25
39.14
0.00
0.00
0.00
0.00
0.00
0.00
Mandarin B16
18.69
15.94
34.97
0.00
0.00
0.00
0.00
0.00
0.00
Mandarin B18
17.90
18.21
35.22
39.24
0.00
0.00
0.00
0.00
0.00
0.00
0.00†
Mandarin B19
20.15
dead
35.60
0.00
0.00
80
0.00
NS
0.00
Table 3.5. ‘Candidatus Liberibacter asiaticus’ titres in stumps of mature mandarin trees (inoculum source) and 2014-curry leaf seedlings used in the acquisition and
transmission study by Diaphorina communis adults in 2014 (Experiment 1).
Seedling
stump/cage
number1
1
and
FAM Ct values of
stumps2
Before2a
After2b
FAM Ct values of test plants (days after the second release of psyllids)
44 d
125 d
210 d
305 d
377 d
458 d
541 d
Curry leaf B3
25.70
18.95
38.18
0.00
0.00
0.00
0.00
0.00
0.00
Curry leaf B6
17.77
16.59
0.00
0.00
0.00
0.00
0.00
0.00
dead
Curry leaf B7
19.46
19.79
0.00
0.00
0.00
0.00
37.81
*
0.00†
Curry leaf B8
18.14
22.77
0.00
0.00
0.00
0.00
0.00
*
0.00
Curry leaf B15
16.93
18.81
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Curry leaf B17
15.50
15.94
36.69
0.00
0.00
0.00
0.00
0.00
0.00
Curry leaf B20
16.78
17.77
34.62
0.00
0.00
0.00
0.00
0.00
0.00
Curry leaf B21
16.10
15.66
35.40
0.00
0.00
0.00
38.38
0.00
0.00
Curry leaf B22
14.18
16.06
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Curry leaf B23
17.06
16.08
0.00
0.00
0.00
0.00
0.00
*
0.00
Curry leaf B24
14.90
16.70
0.00
0.00
0.00
0.00
0.00
*
0.00
Positive controls
25.19
23.93
26.11
33.88
34.60
33.46
33.46
20.03
18.34
2
Common name of test plants followed by the stump/cage number; FAM Ct values for samples obtained from the particular stump number before (2a) and after (2b) the
acquisition and inoculation period; CAL (internal control) Ct value range for above samples: 14.13–29.9 except for the two samples marked †both of which produced CAL
Ct values of 38.89; * produced zero CAL Ct values due to degraded DNA as indicated by dark colour of the eluted DNA.
NS=test plant still alive but hardly any leaves to sample
Ct values of the samples subjected to cPCR are emboldened.
81
Table 3.6. Real-time PCR detection of ‘CLas’ in pooled samples of Diaphorina communis adults
representative of the population released in the cages, and samples collected at the end of the
acquisition and transmission interval (ATI) during Experiment 1 (2014). Five adults from each cage
were tested individually unless specified.
Seedling
and
stump/cage
number
FAM Ct range and number of samples positive for 'CLas'
Before release
End of ATI
‘CLas' +ve1
FAM Ct range†
‘CLas'
+ve1
FAM Ct range†
Mandarin B1
0
0
NA
NA
Mandarin B2
0
0
NA
NA
Mandarin B9
0
0
NA
NA
Mandarin B10
0
0
NA
NA
Mandarin B11
0
0
NA
NA
Mandarin B12
0
0
NA
NA
Mandarin B13
0
0
NA
NA
Mandarin B14
0
0.00 & 38.94
NA
NA
Mandarin B16
0
0
0
0.00 & 34.54–36.93
Mandarin B18
0
0
NA
NA
Mandarin B19
0
0
NA
NA
Curry leaf B3
0
0
0
0.00a
Curry leaf B6
0
0
0
0
Curry leaf B7
0
0
0
0
Curry leaf B8
0
0
0
0.00 & 37.20
Curry leaf B15
0
0
0
0
Curry leaf B17
0
0
0
0.00 & 33.77–35.12
Curry leaf B20
0
0
0
0.00 & 35.12–37.76c
Curry leaf B21
0
0.00b
0
0 & 38.64
Curry leaf B22
0
0
1
0.00 & 30.17–35.86
Curry leaf B23
0
0
1
0.00 & 31.06–35.71
Curry leaf B24
0
0
0
0
1
Numbers of ‘CLas’ positive samples.
† 0.00 indicates all samples had Ct values of zero: 0.00 and a numerical value > 0 indicates at least
one sample produced a Ct value > 0; 0.00 and a range of values indicates at least one sample in the
group produced a Ct = 0, the rest produced Ct values > 0.
a
Two, b three, and c four adults tested from for these cages, respectively.
NA= no adults in the cage.
82
3.4.2 Experiment 2 (2014)
The objective of this experiment was to determine the acquisition and transmission
of ‘CLas’ by Diaphorina communis from 2014-curry leaf seedlings to NSC mandarin
seedling. It was based on the assumption that ‘CLas’ may have been transmitted to
2014-curry leaf seedlings used in Experiment 1 (2014).
Materials and Methods. All eleven 2014-curry leaf seedlings from Experiment 1
(2014) were used. One pathogen-free, NSC mandarin seedling and one 2014-curry
leaf seedling infested with Diaphorina communis eggs and nymphs were placed in
each of eleven BugDorm cages. The numbers of adult psyllids in each cage were
counted five days later and then every 10−14 d for 79 d. Samples of psyllid adults
were then removed from the cages and tested for the presence of ‘CLas’. A total of
13 psyllid samples were tested either singly or in groups of five. The 2014-curry leaf
seedlings and NSC mandarin seedlings were then maintained, in the absence of
psyllids, in larger cages for 107 d (~3.5 months), then transported to NPPC.
Sampling of plant tissues from NSC mandarin seedlings in this experiment
commenced at 79 d after the eclosion of the first adult psyllid (EFAP), and then
continued for 536 d (~18 months).
Results.
‘Candidatus Liberibacter asiaticus’ titres in tissue sampled on seven
occasions from the mandarin seedlings, and the numbers of Diaphorina communis
adults present in each cage when the samples were taken, are presented in Table 3.7.
FAM Ct values for samples collected at 79 d after EFAP ranged from 31.72 to 38.73.
Most subsequent samples showed FAM Ct values equal to zero and few samples
produced FAM Ct values > 36, except for mandarin seedling 5 sampled on day 372
(FAM Ct = 35.73), and mandarin seedlings 6 and 8 (FAM Ct values = 35.95 and
34.25, respectively) sampled on day 536. The FAM Ct values for the positive
controls ranged from 20.03 to 34.60, and the CAL Ct values ranged from 14.13 to
32.15 except for one sample that produced a Ct value of 36.34.
All cages had at least one or more adults present during ~35-71 d of the 71 d over
which adults were present in the cages, with the exception of cages CLB15 and
83
CLB21 housing mandarin seedlings 5 and 8, respectively. The highest numbers of
adults were recorded on mandarin seedling 4 (86 adults) in cage CLB8 followed by
with mandarin seedling 6 (44 adults) in cage CLB17. Adults collected on day 71
after first eclosion had ‘CLas’ FAM Ct values equal to zero.
84
Table 3.7. Real-time PCR results of the experiment to determine transmission of 'CLas' by Diaphorina communis adults from curry leaf to mandarin seedlings at Baychhu
in 2014.
Mandarin seedling1
FAM Ct values of test plants (days after the eclosion of first adult psyllid (EFAP) in the cages on 10 July
2014)
Number of psyllids2
79 d
120 d
160 d
300 d
372 d
453 d
536 d
Mandarin 1 (CLB3)
38.38
0.00
0.00
0.00
0.00
0.00
0.00
1–6
Mandarin 2 (CLB6)
35.99
36.61
0.00
0.00
0.00
0.00
0.00
1–2
Mandarin 3 (CLB7)
38.73
0.00
0.00
0.00
0.00
0.00
0.00
1–3
Mandarin 4 (CLB8)
33.87
0.00
0.00
0.00
0.00
0.00
0.00
4–86
Mandarin 5 (CLB15)
35.24
0.00
0.00
0.00
35.73
37.58
36.09
0.00
Mandarin 6 (CLB17)
35.48
0.00
0.00
0.00
0.00
0.00
35.95
0–44
Mandarin 7 (CLB20)
33.82
0.00
0.00
0.00
0.00
0.00
0.00
2–11
Mandarin 8 (CLB21)
31.72
0.00
0.00
0.00
0.00
0.00
34.25
0.00
Mandarin 9 (CLB22)
34.25
0.00
0.00
0.00
0.00
0.00
36.86
1–13
Mandarin 10 (CLB23)
34.25
0.00
0.00
0.00
0.00
0.00
0.00
5–18
Mandarin 11 (CLB 24)
32.82
0.00
0.00
0.00
0.00
0.00
36.93
0–6
Positive control
24.88
33.88
34.6
33.46
33.46
20.03
21.34
1
Mandarin seedlings caged with 2014- curry leaf seedlings. The 2014-curry leaf seedlings were infested with eggs and nymphs of Diaphorina communis previously caged
with stumps of ‘CLas’-infected mature mandarin stumps.
2
Number of psyllids emerging during the ATI of curry leaf to mandarin. The range indicates the minimum number of psyllids one week after the cage was removed from
the infected stumps and maximum number of adults present during the ATI. A total of 13 psyllids when tested for’ CLas’ either singly or in groups of 2–5 produced FAM
Ct values equal to 0.00.
CAL Ct value ranged from: 14.13 to 32.15, plus one sample having a value of 36.34. Samples subjected to cPCR are emboldened.
85
3.4.3 Experiment 3 (2014)
The objective of this experiment was to further determine host preferences of
Diaphorina communis. The field experiments in 2013 (see Section 3.3.1 to 3.3.5)
and 2014 (see Section 3.4.1 Experiment 1 (2014) at Baychhu indicated that
Diaphorina communis cannot develop on mandarin.
To confirm this, host
preferences of the psyllid were assessed under laboratory conditions with NSC
mandarin seedlings and NPPC curry leaf seedlings at the NPPC in 2014.
Materials and Methods. Diaphorina communis adults were released into BugDorm
cages containing:
a. Two mandarin seedlings
b. One mandarin seedling and one curry leaf seedling
c. Two curry leaf seedlings
The plants were pruned to 120–150 mm in height at least three weeks before
commencing the experiment. All plants had young 1–5 mm buds/flush growth when
the experiment commenced.
Each plant pair was replicated four times.
Fifty
Diaphorina communis adults from psyllid cultures maintained in a rearing room at
NPPC were released into each cage. All cages were maintained in a rearing room at
21–30°C and 40–60% RH. Adults were removed from the cages 11 d after their
release. The plants in the cages were then observed daily for emergence of the next
generation of psyllids. The number of second generation adults was recorded for the
next eight days.
Results: Average numbers of second generation adults developing under laboratory
conditions on mandarin and curry leaf seedlings in the choice and no-choice assays
with paired mandarin, mandarin and curry leaf, and curry leaf seedlings in cages into
which 50 adults were released are shown in Figure 3.7. The average number of
second generation adults in the cages was relatively higher in cages containing two
curry leaf seedlings followed by the cages with paired mandarin and curry leaf
seedlings. The average number of adults in the cages with only paired mandarin
86
seedlings was negligible. On the eighth day average numbers in the cages were 115,
42 and zero, respectively.
Figure 3.7. Average (n = 4) number of second generation Diaphorina communis adults following
their development on curry leaf and mandarin seedlings in the choice and no-choice test conducted in
the laboratory at NPPC in 2014.
3.5
Studies in 2015
For the studies in 2015, similar to studies in 2014, greenhouse-raised curry leaf and
mandarin seedlings were used. Curry leaf seedlings used in the 2015 experiments for
transmission of ‘CLas’ from mandarin to curry leaf are referred to as 2015-curry leaf
seedlings, and the curry leaf seedlings used for transmission of ‘CLas’ from 2015curry leaf seedlings are referred to as 2015B-curry leaf seedlings.
seedlings are referred as NSC mandarin seedlings.
87
Mandarin
Two experiments were conducted. The first was similar to the experiment with
immature flush growth on mandarin stumps, and mandarin or curry leaf seedlings in
2014 (see Section 3.4.1). The aim was to further determine whether Diaphorina
communis can transmit ‘CLas’. The second investigated transmission of ‘CLas’
from curry leaf to mandarin or curry leaf by Diaphorina communis.
Average
ambient temperature and relative humidity at Baychhu in 2014–2015 are shown in
Figure 3.4.
3.5.1 Experiment 1 (2015)
The objective of this experiment was to further determine whether Diaphorina
communis can acquire and transmit ‘CLas’ from ‘CLas’-infected mandarin stumps to
mandarin and curry leaf seedlings.
Materials and Methods. The field component of the experiment was conducted from
6 May 2015 to 17 June 2015. Fourteen infected stumps, including four used in 2014,
were selected from within the orchard.
All other methods were similar to
Experiment 1 (2014) (see Section 3.4.1) except for the following modifications:
In order to confirm that all plants were pathogen-free, leaf and bark samples
were collected from the 2015-curry leaf seedlings from the NPPC and NSC
mandarin seedlings and were subjected to PCR before the plants were placed in
the field.
Two NSC mandarin and 2015-two curry leaf seedlings were placed in each of 14
BugDorm cages, in contrast to the single seedlings of each plant species in 2014.
Forty Diaphorina communis adults were released into each of the cages on 6
May 2015. The placement of both mandarin and curry leaf seedlings in each of
the cages was done to enhance survival of the adult psyllids.
Two seedlings of each species were also placed into two other BugDorm cages
to serve as control plants (Diaphorina communis adults were not released into
these cages) (Table 3.8).
88
Table 3.8. NSC mandarin and 2015-curry leaf seedling allocations in the BugDorm cages used in
2015 for transmission of 'CLas' from infected mandarin stumps by Diaphorina communis.
Cage/stump
number
NSC
mandarin
seedling
identification number
2015-curry leaf seedling identification
number
L1
M1 & M2
CL1 & CL2
L2
M31 & M32
CL42 & CL43
L3
M5 & M6
CL5 & CL6
L4
M7 & M8
CL7 & CL8
L5
M9 & M10
CL9 & CL10
L6
M11 & M12
CL32 & CL41
L8
M13 & M14
CL13 & CL14
L9
M15 & M16
CL15 & CL16
L10
M17 & M18
CL17 & CL18
U1
M23 & M24
CL23 & CL24
B8
M27 & M28
CL34 & CL38
B16
M21 & M22
CL21 & CL22
B20
M29 & M30
CL29 & CL30
B22
M19 & M20
CL19 & CL20
Control
M33 & M44
CL44 & CL45
Diaphorina communis adults were collected from Basochhu, as described in Section
3.3.2, and 40 adults were released into the cages on 6 May 2015.
Unlike in
Experiment 1 (2014) (see Section 3.4.1), numbers of surviving adults in each cage
were counted one day after they were released into the cages, and a second release
was made on 8 May 2015 with 55 adults as more than 50% of adults released on 6
May had died. Subsequently, psyllid colonies in the cages were augmented weekly
so as to maintain populations of ~50 adults per cage for 39 d. The psyllid release
method was slightly modified for this work. The desired number of psyllids were
aspirated into 15 mL glass tubes and then released on the same day with the tubes
left open in the BugDorm cages. Pooled samples of psyllids representative of the
population used in the first three releases (6, 8 and 14 May 2015) were stored in
89
100% ethanol for detection of ‘CLas’, samples tested included adults for the first two
release, and 4th–5th instar nymphs for the third release.
On the fortieth day, the live adults in the cages were collected using an aspirator and
stored in 100% ethanol; psyllid DNA extracts were then prepared from single adults
or samples comprising four or five adults and used to test for ‘CLas’. All seedlings
were removed and transferred to separate cages and maintained under green shade
cloth at Baychhu for 40 d before they were transported to the NPPC. The NSC
mandarin seedlings, each labelled, were caged together in groups of four or five. The
2015-curry leaf seedlings were caged separately to be used in Experiment 2 of 2015
(Section 3.4.6). Tissue samples from the mandarin stumps were collected to test for
the presence of the pathogen in the stumps. Periodic testing for ‘CLas’ in the NSC
mandarin and 2015-curry leaf seedlings commenced 76 d (~ 2.5 months) after the
ATI and sampled two more times during the next 217 d.
Results.
The four stumps that were used in 2014 were not tested prior to the
experiment as they were all ‘CLas’-positive in 2014. The FAM Ct ranges before and
after commencement of the experiment for the 10 stumps selected in 2015 were
27.89–38.57 and 28.65–36.90. The FAM Ct values for the positive control samples
used in the assays were 34.61 and 25.19, and that of the extraction control were zero.
The internal control (CAL) Ct values ranged from 16.64 to 25.19 and 15.0 to 34.21
for samples collected before and after ATI respectively (Table 3.9).
The average numbers of Diaphorina communis adults alive in the cages over the six
weeks from the second release of adults into the cages on 8 May 2015 are shown in
Figure 3.8. All cages had at least 20 adults alive on average during the acquisition
and transmission interval (ATI). The highest number of adults were alive in cage L4
followed by cage L5 and B8, minimum in cage L10 followed by U1.
90
Table 3.9. Ct values of samples of Baychhu mature mandarin tree stumps before and after ATI of
Experiment 1 (2015).
Stump Number 1
Ct values
Before (19 March 2015)
After (17 June 2015)
FAM
CAL
FAM
CAL
B8
NA
NA
31.65
15.49
B16
NA
NA
29.35
15.01
B20
NA
NA
36.90
20.82
B22
NA
NA
29.57
16.75
L4
31.03
20.43
29.56
15.50
L5
34.53
18.13
29.56
15.36
L6
29.79
16.64
31.01
17.62
L7
30.00
20.04
dead
dead
L8
29.71
19.09
29.76
16.31
L9
34.68
18.10
31.32
16.66
L10
28.71
17.58
29.95
15.62
U1
27.89
16.50
28.65
16.97
Positive control
34.61
25.19
34.23
23.14
Extraction control
0.00
18.35
0.00
34.21
1
Stumps B8, 16, 20, and 22 were stumps used in 2014.
NA = all 10 stumps used in 2014 were not tested before the 2014 experiment commenced
91
Figure 3.8. Average numbers of live Diaphorina communis adults in experimental cages (Mean ±
SE) for six weeks after 50 adults were released into each cage on May 8 2015. Populations in the
cages were augmented weekly to the initial number if less than 25 adults were present.
‘Candidatus Liberibacter asiaticus’ titres for pooled samples of Diaphorina
communis adults representative of the populations used for release in the cages
housing ‘CLas’ infected-stumps, 2015-curry leaf and NSC mandarin seedlings, are
presented in Table 3.10. All samples had FAM Ct values equal to zero and the
internal control (wg gene, flurophor = CAL) Ct value range was 25.23–36.01, except
for two samples that had CAL Ct values equal to zero. The ‘CLas’ Ct values for
Diaphorina communis adults collected at the end of ATI in 2015 are presented in
Table 3.11. A total of 109 DNA extracts prepared from single adults or samples
comprising four or five adults were tested. Of these, 16 produced FAM Ct values >
0.00 and seven had FAM Ct values < 32. Samples that showed FAM Ct values < 32
were subjected to cPCR, and a sample that showed a FAM Ct value of 17.03 was
successfully amplified and sequenced. A BLAST search with the sequence obtained
from this sample had a 99% identity with ‘CLas’ isolates from China, India and
Japan.
92
Table 3.10. ‘Candidatus Liberibacter asiaticus’ titres in pooled samples of Diaphorina communis
adults and nymphs representing the samples released in the cages on the first three occasions.
Sample Name
NPE1
Date
Ct values
FAM
CAL3
Adults
DCO 1
1
6 May 2015
0.00
27.15
DCO 2
1
6 May 2015
0.00
26.83
DCO 3
1
6 May 2015
0.00
26.91
DCO 4
1
6 May 2015
0.00
27.48
DCO 5
1
6 May 2015
0.00
26.89
DCO 6
5
6 May 2015
0.00
26.81
DCO 7
5
6 May 2015
0.00
27.46
DCO 8
5
6 May 2015
0.00
26.35
DCO 8
1
8 May 2015
0.00
0.00
DCO 9
1
8 May 2015
0.00
0.00
DCO 10
1
8 May 2015
0.00
28.87
DCO 11
1
8 May 2015
0.00
29.19
DCO 12
1
8 May 2015
0.00
27.94
DCO 13
3
8 May 2015
0.00
30.89
DCO 14
3
8 May 2015
0.00
36.01
DCN12
5
14 May 2015
0.00
25.94
DCN2
2
5
14 May 2015
0.00
25.23
DCN3
2
5
14 May 2015
0.00
25.30
Nymphs
1
NPE = number per extractions.
Nymphs collected during adult collection for third release were used.
3
Wingless gene probe and primer (Manjunath et al. 2008).
2
93
Table 3.11 ‘Candidatus Liberibacter asiaticus’ titres in Diaphorina communis adults collected at the
end of ATI for Experiment 1 (2015).
FAM Ct values2
‘CLas'
+ve3
CAL
0.00 (8)
0
27-30
20 (8)
0.00 (8)
0
27-29
22
20 (8)
0.00 (7); 38.91 (1)
0
27-30
B22
23
20 (8)
0.00 (7); 37.52 (1)
0
27-30
L1
16
15 (7)a
0.00 (5); 33.10-35.32 (2)
0
26-28
L2
18
15 (7)a
0.00 (7)
0
25-28
L3
25
15 (7)a
0.00 (6); 39.70 (1)
0
26-30
L4
44
20 (8)
17.03-32.78 (8)*
7
23-27
L5
29
20 (8)
0.00 (7); 38.3 (1)
0
25-28
L6
25
20 (8)
0.00 (8)
0
27-30
L8
19
19 (8)b
0.00 (7); 37.24 (1)
0
26-30
L9
32
20 (8)
0.00 (8)
0
27-30
L10
25
20 (8)
0.00 (8)
0
25-29
U1
24
20 (8)
0.00 (7); 36.04 (1)
0
27-30
Cage
number
Number of adults
collected
Number of
adults tested
per cage1
B8
22
20 (8)
B16
27
B20
1
Five and three extracts made with one adult and five adults per extraction respectively except:
Two extractions with 5 adults: total extraction numbers are given in parentheses.
b
Two extractions with 5 adults and 1 extraction with 4 adults;
2
FAM Ct values are given without parentheses. Numbers within parentheses indicate the number of
the samples showing the respective values. Only 16 samples produced FAM > 0.00.
3
Number of samples that are ‘CLas’ positive
* Psyllid sample that produced FAM Ct value of 17.03 (isolate L4-1) was sequenced; the rest of the
extracts in the same batch did not yield any amplicon in cPCR.
a
Titres of ‘CLas’ in 2015-curry leaf seedlings collected before and after the ATI are
shown in Table 3.12. All samples collected before the ATI, and the extraction
control which consisted of midrib tissue of a peach plant, showed FAM Ct values
equal to zero while the positive controls produced a FAM Ct value of 33.81. The
highest number of seedlings with FAM Ct values > 0.00 was obtained from plants
sampled on the first occasion. Twelve seedlings produced FAM Ct values ranging
from 33.14–39.40 and three of these had FAM Ct values ≤ 36. None of these
94
samples yielded any amplicons when subjected to cPCR. Hence, the presence of
‘CLas’ could not be confirmed through sequencing. On the second occasion when
seedlings were sampled, the same plants produced FAM Ct values equal to zero or
higher than the FAM Ct of the first sampling except for curry leaf seedlings, CL13
and CL17, that both yielded FAM Ct value of 35.27. On the third occasion, 293 d
(~10 months) after the ATI, all plants yielded FAM Ct values equal to zero. The
internal control Ct values for some of the first samples were zero due to degraded
DNA (DNA elutes appeared dark).
Titres of ‘CLas’ in the NSC mandarin seedlings before and after the ATI are shown
in Table 3.13. As for the curry leaf seedlings above, samples from the mandarin
seedlings were collected before being placed in the experimental cages with infected
mandarin stumps and adult psyllids. Samples were collected at the same intervals as
noted above for the curry leaf seedlings. All mandarin seedling samples collected
before the experiment commenced produced FAM Ct values equal to zero and the
CAL Ct values ranging from 14.01 to 22.45. The highest number of samples with
FAM Ct values > 0.00 was observed for plants, including control plants, sampled on
the first occasion. Ten of 17 plants with FAM Ct values > 0.00 were subjected to
cPCR but none produced amplicons. The number of samples with FAM Ct values >
0.00 decreased in the subsequent samplings. The FAM Ct values for the positive
controls in the assays ranged from 19.66 to 33.80 while those of the extraction
controls were 0.00. The CAL Ct values for the positive and extraction controls
ranged from 14.34–27.36 and 18.57–33.5, respectively.
95
Table 3.12. 'CLas' titres in samples of 2015-curry leaf seedlings collected before and after the ATI of Experiment 1 (2015).
2015-curry
leaf seedling
(cage
number)1
Ct values before ATI
Ct values of samples (days after ATI)
76 d
243 d
293 d
FAM
CAL
FAM
CAL
FAM
CAL
FAM
CAL
CL1 (L1)
0.00
15.95
0.00
0.00
0.00
24.20
0.00
20.71
CL2 (L1)
0.00
16.16
0.00
0.00
0.00
24.33
0.00
21.21
CL5 (L3)
0.00
18.61
0.00
0.00
0.00
24.04
0.00
21.57
CL6 (L3)
0.00
17.33
0.00
0.00
0.00
24.21
0.00
20.52
CL7 (L4)
M
M
0.00
39.63
0.00
22.61
0.00
20.62
CL8 (L4)
0.00
19.18
0.00
0.00
0.00
25.86
0.00
20.01
CL9 (L5)
0.00
18.41
34.45
27.53
0.00
28.22
0.00
21.32
CL10 (L5)
0.00
17.16
33.14
25.99
39.63
28.37
0.00
21.10
CL13 (L8)
0.00
18.00
39.40
25.72
35.27
27.76
0.00
19.03
CL14 (L8)
0.00
18.05
0.00
24.32
0.00
27.07
0.00
19.84
CL15 (L9)
0.00
17.37
37.67
23.83
0.00
24.85
0.00
31.64
CL16 (L9)
0.00
16.87
0.00
0.00
0.00
26.31
0.00
0.00
CL17 (L10)
0.00
17.06
37.97
24.53
35.27
25.42
0.00
0.00
CL18 (L10)
0.00
17.41
36.00
29.20
0.00
24.55
0.00
34.26
CL19 (B22)
0.00
17.72
37.99
29.84
0.00
25.19
0.00
0.00
CL20 (B22)
0.00
20.22
0.00
27.83
0.00
24.67
0.00
0.00
CL21 (B16
0.00
17.97
37.45
29.18
0.00
24.47
0.00
0.00
96
2015-curry
leaf seedling1
Ct values before ATI
Ct values of samples (days after ATI)
76 d
243 d
293 d
FAM
CAL
FAM
CAL
FAM
CAL
FAM
CAL
CL22 (B16)
0.00
19.61
0.00
28.11
0.00
25.54
0.00
21.75
CL23 (U1)
0.00
19.45
37.68
25.13
0.00
24.84
0.00
0.00
CL24 (U1)
0.00
17.76
36.90
24.82
0.00
23.62
0.00
0.00
CL29 (B20)
0.00
17.75
0.00
27.99
0.00
25.54
0.00
0.00
CL30 (LB20)
0.00
17.99
0.00
28.27
0.00
23.85
0.00
0.00
CL32 (L6)
0.00
17.96
36.07
27.80
0.00
0.00
0.00
0.00
CL34 (B8)
0.00
15.45
0.00
25.18
0.00
24.82
0.00
0.00
CL38 (B8)
0.00
16.30
36.61
26.51
37.41
24.34
0.00
0.00
CL41 (L6)
0.00
16.46
0.00
31.74
0.00
23.47
0.00
0.00
CL42 (L2)
0.00
17.11
0.00
28.12
38.63
28.22
0.00
0.00
CL43 (L2)
0.00
19.67
M
M
0.00
27.57
0.00
0.00
CL44 (C)
0.00
15.79
M
M
0.00
38.03
0.00
0.00
CL45(C)
0.00
16.99
M
M
0.00
26.70
0.00
0.00
PC
33.81
24.27
19.95
22.72
18.89
21.25
19.66
14.34
EC
0.00
33.50
0.00
27.00
0.00
23.74
0.00
18.57
1
Cage numbers are given in parentheses.
ATI = Acquisition and transmission interval.
‘C’ indicates control cage which was set up without psyllids or inoculum source with test plants CL44 & CL45.
FAM = ‘CLas’ DNA, CAL = internal control;
M = missing samples; PC = positive controls; EC = extraction control (peach midribs).
Samples subjected to cPCR are shown with emboldened FAM Ct values.
97
Table 3.13. ‘CLas’ titres in NSC mandarin seedlings before and after the ATI of the Experiment 1 (2015)
NSC
mandarin
seedling1
Ct values before ATI
Ct values of samples collected after ATI
76 d
243 d
293 d
FAM
CAL
FAM
CAL
FAM
CAL
FAM
CAL
M1 (L1)
0.00
17.61
0.00
18.77
0.00
21.58
0.00
26.94
M2 (L1)
0.00
16.31
34.15
18.36
0.00
21.59
37.84
25.95
M5 (L3)
0.00
17.21
0.00
18.67
0.00
21.92
0.00
25.19
M6 (L3)
0.00
16.11
0.00
18.84
0.00
24.11
0.00
25.01
M7 (L4)
0.00
16.29
0.00
18.47
0.00
24.34
0.00
25.94
M8 (L4)
0.00
17.84
36.52
18.86
0.00
27.50
39.02
24.74
M9 (L5)
0.00
18.04
0.00
19.40
0.00
23.80
0.00
26.43
M10 (L5)
0.00
21.48
38.32
19.30
0.00
21.62
0.00
24.20
M11 (L6)
0.00
15.21
38.75
19.12
38.87
17.27
0.00
27.34
M12 (L6)
0.00
15.13
37.10
20.24
36.80
18.53
0.00
28.52
M13 (L8)
0.00
16.40
38.82
18.27
0.00
18.40
38.49
23.42
M14 (L8)
0.00
15.32
0.00
21.27
0.00
19.75
0.00
24.92
M15 (L9)
0.00
22.45
0.00
19.33
0.00
17.16
0.00
26.56
M16 (L9)
0.00
15.88
0.00
21.63
0.00
18.03
0.00
27.25
M17 (L10)
0.00
15.39
0.00
24.63
0.00
16.79
0.00
24.79
M18 (L10)
0.00
14.61
36.86
21.17
37.05
15.64
0.00
27.36
M19 (B22)
0.00
14.54
M
M
38.56
15.87
0.00
27.43
M20 (B22)
0.00
14.01
36.30
18.65
37.99
17.33
36.55
24.58
M21 (B16)
0.00
14.34
0.00
20.14
0.00
23.23
0.00
27.18
98
NSC
mandarin
seedling1
Ct values before ATI
Ct values of samples collected after ATI
76 d
FAM
CAL
243 d
293 d
FAM
CAL
FAM
CAL
FAM
CAL
M22 (B16)
0.00
15.16
0.00
19.16
0.00
22.81
0.00
25.45
M23 (U1)
0.00
16.06
38.54
20.15
0.00
23.45
0.00
27.58
M24 (U1)
0.00
14.72
38.92
19.20
0.00
22.72
0.00
23.58
M26 (B11)
0.00
14.01
38.05
18.12
0.00
24.79
0.00
23.58
M27 (B8)
0.00
15.58
37.61
18.33
M
M
37.00
24.50
M28 (B8)
0.00
14.93
37.09
18.36
0.00
23.59
0.00
25.81
M24 (U1)
0.00
14.72
38.92
19.20
0.00
22.72
0.00
23.58
M29 (B20)
0.00
14.35
0.00
19.80
0.00
24.56
0.00
24.90
M30 (B20)
0.00
15.65
38.36
23.95
0.00
21.51
0.00
24.10
M31 (L2)
0.00
19.54
0.00
21.86
35.67
28.92
dead
dead
M32 (L2)
0.00
15.88
37.77
18.26
0.00
24.46
0.00
24.50
M33 (C)
0.00
16.45
35.87
19.03
M
M
0.00
25.52
M34 (C)
0.00
14.81
36.58
18.42
M
M
0.00
23.22
PC
33.80
24.27
26.51
27.36
19.66
14.34
20.62
0.00
EC
0.00
33.50
0.00
21.31
0.00
18.57
0.00
24.10
1
Cage numbers are given in parentheses; ‘C’ indicates control cage that was set up without psyllids or inoculum source with test plants M33 & M44.
Sampling commenced ~75d after start of incubation and subsequent sampling conducted at the same interval; FAM = ‘CLas’ DNA; CAL = internal control;
M = missing samples; PC = Positive controls; EC = Extraction controls (peach midribs).
Ct value of the sample subjected to conventional PCR is emboldened.
2
99
3.5.2 Experiment 2 (2015)
The objective of this experiment was to further determine possible transmission of
‘CLas’ by Diaphorina communis adults eclosing from nymph-infested 2015-curry
leaf seedlings to initially psyllid-free NSC mandarin seedlings and 2015B-curry leaf
seedlings.
It was undertaken on the assumption that ‘CLas’ may have been
transmitted to the 2015-curry leaf seedlings from the ‘CLas’-infected mandarin
stumps.
Materials and Methods. Fourteen BugDorm cages were used. Pairs of 2015-curry
leaf seedlings infested with eggs and nymphs were placed in each of the 14 cages on
17 June 2015 in which one ‘CLas’-free NSC mandarin seedling and one ‘CLas’-free
2015B-curry leaf seedling were also placed eight days later (25 June 2015) and
maintained for 82 d; by this time, Diaphorina communis adults eclosed in some
cages. Numbers of Diaphorina communis eggs, nymphs and adults present on 2015curry leaf seedlings were recorded.
Titres of ‘CLas’ in the NSC mandarin, 2015B-curry leaf seedlings and psyllids were
assessed. The first plant samples were collected at 35 d after the ATI (on 15 October
2015), two subsequent samples were collected at 81–83 d. Representative psyllid
samples were collected on 8 July 2015 which corresponded to ~13 d after the
eclosion of first adult psyllids (EFAP) and at the end of the acquisition and
transmission interval (ATI), which lasted for 82 d (i.e., 90th d after commencement of
experiment).
Results. Numbers of Diaphorina communis eggs, nymphs and adults present on
2015-curry leaf seedlings when they were removed from the 14 cages with ‘CLas’infected mandarin stumps and Diaphorina communis adults are presented in Table
3.14. Nymphs were present on three of the plants on the day after removal from the
cage with infected stumps (17 June 2015). Adults were present in five cages eight
days after the cages were removed from the mandarin stumps. Adults were present
in seven of the cages 21 d after the cages were removed from the stumps and most
adults survived until removal on day 90.
100
Titres of ‘CLas’ in Diaphorina communis adults, and the NSC mandarin and 2015Bcurry leaf seedlings during the experiment are presented in Table 3.15. All samples
of psyllid adults collected at 13 d after eclosion of first adult psyllids produced FAM
Ct value of zero except for two samples that produced FAM Ct values of 37.23 and
37.44. Psyllid samples collected at the end of ATI produced FAM Ct values of zero.
The CAL Ct values for the two sample dates ranged from 24.5–26.66 and 24–28,
respectively. Samples of all NSC mandarin and 2015B-curry leaf seedlings before
they were placed in cages with the egg-infested 2015-curry leaf seedlings produced
FAM Ct values equal to zero. Analysis of samples collected at 35 and 118 d after the
ATI resulted in some samples producing FAM Ct values between 34.09 and 39.89
for both the mandarin and curry leaf seedlings used to test transmission. Attempts to
amplify samples with FAM Ct values ranging from 34 to 36 with A2/J5 primers did
not yield any amplicons. Samples collected at 199 d after the ATI resulted in only
one mandarin sample with FAM Ct value > 0.00 (= 38.08) and all curry leaf
seedlings produced FAM Ct values equal to zero.
101
Table 3.14. Number of eggs, nymphs and adults present on 2015-curry leaf seedlings when (17 June 2014) they were removed from the cages with ‘CLas’ infected
mandarin stumps and Diaphorina communis, used to determine transmission of ‘CLas’ from them by eclosing Diaphorina communis adults to NSC mandarin and 2015Bcurry leaf seedlings from 17 June 2015 to 15 September 2015.
Original
cage
number1
Set A curry
leaf seedling
number
NSC
Mandarin
Set B
curry
leaf
seedlings
Presence of eggs and nymphs on 2015-curry leaf seedlings and number of adults emerged during ATI
Day 0 (17 June 2015)
Eggs
nymphs
adults
Day 8 (25 June 2015)
eggs
Day 21 (8
July 2015)
Day 37 (24
July 2015)
Day 90 (15
Sept 2015)
nymphs
adults
adults2
adults
adults2
L1
CL1 & CL2
M8a
CL8a
++
--
0
--
+/-
0
8
1
0
L2
CL42 & CL43
M7a
CL7a
++
--
0
++*
++*
0
4
0
0
L3
CL5 & CL6
M13a
CL13a
+/-
-/+
0
--
--
5
40
13
45
L4
CL7 & CL8
M10a
CL10a
++
++
0
--
--
13
30
5
7
L5
CL9 & CL10
M9a
CL9a
++
--
0
--
--
4
17
2
0
L6
CL32 & CL41
M6a
CL6a
++
--
0
--
+/-
0
1
0
0
L8
CL13 & CL14
M3a
CL3a
++
--
0
--
++
0
4
1
0
L9
CL15 & CL16
M14a
CL14a
++
--
0
++
+/-
1
52
15
22
L10
CL17 & CL18
M11a
CL11a
++
--
0
++
--
3
49
17
6
U1
CL23 & CL24
M12a
CL12a
++
--
0
--
--
0
0
0
0
B8
CL34 & CL38
M2a
CL2a
++
--
0
--
++
0
0
0
0
B16
CL21 & CL22
M1a
CL1a
++
--
0
--
++
0
16
12
25
B20
CL29 & CL30
M5a
CL5a
+/-
--
0
--
++
0
0
0
0
B22
CL19 & CL20
M4a
CL4a
++
-0
-++
0
11
2
0
1
Cage number refers to the experimental cage with infected stumps for transmission for ‘CLas’ from the mandarin stumps to mandarin and Set A curry leaf seedlings; 2
Representative psyllids collected and tested for ‘CLas’; *dried and dead; ++ or --indicate the presence/absence of eggs/nymphs on both inoculum source plants respectively.
+/- indicates presence on one of the source plants but not the other.
102
Table 3.15. ‘CLas’ titres in Diaphorina communis adults, and the NSC mandarin and 2015B-curry leaf
seedlings during the experiment to determine transmission of ‘CLas’ by Diaphorina communis adults
from 2015-curry leaf seedlings.
FAM Ct range of psyllids collected at 13 d after the eclosion of first adult
psyllids1
0.00–37.44*
CAL Ct range of psyllids collected at 13 d after the eclosion of first adult
psyllids1
24.5–26.66
FAM Ct range of psyllids at end of ATI2
0.00
CAL Ct range of psyllids at end of ATI2
24–28
FAM Ct range of the 14 NSC mandarin seedlings before caging with 2015curry leaf seedlings.
0.00
FAM Ct range of 14 2015B-curry seedlings before caging with 2015-curry
leaf seedlings.
0.00
Number of NSC mandarin seedlings with FAM Ct values > 0.00 and their Ct range. Samples collected
on three occasions:
1.
35 d after ATI
7 & 34.95–38.90**
2.
118 d after ATI
4 & 37.06–39.72
3.
199 d ATI
1 & 38.08
Number of Set B curry leaf seedlings with FAM Ct values > 0.00 and their Ct range:
1.
35 d after ATI
10 & 34.09–39.89**
2.
118 d after ATI
5 & 36.51–39.39
3.
199 d after ATI
0
1
16 samples of adult Diaphorina communis and each sample extracted from composite of two adults;
38 samples comprising of 22 extracts made from single adults and 16 extracts made from composite
of five adults per extract;
* Only two samples with FAM Ct of 37.23 & 37.44;
** Samples with FAM Ct between 34 and 38 subjected to cPCR with primers A2/J5, but did not yield
any amplicon.
2
103
3.6
Discussion
3.6.1 Preliminary studies in 2013
In the preliminary experiments, Diaphorina communis nymphs failed to survive on the
NSC mandarin seedlings and no oviposition took place when adults were caged on
immature, fully-expanded flush growth that developed on skeletonised branches of
‘CLas’-infested mandarin trees. However, survival of adults was better than nymphs.
When adults were placed on branches of mandarin plants with young buds after the
leaves had been stripped off, one egg was observed but the nymph that hatched from it
did not survive to the second instar.
These observations suggest that Diaphorina communis does not develop, or rarely
develops, on mandarin and possibly other species of citrus.
Therefore, further
observations were made with regards to number of eggs, nymphs and adult emergence
in cages in the field as well as under laboratory conditions. These observations
confirmed that curry leaf is the preferred host of Diaphorina communis, and complete
development takes place, possibly exclusively, on curry leaf. In the field cages at
Baychhu, curry leaf plants had an average of 71 eggs per plant compared to one egg
laid on mandarin. In the following weeks, higher numbers of eggs (65) were found on
curry leaf compared to mandarin (0), and many nymphs had emerged on curry leaf by
the second week.
However, the proportion of surviving adults was comparable
between mandarin and curry leaf, i.e., adult psyllids can survive on mandarin but
rarely reproduce. A similar trend was observed in the laboratory when adults of
Diaphorina communis were provided with curry leaf and mandarin plants in the
choice and no choice experiment. No psyllids developed if curry leaf seedlings were
not provided (Figure 3.6). This outcome provided further evidence that Diaphorina
communis does not develop on mandarin. In the field, adults reared from nymphs
collected from curry leaf shrubs were always Diaphorina communis (pers. obs.).
However, adults were often observed at Baychhu on mature, seedling mandarin trees,
Murraya elongata (see Chapter 7), and a species of Zanthoxylum.
Diaphorina
communis was originally described from curry leaf (Mathur 1935; 1975), and Tara &
Sharma (2010a; 2010b) have more recently reported it as a host plant of the psyllid.
104
My results suggest that Diaphorina communis has a very narrow host range, in
contrast to Diaphorina citri, which is known to colonise a wide range of hosts (Aubert
1990; Manjunath & Halbert 2004; Nava et al. 2007; Beattie & Barkley 2009;
Westbrooke et al. 2011). The most common theory applied to insect host plant
preference is the ‘optimal oviposition’ theory, which takes into account the suitability
of the host plant for the development of sessile offspring (Prager et al. 2014). The
factors that influence the preference for curry leaf by Diaphorina communis remain to
be determined. Insect host preference is influenced by many factors such as stimulant
or deterrent host plant metabolites, and involves visual, olfactory, gustory and tactile
cues and mechanisms (Nishida 2014).
Testing for ‘CLas’ in adult Diaphorina communis caged on infected branches of
mature mandarin trees for 54 d in the preliminary experiment (Section 3.3.2) showed
FAM Ct values ranging from 20.58 to 39.5 and 21.53 to 38.11 in single and composite
samples of adults, respectively. In total, 14 samples produced FAM Ct value between
0.00 and 32, while 41 samples out of the total 53 samples produced FAM Ct values
between 0.00 and 40. Selected samples have been successfully sequenced. This result
supports an earlier record of acquisition of ‘CLas’ by Diaphorina communis from
mandarin (Donovan et al. 2012a; 2012b).
3.6.2 Studies in 2014 and 2015
Studies on acquisition of ‘CLas’ by Diaphorina communis commenced in 2013, and
possible transmission was assessed in 2014 and 2015 using greenhouse-grown curry
leaf and mandarin seedlings with naturally-infected mandarin stumps as a source of
‘CLas’. Possible acquisition and transmission of ‘CLas’ by Diaphorina communis
from curry leaf seedlings held with ‘CLas’-infected mandarin stumps to greenhousegrown mandarin and curry leaf seedlings was also assessed.
Although the mandarin stumps used as inoculum sources in 2014 all tested positive
for ‘CLas’ (Table 3.5), few psyllids collected at the end of the ATI tested positive for
‘CLas’ (Table 3.6) when the FAM Ct threshold was set at 32. Some samples of
psyllids collected at the end of the ATI on 5 July 2014 produced FAM Ct values > 32
105
in contrast to samples tested before their release into the cages with ‘CLas’ infected
mandarin stumps that mainly produced FAM Ct value of zero. This questions the
reliability of the Ct cut-off value. A value of zero indicates that the bacterium is not
detected, whereas the results of samples with FAM Ct values in the range of 32 <
FAM Ct ≤ 40 are ambiguous, as these high Ct values can be interpreted as the insects
having low titres of the bacterium or due to some nonspecific amplification during the
assay.
However, non-specific amplifications seem quite unlikely given that all
psyllids samples produced FAM Ct value of zero when tested before the psyllids were
released in cages with ‘CLas’-infected mandarin infected stumps (Tables 3.6).
For NSC mandarin and 2014-curry leaf seedlings used to test transmission of the
pathogen by Diaphorina communis in 2014, FAM Ct values in the range of 0.00 <
FAM Ct < 36 were recorded in five plant samples collected at 44 d after ATI (Table
3.4 & 3.5). These values indicated that the plant samples contained ‘CLas’ as a result
of acquisition and transmission of the pathogen from ‘CLas’-infected mandarin
stumps by Diaphorina communis. However, with one exception (mandarin B1 at 377
d), subsequent samples from all seedlings produced FAM Ct values equal to zero or in
the range of 36 < FAM Ct < 40. Further assessment with cPCR of extracts from
plants with FAM Ct values from 34 to 37 did not yield any amplicons. Similar results
were obtained with the attempt to inoculate NSC mandarin seedlings from the 2014curry leaf seedlings (Experiment 2 of 2014, Table 3.7). These samples could not be
‘CLas’ positive. The FAM Ct values in the range of 0.00 < FAM Ct < 36 during the
first sampling of both mandarin and curry leaf samples of Experiment 1 and mandarin
samples of Experiment 2 of 2014 could have been due to contamination during the
extraction process in the laboratory.
Some of the samples processed prior to
November 2014 were chopped with the same blade. Use of disposable blades and
sterile glassware were strictly observed after observation of such PCR results in
November 2014. Moreover, a very low number of adults survived in the cages after
50 were released into each cage in the experiment, with most of these adults dying
shortly after they were released. Populations in the cages were supplemented by the
release of additional adults on two occasions, the last 16 d before ATI. Adult survival
was higher in the cages with curry leaf seedlings (Tables 3.2 & 3.3). For Experiment
106
2 of 2014, it is worthy to note that the samples of the NSC mandarin seedlings
mandarin 5 and 8 in cages CLB15 and CLB21 collected at 372 and 536 d after ATI
produced FAM Ct values ≤ 36 but these cages did not even have any adult eclosed
during the entire 71 d period (Table 3.7).
The outcome of studies on acquisition and transmission of ‘CLas’ by Diaphorina
communis in 2015 (Tables 3.9–3.15) did not differ from the outcomes in 2014. The
experiment in 2015 addressed factors overlooked in 2014 by increasing the number of
test seedlings; using both curry leaf and mandarin seedlings so as to enhance survival
of psyllid adults in the cages; sampling curry leaf and mandarin seedlings for ‘CLas’
before and after psyllid adults fed on them; including curry leaf and mandarin
seedlings not exposed to Diaphorina communis and ‘CLas’-infected mandarin stumps
as controls; counting and augmenting psyllid populations in cages weekly; and
including DNA extraction controls using leaf tissue of either apple or peach to detect
any contamination during extraction. However, sampling of mandarin and curry leaf
seedlings used in the 2015 experiment was only feasible on three occasions up to 293
d after the ATI. As in 2014, all psyllid samples representing populations released into
the cages produced FAM Ct values equal to zero.
In contrast, psyllid samples
collected from the cages at the end of ATI produced FAM Ct values ranging from
0.00 to 39.7, and seven samples had FAM Ct values ≤ 32 (Table 3.11). Although one
psyllid sample with a FAM Ct value of 17.03 produced an amplicon that was
successfully sequenced using primers A2/J5, other extracts with FAM Ct values >
0.00 failed to produce amplicons.
The current data on the detection of ‘CLas’ in Diaphorina communis indicates a low
acquisition rate of ‘CLas. In addition, the experiments in 2014 and 2015 indicate that
although Diaphorina communis can acquire ‘CLas’, it does not appear to be able to
transmit it, and the pathogen may not multiply within the insect. Although some of
the test plants produced FAM Ct values ≤ 36 (Tables 3.4 & 3.5, 3.7, 3.12 & 3.13),
these plants cannot be interpreted as ‘CLas’ positive, because no sequencing results
could be achieved. Moreover, it is difficult to know where to set the Ct threshold,
107
given that Ct thresholds vary in the literatures and there appears to be an inconclusive
region above the threshold.
3.7
Highlights of the study:
Diaphorina communis adults can acquire ‘CLas’ but at a low rate.
Diaphorina communis feeds and develops exclusively on curry leaf when given
the choice. However, a low to negligible rate of development of Diaphorina
communis is observed on mandarin under no-choice conditions
There is no evidence that curry leaf can harbour ‘CLas’.
No evidence of transmission of ‘CLas’ by Diaphorina communis from infected
mandarin to mandarin or curry leaf could be established.
108
Chapter 4: Comparison of parasitoids of Diaphorina
citri Kuwayama and Diaphorina communis Mathur
(Hemiptera: Sternorrhyncha: Liviidae)
____________________________________________________________________
4.1
Introduction
Tamarixia radiata (Waterson) (Hymenoptera: Eulophidae) is an ectoparasitoid
attacking nymphs of the Asiatic citrus psyllid (ACP), Diaphorina citri and was first
described as Tetrastichus radiatus by Waterston (1922). The Asiatic citrus psyllid is
the primary vector of ‘CLas’, the causal agent of huanglongbing (HLB) (Aubert 1987;
Halbert & Manjunath 2004; Bové 2006; Hall et al. 2013). As mentioned in Chapter 1,
HLB is a serious disease of citrus for which there is no cure. Disease mitigation
requires integrated programs including the suppression of psyllid populations through
various methods. Biological control is one approach to limiting psyllid populations,
particularly in homegarden, suburban situations or where the use of insecticides is
problematic.
As such, Tamarixia radiata has been introduced intentionally or
unintentionally to Réunion and Mauritius (Aubert et al. 1996), China, including
Taiwan (Chiu et al. 1988; Tang 1988; Chien & Chu 1996), the Caribbean and widely
within the citrus growing regions of North, Central and South America where
Diaphorina citri now occurs (Étienne et al. 2001; Parra et al. 2016).
As mentioned in the earlier chapters, the black psyllid, Diaphorina communis, was
recorded by Mathur (1935) on curry leaf (Bergera koenigii L.) at Dehradun in
northern India, but not described until 1975 (Mathur 1975). Heavy infestations of
nymphs cause leaves to wilt and fall, and young growth can be killed and branches
deformed (Mathur 1935).
Aubert (1987) briefly mentioned that Diaphorina
communis and two other psyllids, Diaphorina auberti Hollis and Trioza litseae
Bordage, harbour ‘chalcidoid parasite insects identical to those attacking either Trioza
erytreae or Diaphorina citri’. ‘Candidatus Liberibacter asiaticus’ was recorded in the
adult psyllid by Donovan et al. (2012a;2012b). The pathogen was also recorded in
Diaphorina communis in my studies (Chapter 3).
109
In Bhutan, parasitised nymphs of Diaphorina communis were first observed in a small
mandarin orchard at Baychhu (27.29749ºN, 89.96691ºE, 784 m ASL), Wangdue
Phodrang Dzongkhag, in April 2013 on curry leaf. Although the percent parasitism in
the orchard was not determined, parasitised nymphs were common. These nymphs
exhibited exit holes on the thorax similar to holes left by emerging Tamarixia radiata
adults in mummified Diaphorina citri nymphs. Parasitised nymphs of Diaphorina
citri were also observed in another location on Murraya paniculata (L.) Jack (syn.
Murraya exotica L.) (see Mabberley 2016). Subsequently, samples were collected to
determine the parasitoids associated with the two species of psyllid.
Molecular characterisation is necessary to complement morphological descriptions
and is crucial if cryptic species are present in a genus.
Moreover, the proper
identification or characterisation of a parasitoid is important in biocontrol programs to
ensure that any introduced agent is a primary parasitoid and not a hyperparasitoid, as
hyperparasitism can reduce the effectiveness of biocontrol agents (Aubert 1987).
Therefore, in this study, the mitochondrial cytochrome oxidase subunit I (COI) gene
and nuclear internal transcribed spacers (ITS1 & ITS2) of the rRNA gene were used to
investigate the genetic diversity and phylogenetic relationships between the two
parasitoids of Diaphorina citri and Diaphorina communis. All three regions have
been extensively used for molecular studies of insects (Lunt et al. 1996; Hebert et al.
2003; Ji et al. 2003).
The specific aims of this section of study were to:
describe the molecular and morphological characteristics of the parasitoid of
Diaphorina communis; and
to study the phylogenetic relationships between the parasitoids collected from
Diaphorina communis and Diaphorina citri.
110
4.2
Materials and methods
4.2.1 Collection of parasitoids
Although parasitism was recorded in April 2013, only specimens collected in 2014
were used for molecular studies.
In this year, parasitoids emerging from single
nymphs were individually collected. A total of 230 parasitoids from Diaphorina
communis and 14 parasitoids from Diaphorina citri were collected from April to June
2014 in Bhutan. Parasitised nymphs of Diaphorina communis were collected from
curry leaf in Baychhu and nearby Basochhu (27.36463°N, 89.91241°E, 1036 m ASL)
while parasitised nymphs of Diaphorina citri were collected from Murraya paniculata
(as identified in this study) found in Zangtopelri Park (26.86212°N; 89.38288°E, 221
m ASL) in Phuentsholing, Chukhha Dzongkhag. Each parasitised nymph was excised
along with the leaf tissue and placed in a gelatine capsule on the evening of the
collection day. The capsules were then placed in plastic containers and kept at room
temperature (except during transportation) from the field to the laboratory. Capsules
were examined daily for emergence of adult parasitoids. Upon emergence, adults
were immobilised by freezing whole capsules at –20°C for > 3 h, preserved in 100%
ethanol and then stored at –20°C until further use.
4.2.2 Molecular and phylogenetic analyses
4.2.2.1
Parasitoid DNA extraction
DNA was then extracted from whole insects using ISOLATE II Genomic DNA kits
(Bioline) following the manufacturer’s instructions but without crushing the samples
(non-destructive method). This method allowed the same specimen to be used for
microscopy. The pre-lysis step of the protocol was done overnight in a shaking,
thermal block at 56°C and 300 rpm (Thermomixer Comfort, Eppendorf).
The
specimens were taken out before the DNA binding step, and stored in 80% ethanol at
−20°C. DNA samples were stored at −20°C till further work.
111
4.2.2.2
Amplification of DNA and sequencing
The COI gene and ITS1 and ITS2 regions from the parasitoids were amplified using
the polymerase chain reaction (PCR). For the COI gene, the primer pair, CI-J-1718
(5’-GGA GGA TTTG GAA ATT GAT TAG TTC C-3’) and C1-N-2191 (5’-CCC
GGT AAA ATT AAA ATA TAA ACT TC-3’) (Simon et al. 1994; Barr et al. 2009),
was used. The ITS1 and ITS2 regions were amplified using primers ITS1-F/18sF (5’GTGAACCTGCGAAGGA-3’) with 5.8sR (5’-GTT CAT GTC CTG CAG TTC
ACA-3’), and 5.8sF (5’-TGT GAA CTG CAG GAC ACA TGA AC-3’) with 28sR
(5’- ATG CTT AAA TTT AGG GGG TA-3’), respectively. Reactions were set up as
in Section 2.2.2 (Chapter 2).
For all three regions, amplification cycling parameters were taken from Barr et al.
(2009). For COI: initial denaturation at 94°C for 3 min followed by 39 cycles of
denaturation at 94°C for 20 sec, annealing at 50°C for 20 sec, extension at 72°C for 30
sec and a single cycle of final extension at 72°C for 5 min. For ITS1 region: initial
denaturation at 94°C for 3 min and 35 cycles of denaturation at 94°C for 30 sec,
annealing at 55°C for 30 sec, and extension at 72°C for 50 sec followed by a single
cycle of final extension at 72°C for 7 min. The same conditions were applied to the
ITS2 region except the annealing temperature was 50°C. Post PCR procedures were
conducted as in Section 2.2.3 (Chapter 2).
4.2.2.3
Sequence analyses
Contiguous sequences of DNA were assembled at first with DNA Baser Sequence
Assembler software (Version 4, Heracle Biosoft) and later with Geneious (Version 8,
Kearse et al. 2012). Homologous sequences in GenBank (Table 4.1) were obtained
through BLAST searches (NCBI, Maryland, USA). Only sequences that showed >
70% identity and coverage were selected from the data base. The sequences obtained
from this study and from the database were aligned using Muscle as enabled in
MEGA (Version 6, Tamura et al. 2013). Each alignment was cross checked to verify
base callings and for the presence of indels.
112
For each gene or region, the number of variable sites and genetic distances (p) of the
aligned sequences were calculated using MEGA 6. For COI, nucleotide sequences
were translated to amino acid sequences using the invertebrate mitochondrial genetic
code in MEGA 6 and also with the program ExPASy-translate tool (Gasteiger et al.
2003) to check the open reading frame (ORF).
113
Table 4.1. GenBank sequences used in molecular analyses of sequence data obtained from different populations of parasitoids of Diaphorina communis and Diaphorina citri
collected in Bhutan.
Gene
Species
Accession number
Reference
COI
Tamarixia radiata
FJ807949
de León & Sétamou (2010)
Tamarixia radiata
GQ912277
de León & Sétamou (2010))
Tamarixia radiata
KT253023
Guzman-Larralde et al. (unpublished)
Tamarixia triozae
GQ912287
de León & Sétamou (2010)
Aprostocetus aethiops
HM573652
Kaartinen et al. (2010)
Aprostocetus cerrricola
JQ416730
Stone et al. (2012)
Aprostocetus cerrricola
HM573863
Kaartinen et al. (2010)
Aprostocetus forsteri
KR882328
Hebert et al. (2016)
Aprostocetus gala
KJ701414
Shylesha & Abraham (unpublished)
Aprostocetus meltofei
KR805635
Hebert et al. (2016)
Aprostocetus meltofei
KU374349
Wirta et al. (2016)
Tetrastichus schoenobii
KJ627790
Reetha et al. (unpublished)
Tamarixia radiata
FJ807955
de León & Sétamou (2010)
Tamarixia radiata
GQ401991
Barr et al. (2009)
Tamarixia radiata
JF924972
Tanner et al. (2011)
Tamarixia radiata
GQ401789
Barr et al. (2009)
Tamarixia radiata
JF925003
Tanner et al. (2011)
Aprostocetus aethiops
HM573653
Kaartinen et al. (2010)
Aprostocetus gala
KF958278
Reetha et al. (unpublished)
ITS1
ITS2
114
4.2.2.4
Phylogenetic relationships
Phylogenetic relationships between the parasitoids of Diaphorina communis and
Diaphorina citri were inferred based on the COI gene and ITS1 and ITS2 regions
using maximum likelihood (ML).
Before analysis, the appropriate evolutionary
model was determined for each gene. For the COI gene, General Time Reversible
model with a discrete gamma distribution (GTR + G) (Nei & Kumar 2000) was used
to model the evolutionary rate differences among sites. For the ITS1 and ITS2
regions, the Kimura 2-parameter model (Kimura 1980) was used. For all three
genes, initial trees for the heuristic search were obtained automatically by applying
Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated
using the maximum composite likelihood approach, and then selecting the topology
with superior log likelihood value. All analyses were performed with 1000 bootstrap
replicates and were conducted in MEGA6 (Tamura et al. 2013).
For COI, sequences of Tetrastichus schoenobii Ferrière (Reetha et al. unpublished)
was used as an outgroup. Analyses of ITS1 did not include any outgroup sequences
from GenBank, as the available sequences were much longer than the sequences of
the parasitoids from this study (≈235–451 bp differences), as alignment of sequences
that differ this number of nucleotides would give inaccurate results (de León &
Sétamou 2010). Details of specimens obtained from the current study are shown in
Table 4.2.
115
Table 4.2. Specimens of parasitoids used for molecular analysis and the GenBank accession numbers of the three genetic regions analysed. N/D = no data.
Genetic regions
ITS1
ITS2
Accession Number
Host
Location
PBT.1
Diaphorina communis
Baychhu
KX986284
KY047789
KY047810
PBT.2
Diaphorina communis
Basochhu
KX986285
N/D
KY047799
PBT.3
Diaphorina communis
Baychhu
KX986286
KY047784
KY047800
PBT.4
Diaphorina communis
Baychhu
KX986287
KY047785
KY047803
PBT.5
Diaphorina communis
Baychhu
KX986288
KY047786
KY047804
PBT.6
Diaphorina communis
Baychhu
KX986289
KY047787
KY047812
PBT.7
Diaphorina citri
Phuentsholing
KY026089
KY047795
KY047813
PBT.8
Diaphorina citri
Phuentsholing
KY026090
KY047798
KY047814
PBT.9
Diaphorina citri
Phuentsholing
KY026091
KY047797
KY047816
PBT.10
Diaphorina citri
Phuentsholing
KY026092
KY047796
KY047815
PBT.11
Diaphorina communis
Basochhu
KX986290
KY047793
KY047805
PBT.12
Diaphorina communis
Basochhu
KX986291
KY047794
KY047817
PBT.13
Diaphorina communis
Basochhu
KX986292
N/D
KY047806
PBT.14
Diaphorina communis
Basochhu
KX986293
KY047792
KY047807
PBT.15
Diaphorina communis
Basochhu
KX986294
KY047791
KY047808
PBT.16
Diaphorina communis
Basochhu
KX986295
KY047790
KY047809
PBT.17
Diaphorina communis
Basochhu
N/D
KY047788
KY047811
PBT.18
Diaphorina communis
Basochhu
KY026088
N/D
KY047801
PBT.19
Diaphorina communis
Basochhu
N/D
N/D
KY047802
COI
116
4.2.3 Morphology
Morphological examinations and descriptions were carried out by Dr Zoya A.
Yefremova (Department of Zoology, The Steinhardt Museum of Natural History, Tel
Aviv University, Aviv, Israel) and Ekaterina N. Yegorenkova (Department of
Geography and Ecology, Ulyanovsk State Pedagogical University, Ulyanovsk,
432700, Russia). The following terminologies and methods were applied in the
descriptions:
Morphological terminology follows that of Bouček and Askew (1968), Graham
(1991) and Gibson et al. (1997): F1 – first segment of antennal funicle; F2 –
second segment; F3 – third segment; F4 – fourth segment; C1 – first segment
of clava; C2 – second segment of clava; C3 – third segment of clava; SMV –
submarginal vein; MV – marginal vein; PMV – postmarginal vein; SV –
stigmal vein; POL – the minimum distance between the posterior ocelli; OOL –
the minimum distance between the eye margin and the adjacent posterior
ocellus.
Absolute measurements in millimeters (mm) were used for body and forewing
length.
For all other dimensions, relative measurements were used.
Observations and measurements were made using a Leica M80, and the
photographs were taken with a Nikon 1V1 mounted on a Leitz Z16 APO
macroscope by Zoya A. Yefremova.
117
4.3 Results
4.3.1 Field observation of parasitism
No formal survey was made to determine the distribution and percent parasitism in
Bhutan. During the course of this study, both nymphs of Diaphorina citri and
Diaphorina communis were found to be parasitised. In March 2013, parasitized
nymphs of Diaphorina communis were found on Bergera koenigii in Baychhu,
Wangdue Phodrang Dzongkhag. Thereafter, parasitism of Diaphorina communis
was observed in the Basochhu area (~10 km from Baychhu). Parasitised nymphs of
Diaphorina citri were found on Murraya paniculata (as identified in this study) in
Zangtopelri Park in Phuenthsoling, Chukhha dzongkhag. The parasitised nymphs
were sessile and appeared dark brown. Both types of nymphs exhibited exit holes
through the thorax.
Figure 4.1 shows the parasitised nymphs of Diaphorina
communis and Diaphorina citri.
4.3.2 Molecular and phylogenetic analyses
4.3.2.1 COI region
A BLAST search for homologous sequence from GenBank using the sequences of
parasitoids of Diaphorina citri from Bhutan showed 99% identity with Tamarixia
radiata. Three sequences of Tamarixia radiata from different studies (Table 4.1)
were selected from GenBank for comparison with the parasitoid sequences in this
study. Searches using the sequences from the parasitoids of Diaphorina communis
returned sequences from Tamarixia radiata; however, the sequences only had 91–
93% identity with those from Tamarixia radiata.
The unaligned length of the multiple COI nucleotide sequences of the parasitoids of
Diaphorina citri and Diaphorina communis collected in Bhutan ranged from 450–
497 bp which, when aligned and trimmed, resulted in 447 bp. Out of the aligned 447
118
Figure 4.1. Parasitised psyllid nymphs and thoraxic exit holes (A-C); Diaphorina communis on
Bergera koenigii (A & B); Diaphorina citri nymphs on Murraya paniculata. (C); Adult parasitoid
laying eggs on Diaphorina citri nymphs on Murraya paniculata (D) (Photos: N. Om & GAC Beattie).
bp, 39 sites were variable with 36 sites being parsimoniously informative (Table 4.3).
Variation was also observed between individuals within each group with four and
eight variable sites and pairwise sequence divergence of 0–1% and 0–2% within the
Diaphorina citri and Diaphorina communis groups, respectively (Table 4.3). The
number of nucleotide differences between the two groups was 31–36 giving in 7–8%
sequence divergence. Both the Diaphorina citri and Diaphorina communis groups
showed ~10% sequence divergence from Tamarixia triozae. The translated COI
amino acid sequences did not have any stop codons nor were there any frameshifts.
Figure 4.2 shows the phylogenetic analysis of the COI gene from 13 individuals
obtained from Diaphorina communis and four individuals obtained from Diaphorina
citri.
Three different clades are formed with strong bootstrap support (> 90).
Parasitoids collected from Diaphorina communis formed a monophyletic clade
except for one individual (PBT.12) that grouped with Aprostocetus. PBT.12 was
119
also morphologically different from the rest of the parasitoids collected in Bhutan.
The second clade comprised Tamarixia radiata isolates from the GenBank and
individuals collected from Diaphorina citri in Bhutan. Tamarixia triozae formed a
sister clade to Tamarixia radiata and the parsitoids of Diaphorina communis.
Variation among the members of the two Tamarixia clades was observed. However,
it was not location-specific; this is more apparent within the parasitoids collected
from Diaphorina communis.
Sequences for the parasitoids collected from
Diaphorina communis were obtained from 12 individuals collected from two sites,
Baychhu and nearby Basochhu, and all subclades contained individuals from both of
these sites.
120
Table 4.3. Sequence polymorphisms within and between populations of parasitoids of Diaphorina citri and Diaphorina communis.
Genetic Region
Analysis
COI
Range of sequence length
450–497
Number of variable sites between and within the accessions of parasitoids from
ITS1
496–530
ITS2
462–514
39 (4; 8)
49 (1; 0)
37 (0; 2)
36 (1; 6)
48 (0; 0)
36 (0; 1)
Diaphorina citri & Diaphorina communis
Number of parsimonious sites between and within the accessions of
parasitoids from Diaphorina citri & Diaphorina communis
Range of number of nucleotide difference between and within the accessions
31–36 (0–3; 0–7)
48–49 (0–1; 0)
35 (0; 0–2)
of parasitoids from Diaphorina citri & Diaphorina communis
Percent sequence divergence between and within parasitoids from
7–8 (0–1; 0–2)
10–11 (0; 0)
8 (0; 0–0.2)
Diaphorina citri & Diaphorina communis
Percent sequence divergence of Diaphorina citri & Diaphorina communis
10–11; 10
*
*
from Tamarixia triozae
Percent sequence divergence of the parasitoids from Diaphorina citri &
0–1; 8
Diaphorina communis from Tamarixia radiata from GenBank
Within group differences are shown in parentheses. Values for Dipahorina communis are embolden; *no sequence available
121
0–1; 11–13
0; 8–9
Figure 4.2. Phylogenetic relationships among the parsitoids of Diaphorina communis and
Diaphorina citri collected in Bhutan based on COI as inferred by maximum likelihood using the
General Time Reversible model with a discrete gamma distribution (GTR + G) (Nei & Kumar 2000).
All parasitoids from Diaphorina communis and Diaphorina citri were collected in Bhutan, and the site
of collection is given next to the accession code. The sequences from the remaining individuals were
obtained from GenBank. The tree with the highest log likelihood is shown. The percentage of trees
from 1000 bootstrap replicates in which associated taxa clustered together is shown next to the nodes.
The tree is rooted using Tetrastichus schoenobii.
122
4.3.2.2 ITS1 region
The length of the ITS1 sequences from the parasitoids of Diaphorina citri and
Diaphorina communis ranged from 496 to 530 bp. There were 49 variable sites with
48 parsimony informative sites between the two groups. However, no variation was
observed within each group. Sequence divergence between the two groups was 10–
11% while zero divergence was observed within each group. For the Diaphorina
citri group, sequence divergence from Tamarixia radiata was 0–1% compared to that
of the Diaphorina communis group which showed had 8% divergence (Table 4.3).
This observation is further supported by the phylogenetic analysis conducted on the
ITS1 region (Figure 4.3) as inferred using maximum likelihood. Similar to the COI
analysis, the parasitoids collected from Diaphorina communis formed a clade
separate from the parasitoids collected from Diaphorina citri. Individuals collected
from Diaphorina citri grouped with Tamarixia radiata isolates taken from GenBank.
Sequences obtained from PBT.12 did not fall into either of the groups.
123
Figure 4.3 Molecular analysis of the ITS1 region of parasitoids collected from Diaphorina citri and
Diaphorina communis in Bhutan as inferred by maximum likelihood method and using the Kimura 2parameter model of evolution (Kimura 1980). The tree with the highest log likelihood (-1022.5155) is
shown. The percentage of trees from 1000 bootstrap replicates in which the associated taxa clustered
together is shown next to the nodes. Site of collection is given next to the accession code for
sequences obtained in this study. Accession numbers are given in parenthesis for sequences obtained
from the GenBank.
124
4.3.2.3 ITS2 region
Sequences of the ITS2 region of individuals from Diaphorina citri and Diaphorina
communis ranged from 462–514 bp. Sequence analysis showed 37 variable sites
with 36 of them being parsimoniously informative (Table 4.3). No variation was
observed within the group of individuals from Diaphorina citri collected in Bhutan
or with the sequences from the database. Within the Diaphorina communis group,
there were two variable sites, of which one was parsimoniously informative. Similar
to the ITS1 region, results showed that sequence divergence between the two groups
was higher (8%) than within groups (zero). The Diaphorina citri group showed no
sequence divergence from Tamarixia radiata while the Diaphorina communis group
had 8–9% divergence.
The phylogenetic analysis of the ITS2 region is shown in Figure 4.4. All individuals
collected from Diaphorina communis formed one clade except for PBT.12 that
grouped with Aprostocetus aethiops. Individuals from Diaphorina citri formed a
distinct, separate clade from individuals of Diaphorina communis with strong
bootstrap support (100%).
125
Figure 4.4. Phylogenetic analysis of the ITS2 region of parasitoids of Diaphorina citri and
Diaphorina communis inferred by maximum likelihood using the Kimura 2-parameter model of
evolution (Kimura 1980). The tree with the highest log likelihood (-1047.2999) is shown. The
percentage of trees from 1000 bootstrap replicates in which the associated taxa clustered together is
shown next to the nodes. Site of collection is given next to the accession code for sequences obtained
in this study. Accession numbers are given in parenthesis for sequences obtained from the GenBank.
126
4.3.3 Morphological description
Name: Tamarixia drukyulensis sp. n. Yefremova & Yegorenkova
Etymology: The specific name is derived from the Bhutanese, drukyul, meaning
‘land of the dragon’, i.e., Bhutan, where type materials were collected.
Material examined: Holotype: ♀, Bhutan, reared from Diaphorina communis
(deposited in TAUI). Paratypes: 32 ♀, 26 ♂ with the same labels (deposited in
TAUI); DNA was extracted from two females and two males. The male is almost
identical to the female in colour but differs in the structure of the antennae and
forewings.
Female (Figure 4.5a)
Length: 0.9–1.2 mm, forewing 0.90–1.25 mm.
Colour: Body mostly brown. Head brown. Eyes dark red. Ocelli red. Antenna dark
yellow. Tegulae from dark yellow to brown. Gaster brown with yellow spot in
middle of T1 and T2, ventrally yellow. Legs yellow (except brown coxae).
Head: 2.2 times as wide as long and 1.1 times as tall. Face smooth. POL 1.3 times as
OOL. Malar space 0.66 length of eye. Toruli placed at level of the lower margin of
eyes. Antenna with scape 3.3 times as long as broad; pedicel 2.9 times as long as
broad; and one discoid anellus, and funicle. F1 2.2 times as long as broad; F2 2.3
times as long as broad and 1.1 shorter than F1; F3 1.8 times as long as broad and
1.05 shorter than F3; clava 3-segmented 3.1 times as long as broad and 2.4 times as
long as F3, with apical sensillum. Ratio length of clava to length of funicle = 1.05.
Mesosoma: Thorax 1.1 times as long as broad and 1.2 times as long as gaster.
Pronotum short. Mesoscutum 1.4 times as long as broad, with median complete line,
with 2 short dark pairs adnotaular setae. Scutellum 1.6 times as long as broad, with
submedian and sublateral lines. Sculpture of scutellum the same as in mesoscutum.
Propodeum 1.6 times as long as dorsellum, smooth, with median carina, spiracle
round with paraspiracular carina near metanotum, callus with 1 dark long seta. Fore
127
wings 2.2 times as long as broad. Speculum narrow extending along ⅓MV, closed.
MV 1.6 times as long as SMV; MV 4.4 times as long as STV. SMV with 1 seta, MV
with 8–9 setae. Hind wing apically acute.
Metasoma: Gaster 1.3–1.4 times as long as broad.
Figure 4.5. Micrographs of Tamarixia drukyulensis: (a) female; (b) male (Photos: Z. Yefremova).
128
Male (Figure 4.5b).
Length: 0.8–0.9 mm.
Colour: Body mostly brown. Eyes dark red. Ocelli red. Antenna brownish. Tegulae
dark yellow. Legs yellow (except brown coxae), hind legs with a brown 4th
tarsomere.
Antenna: scape 3.5 times as long as broad with ventral plaque (0.27 length of scape)
situated in middle of scape; pedicel 1.5 times as long as broad, one anellus; F1 1.5 as
long as broad; F2 1.8 times as long as broad, and 1.1 times longer than F1; F3 2.2
times as long as broad and 1.2 times as long as F2; F4 3.0 times as long as broad and
1.1 times as long as F3; clava 3-segmented with apical sensillum, 5.8 times as long
as broad and 2.4 times as long as F4. Funicle with 4 segments with whorled setae;
whorled setae of F1 reaching top of C1; whorls of F2 reaching top of C2; whorls of
F3 reaching middle of C3; whorls of F4 reaching middle of C3.
Forewings: 2.3 times as long as broad. Speculum small, closed, extending along
slightly more ⅓ of MV.MV1.5 times as long as SMV and 4.2 times as long as SV.
SMV with 1 seta; MV with 7–8 setae. Hind wing apically acute.
Genitalia: Gaster 2.0 times as long as broad. Aedeagus ½ of gaster length and 3.6
times as long as digitus; digitus with one spine. Of the 26 male paratypes, seven have
digiti parallel with the aedeagus, eight have digiti located at 45°angles to the
aedeagus and in five males digiti are recurved from the aedeagus.
Host: Diaphorina communis Mathur.
Distribution: Bhutan.
Comments: The new species is similar to Tamarixia radiata. Females of the two
species can be distinguished based on the width to length ratios of the 1st to 3rd
flagellomeres, the length to width ratio of the clava, the width to length ratio of the
clava to funicle as well as body size (Table 4.4). Males of the two species can be
distinguished based on the width to length ratios of the 1st flagellomere, the clava to
4th flagellomere, the 4th flagellomere and of the clava to funicle. Body size and size
129
of the plaque are also differential (Table 4.5). In addition, males of Tamarixia
drukyulensis also differ from males of Tamarixia radiata in the following characters:
F3 1.2 times as long as F2; clava 5.9 times as long as broad (F3 equal to F2, clava 5.0
times as long as F3 in Tamarixia radiata); whorled setae of F1 reaching top of C1;
whorls of F2 reaching top of C2 (whorled setae of F1 reaching top of F4; whorls of
F2 reaching middle of C2 in Tamarixia radiata).
Table 4.4. Comparative characters of females of Tamarixia drukyulensis sp. n. with females of
Tamarixia radiata.
Characters/species
Tamarixia drukyulensis sp.n.
Tamarixia radiata (original
description Waterston, 1922)
Width to length ratio of 1st
flagellomere
2.2
1.7
Width to length ratio of 2nd
flagellomere
2.4
1.4
Width to length ratio of 3rd
flagellomere
1.8
Subquadrate
Length to width ratio of the clava
3.07
2.0
Width to length ratio of clava to
1.05
1.4
1.2
1.3
funicle
Body size (mm)
Table 4.5. Comparative characters of males of Tamarixia drukyulensis sp. n. with males of Tamarixia
radiata.
Characters/species
Tamarixia drukyulensis sp.n.
Tamarixia radiata (original
description Waterston, 1922)
Width to length ratio of 1st
flagellomere
1.5
subquadrate
Width to length ratio of clava to
4th flagellomere
2.4
2.0
Width to length ratio of 4th
flagellomere
3.1
2.0
Width to length ratio of clava to
funicle
1.4
1.8
Size of plaque
0.27
0.11
Body size (mm)
0.8–0.9
1.1
130
4.4 Discussion
The COI gene and the ITS regions have been employed in molecular studies on a
range of insects. COI has also been used to separate closely related Encarsia spp.
(Hymenoptera: Aphelinidae), a genus of minute parasitic wasps with subtle
morphological differences (Monti et al. 2005), reduviid bug (Hemitptera:
Reduviidae) (Pfeiler et al. 2006) and white fly (Hemiptera: Aleyrodidae) (Hu et al.
2011).
The internal transcribed spacers have been used to study phylogenetic
relationships and to resolve taxonomic controversies in species of other insects such
as Culex (Diptera: Culicidae) and Drosophila (Diptera: Drosophilidae) (Miller et al.
1996; Young & Coleman 2004;). Molecular studies of Tamarixia radiata are mostly
related to genetic diversity within the species using COI and ITS1 and 2 (Barr et al.
2009; de León & Sétamou 2010; Peña-Carrillo et al. 2014; Peña-Carrillo et al. 2015).
The only sequence data available for other species of Tamarixia is for Tamarixia
triozae (de León & Sétamou 2010).
In this study, the COI gene and ITS regions were used to determine molecular
differences between and within populations of the parasitoids of Diaphorina citri and
Diaphorina communis occurring in Bhutan. Sequence analyses of the two groups
showed high genetic divergence between the two groups of parasitoids (Table 4.3).
The Diaphorina communis group showed 7–8% and 10% sequence divergence to
those from Diaphorina citri group and to Tamarixia triozae, respectively. This result
is similar to that obtained by de León & Sétamou (2010) with Tamarixia radiata
showing 9–10.3% sequence divergence from its sibling species, Tamarixia triozae.
These ranges fall within the observations by Hebert et al. (2003) who reported
sequence divergence of > 8% in the COI gene between congeneric species in 79% of
13320 animal species pairs. Hebert et al. (2003) also reported that 93% of the
Hymenopteran species pairs showed genetic divergences of 8–16%. These results
strongly suggest that the parasitoid of Diaphorina communis from Bhutan is a
different species of Tamarixia from that found in Diaphorina citri. This is further
supported by the phylogenetic relationships inferred from the COI gene and ITS
131
regions. In these phylogenetic studies, the two parasitoids fell into two separate,
monophyletic clades with strong bootstrap support (Figures 4.2, 4.3 & 4.4).
The morphological examination of the parasitoid of Diaphorina communis shows
that it is a previously unrecorded species. The species is closely related to, and
resembles, Tamarixia radiata. However, females of Tamarixia drukyulensis are
bigger than those of Tamarixia radiata, and the two species can be distinguished
from each other based on width to length ratios of the antennal flagellomeres and
clava and the relative size of the clava to funicle (Table 4.4). Males of the two
species can be distinguished on antennal characteristics, body size and size of the
plaque (Table 4.5).
Analyses of the three regions (Figures 4.2, 4.3 & 4.4) also separated one insect
(PBT.12) collected from Diaphorina communis from all the other individuals. In the
analysis of the COI sequences, this insect clustered with Aprostocetus cerricola,
Aprostocetus gala, Aprostocetus forsteri, and Aprostocetus aethiops and, using the
ITS2 region, with Aprostocetus aethiops and another species of Eulophidae. This
insect in the current study clustered most closely with the eulophid hyperparasitoid,
Aprostocetus sp., and could be a hyperparsitoid of Diaphorina communis. The
presence of hyperparasitoids had been observed for Tamarixia radiata and
Tamarixia triozae. Husain & Nath (1924) reported two unidentified hyperparasitoids
of Tamarixia radiata (cited as Tetratischus radiatus). Waterhouse (1998) compiled
a list of hyperparasitoids belonging to six hymenopteran families occurring in China,
Philippines, and Taiwan that attack both Tamarixia radiata and Diaphorencyrtus
aligarhensis (Shafee, Alam and Agarwal) (Hymenoptera: Encyrtidae). In addition to
Marietta leopardina Motschulsky (Hymenoptera: Aphelinidae) as listed in
Waterhouse (1998), Aprostocetus (Aprostocetus) sp., and Psyllaphycus diaphorinae
Hayat (Hymenoptera: Encyrtidae) were demonstrated to be hyperparasitoids of
Tamarixia radiata and Diaphorencyrtus aligarhensis (Hoddle et al. 2013; Hoddle et
al. 2014; Bistline-East & Hoddle 2016). An Encarsia spp. has been reported to
attack Tamarixia triozae (Butler & Trumble 2011) in addition to being a
132
hyperparsitoid of Tamarixia radiata and Diaphorencyrtus aligarhensis (Aubert
1987; Waterhouse 1998).
4.5 Highlights of the study
This is the first record of a parasitoid of Diaphorina communis and to assess its
genetic variability and phylogenetic relationship with the parasitoid of
Diaphorina citri based on COI, ITS1 and ITS2 regions. The molecular analyses
suggest that the ectoparasitoid of Diaphorina communis is a new species of
Tamarixia.
This study also provides the first morphological descriptions of the parasitoid of
Diaphorina communis and complements the results of the molecular study again
showing it to be a different species from Tamarixia radiata. The name of the
new species has been proposed as Tamarixia drukyulensis sp. n.
The molecular data and the phylogenetic studies also suggest the occurrence of a
hyperparasitoid of Diaphorina communis belonging to the genus, Aprostocetus.
133
Chapter 5: Incidence of psyllids and ‘Candidatus
Liberibacter asiaticus’ in mandarin orchards at
different elevations in Tsirang
___________________________________________________________________
5.1
Introduction
Bhutan is characterised by mountains separated by river valleys. Citrus is grown at
different altitudes within this landscape. An altitudinal gradient is characterised by
rapid changes in environmental conditions within short horizontal distances, and
factors such as temperature, precipitation, UV radiation, partial pressure, and wind
speed can interactively affect the distribution of insects along the gradient
(Hodkinson 2005). Prior to my studies, Diaphorina citri had been recorded in
Bhutan at elevations ranging from 200 m ASL in the south to ~1350 m ASL at
Richina (27.6421°N, 89.8624°E) in the west-central region of Punakha (Figure 5.1)
and, as nymphs, in an orchard (27.0279°N, 90.1210°E) in Dzamling Zor Gewog
(Upper Suntolay) at 1380 m in Tsirang. The latter records were made during an
NPPC-ACIAR survey in May 2009 (Donovan et al. 2012b). Elevations of 1350 m in
Punakha are known to receive occasional frosts during winter.
Mandarins in Tsirang Dzongkhag are grown at elevations ranging from 300 m to
~1550 m ASL. Plantings of seedling trees sourced from unprotected nurseries at
elevations below 400 m in India, Sarpang and/or Phuentsholing regions of Bhutan
may have commenced in the mid-1960s, as trees sampled for morphological studies
by Dorji & Yapwattanaphus (2011) in 2009–2010 were 45 years old.
Huanglongbing and Diaphorina citri were known to be present in south-western
Bhutan (Phuentsholing), adjacent regions in India (Assam and Sikkim), Bangladesh
and Nepal in the 1970s (Catling 1968, 1978; Knorr et al. 1970; Lama & Amtya 1991;
Ghosh 1993, Lama & Amatya 1993). Presence of the pathogen was confirmed in
2002 (Garnier & Bové 2002; Ahlawat et al. 2003; Doe Doe et al. 2003; Bové 2014).
In 2003, there were some 147,500 trees within Tsirang’s 12 gewogs (NPPC 2003).
In 2007, approximately 57% and 87% of the trees were more than 20 years old and
134
10 years old, respectively (NPPC-ACIAR). The first official record of Diaphorina
citri in Tsirang was in two of the 12 gewogs, Gosarling and Phutenchhu, in 2003
(NPPC Survey 2003), but the elevations were not recorded. It was subsequently
recorded in Gosarling on mature trees in mandarin orchards in Pemathang Chiwog
(Lower Suntolay) between 950 m and 1150 m ASL, and, as noted above, in an
orchard in Dzamling Zor Chiwog at 1380 m, during an NPPC-ACIAR survey for the
psyllid and HLB in May 2009 (Figure 5.2). The trees were 20 or more years old and
in rapid decline. Symptoms of HLB were present on all these trees, and many trees
in these orchards were dead. However, symptoms of HLB were not common at
elevations above 1200 m, and trees above this elevation were bearing fruit despite
impacts of powdery mildew, phytophthora and borers.
The NPPC-ACIAR
observations in 2009 were in stark contrast to those reported by Bové (2004) who
found no evidence of HLB in the Damphu/Mendrelgang region of Tsirang, and PCR
tests for tissues sampled by him were negative (Bové 2004).
Figure 5.1. Symptoms of huanglongbing on a ‘Candidatus Liberibacter asiaticus’-infected seedling
mandarin tree at Richina (27.6421N, 89.8624E, 1322–1360 m) (left) and Paul Holford and ‘Citrus’
Dorji (centre, right) inspecting the tree for Diaphorina citri adults and nymphs on 20 May 2009
(Photos: GAC Beattie).
135
Figure 5.2. Dying mature mandarin trees (top left), stumps of dead seedling mandarin trees (top
right), lime leaves with symptoms of huanglongbing (lower left) and Diaphorina citri adults on
mandarin leaves (lower right) at the Waklay orchard (27.0358N, 90.1093E, 950 m), Pemathang
(Lower Suntolay), Gosarling, Tsirang, on 22 May 2009 (Photos: GAC Beattie).
During the NPPC-ACIAR survey in 2009, Diaphorina communis was observed on
curry leaf plants and mandarin seedlings at Kamichhu (650 m), and in Tsirang at
elevations up to ~1050 m, the elevation above which curry leaf does not appear to
grow or is uncommon in Tsirang, although it has been recorded up to 2850 m
elsewhere in Bhutan (Grierson 1991).
Cacopsylla heterogena (as identified in Chapter 6) was first recorded in Bhutan on
19 April 2013 in Tsirang, shortly after my studies commenced: initially in an orchard
(27.0082°N, 90.1391°E, 1260 m ASL) in Dekidling Chiwog, Kilkhorthang Gewog,
when adults were tapped from a mature mandarin tree into a white enamel tray, and
subsequently on the same day at Riserboo ‘A’ (Kamigaon) (26.9532°N, 90.1347°E,
1436 m ASL) in Mendrelgang Gewog when a larger number of adults were tapped
136
from immature and mature seedling mandarin trees on which nymphs were also
present.
The objectives of the studies reported in this Chapter were to determine relationships
between ambient temperatures, relative humidity, rainfall and elevation on the
incidence of Diaphorina citri, Diaphorina communis and huanglongbing.
I
hypothesised that the incidence of the psyllids, particularly Diaphorina citri, and the
disease, were primarily influenced by higher leaf temperatures and reduced plant
growth at the higher altitudes. The scope of the study was widened to include
Cacopsylla heterogena after it was discovered.
5.2
Materials and methods
5.2.1 Site description and experimental plots
This study was conducted in Tsirang Dzongkhag to determine the effect of altitude,
temperature, rainfall and humidity on leaf temperatures of mandarin on ‘CLas’ levels
and psyllid populations. Eight sites ranging from ~800 to 1500 m ASL and separated
by approximately 100 m in altitude were selected. The lowest altitude site was at
Phuensoomgang in Gosarling Gewog and the highest altitude at Droopchhugang in
Tsholingkhar Gewog.
The collective name of the sites will be referred as
Droopchhugang-Phuensoomgang (‘gang’ is a ridge in Bhutanese). Details of each
site are given in Table 5.1 and aerial views are presented in Figures 5.3 and 5.4.
Site preparations and planting of mandarin seedlings were completed from 25–29
May, 2013 in these eight sites using two years old mandarin seedlings ‘assumed to be
disease-free’ (see discussion) obtained from the National Seed Centre (NSC), Bhur
on 26 May 2013. The seedlings were pruned to uniform heights of 500 mm, and
then carried to experimental sites in woven plastic bags to prevent any psyllids
landing on them during transport. At each elevation, three sets of four mandarin
seedlings were planted. Each set of seedlings was planted in 300 mm wide × 400
mm deep holes in a 1 × 1 m plot (Figure 5.5B). The plots were separated by ~30 m.
The plots were fenced with bamboo poles and rails to prevent the ingress of cattle,
137
goats, and wild pigs. Plants in these plots were used for the assessment of plant
growth, leaf temperatures, psyllid incidence and ‘CLas’ titres.
138
Table 5.1. Locations of experimental sites along Droopchhugang-Phuensoomgang in Tsirang.
Farmer
Chiwog1
Gewog
Altitude
(m ASL)
GPS coordinates
2
‘Meme’ Tshering
Phuensoomgang (Garigaon A)
Gosarling
783 (800)
27.0393°N, 90.1110°E
Raj Mal Tamang
Phuensoomgang (Garigaon A)
Gosarling
883 (900)
27.0364°N, 90.1111°E
Lal Bdr. Wakley
Pemathang (Lower Suntolay)
Gosarling
953 (1000)
27.0359°N, 90.1095°E
Mandoge Subba
Pemathang (Lower Suntolay)
Gosarling
1053 (1100)
27.0333°N, 90.1126°E
Renuka Pradhan
Pemathang (Lower Suntolay)
Gosarling
1175 (1200)
27.0302°N, 90.1155°E
Devi Maya Khati
Dzamling Zor (Upper Suntolay)
Gosarling
1273 (1300)
27.0289°N, 90.1181°E
Pema Lham
Dzamling Zor (Upper Suntolay)
Gosarling
1378 (1400)
27.0242°N, 90.1197°E
Dorji Rinchen
Droopchhugang (Harpani)
Tsholingkhar
1478 (1500)
27.0142°N, 90.1183°E
1
Chiwogs refer to administrative divisions below a gewog, and are equivalent to municipalities comprised of groups of villages.
Former names are given in parentheses.
2
Figures in parentheses represent altitude rounded to the nearest hundred/thousand
139
Figure 5.3. Google Earth view of locations of experimental sites along DroopchhugangPhuensoomgang in Tsirang. View facing north from directly above Droopchhugang-Phuensoomgang.
Figure 5.4. Google Earth view of locations of experimental sites along DroopchhugangPhuensoomgang in Tsirang. View facing east from directly above Droopchhugang-Phuensoomgang.
140
Figure 5.5. The experimental site at 783 m ASL at ‘Meme’ Tshering’s orchard at Phuensoomgang,
Gosarling, Tsirang (A) and a plot with four mandarin seedlings at 1053 m ASL at Mandoge Subba’s
orchard in Pemathang, Gosarling, Tsirang (B). (Photos: N. Om).
5.2.2 Plant growth assessment
Growth assessments were based on the numbers of leaves produced on four, topmost, young shoots on each plant in each plot giving 48 shoots assessed per
elevation. The shoots were tagged. Leaves were counted in May 2014 and April
2015.
5.2.3 Soil sample analysis
Soil samples were taken from each plot from 12−14 June 2015. Soil samples from
each elevation consisted of six subsamples, two samples collected from next to each
plot (not within), at a depth of 150 mm.
Subsamples were combined, mixed
thoroughly and a 200 g taken from each elevation. Samples were air dried at room
temperature, transported to Australia where they were irradiated upon arrival, and
then sent to the Department of Science, Information Technology and Innovation
(DSITI), EcoSciences Precinct, Dutton Park Queensland, Australia. The following
parameters were tested:
Extractable soil cations: calcium, potassium, magnesium and sodium
Soil pH (CaCl2)
Soil pH (water)
Electrical conductivity
141
Total carbon
Total nitrogen
Aluminium saturation percentage
Effective cation exchange capacity
Exchangeable sodium percentage
Exchangeable acidity
Exchangeable aluminium
5.2.4 Leaf temperature measurements
Leaf temperatures of mandarin plants in the mini-plots were recorded monthly from
May to August 2014 between 1100–1400 hours using a hand-held, infrared
thermometer (EXTECH, Instrument Choice, South Australia). Readings were taken
within 3 h on the same day by two groups of NPPC staff, each undertaking the
measurements at 3–4 sites. At each elevation, four leaves on each of the four plants
in each plot were measured (48 readings per elevation).
5.2.5 Incidence of psyllid infestations
The incidences of Diaphorina citri and Cacopsylla heterogena at each elevation
were recorded from March to August 2014, and in April 2015. For adult psyllids,
plants in each plot were initially inspected visually, without disturbance, to observe
the number of adults present. Then shoots from each plant were tapped onto a white
enamel tray to detect adults falling on the tray that could have been missed during the
visual inspections. The incidence of adults of each psyllid species on each plant at
each altitude was recorded on a scale of 0 to 5:
0 = no adults
1 = 1 to 5 adults
2 = 6 to 10 adults
3 = 11 to 15 adults
4 = 16 to 20 adults
5 = more than 20 adults
142
The presence or absence of Diaphorina citri eggs and nymphs was assessed nondestructively by examining immature flushes on each of the four plants in each plot
at each altitude on each sampling date. For Cacopsylla heterogena, numbers of a
‘pouch galls’ per plant were counted. A similar scale to that used for the assessment
of adults was used:
0 = no shoots with eggs or nymphs
1 = 1 to 5 shoots with Diaphorina citri eggs or nymphs Cacopsylla heterogena
pouch galls
2 = 6 to 10 shoots
3 = 11 to 15 shoots
4 = 16 to 20 shoots
5 = more than 20 shoots
5.2.6 Recording of weather data
Tinytag data loggers (PLUS 2-TGP-4500, Omni Instruments Australia Pty Ltd) set to
log every 30 min were used to record ambient temperatures and relative humidity at
each site. Polyvinyl chloride (PVC) pipes (110 mm wide and 360 mm long) painted
white and with six 20 mm diameter holes below the caps (for air flow) were used to
house the data loggers. Thin wire was used to suspend each data logger about half
way down each pipe. Each pipe was hung so that the Tinytag was 1.5 m above the
ground level within mature tree canopies (Figure 5.6A).
Rainfall data at each elevation was recorded using Davis rain gauges equipped with
tipping buckets with magnetic reed switch sensors (Davis Instruments, 3465 Diablo
Avenue, Hayward, CA 94545 USA) connected to a EasyLog USB data loggers
(Lasker Electronis, 4258 West 12th Street, Erie, PA 16505 USA). The gauges were
installed according to the manufacturer’s instructions and mounted 0.9 m above
ground level on wooden posts (Figure 5.6B) and were set to log every hour
measuring 0.2 mm of rain for every bucket tip which was recorded as number of
events.
143
Figure 5.6. A PVC pipe containing a Tinytag data-logger (A) and a Davis rain gauge (B).
5.2.7 Prevalence of ‘CLas’ in plant samples at different altitudes
Detection of ‘CLas’ was conducted by collecting samples from:
mature mandarin trees along Droopchhugang-Phuensoomgang and also from
different elevations at Mendrelgang Gewog; and
young mandarin seedlings planted in the plots at elevations from 800 to 1500 m
ASL along Droopchhugang-Phuensoomgang.
5.2.7.1 Sample collection from mature trees
In June 2014, bark samples were collected from the eight sites along
Droopchhugang-Phuensoomgang.
At each site, 10 mature mandarin trees were
randomly selected, and bark samples were collected using an 8 mm diameter leather
hole punch. At least 10–12 bark plugs were collected from each tree. Subsequent
samplings of bark were made in April 2015, and March 2016. Punchers were
cleaned either by dipping in 4% bleach or by flaming after each tree. Root samples
were also taken on these occasions. The root samples were collected by digging
holes on four sides of a tree under the canopy and collecting the fibrous roots (~5 to
10 mm in circumference). During April 2015, bark, root and leaf samples of mature
mandarin trees were collected from different elevations in the Mendrelgang Gewog
in Tsirang. Samples were stored at 4°C until DNA extraction.
144
5.2.7.2 Sample collection from young plants in the plots
Both bark and root samples were collected from all four plants in each plot at each
elevation. Bark strips of various lengths were stripped off the shoots and placed in
plastic bags. Fibrous roots were dug out as described above.
5.2.7.3 DNA extraction and amplification
Extraction of ‘CLas’ from plant tissues was performed in a similar manner to the
methods described in Section 3.2.3 (Chapter 3) except that the scaly epidermis on the
root and bark was scraped off with disposal razor blades, and any wood and pith
removed before washing and chopping the sapwood. All samples were extracted at
the NPPC.
Samples were tested using real-time PCR (qPCR), selected samples were subjected
to cPCR, and successful amplifications were sequenced as described in Section 3.2.5
(Chapter 2).
145
5.3
Results
5.3.1 Plant growth assessment and soil analysis
Table 5.2 shows the average number of leaves produced on the four terminal-shoots
in May 2014 and April 2015. The results indicate that, one year after planting,
seedlings planted at 800–1000 m ASL produced more leaves than seedlings planted
at 1100 and 1500 m ASL. A similar trend was observed in April 2015 with plants at
800 and 1000 m ASL producing almost 8–10 times higher numbers of leaves
compared to plants at 1100 and 1500 m ASL.
Plant at other elevations had
intermediate growth.
The results of the soil nutrient analysis are given in Figure 5.7. Cation extractable
for magnesium, potassium and calcium were lowest at 1500 m followed by 1100 m
and 1200 m, and the highest at 1000 m ASL (Figure 5.7A). Extractable sodium was
the same (< 0.08 cmol_c/kg) at all sites (data not shown). The soil at all locations
was acidic but not saline as indicated by the low electrical conductivity and CaCl2
pH values (Figure 5.7B & 5.7D). The C/N ratio was within the normal range but
total carbon (TC) was lowest at 1100 m followed by 1200 m ASL and highest at
1300 m followed by 1400 m and 1000 m ASL (Figure 5.7C).
5.3.2 Ambient
temperatures,
relative
humidity
and
leaf
temperatures
Generally, leaf temperatures were lower than ambient temperature by 1−2°C in May,
and by 2−7°C in July−August but rarely over 30°C (Figure 5.8). The mean leaf
temperatures at ≥ 1200 m were lower than leaf temperatures at 1100 m and below.
As for the ambient temperatures, based on the mean minimum temperature, lowest
temperatures observed were in December and January with minimum temperatures
ranging from 7.3°C to 11.1°C at 800–1100 m, and 5.7°C to 8.1°C at 1200–1500 m
ASL. The hottest months were May to September. Maximum temperatures during
these months rarely exceeded 30°C at elevations above 1000 m and ranged between
146
30°−32°C at 800–1000 m ASL and 23−30°C at 1100–1500 m ASL (Figure 5.9B).
Mean minimum relative humidity was comparatively higher from June to September
with a range of 51%−73% for 800–1100 m ASL, and 58%−85% for 1200–1500 m
ASL (Figure 5.10).
Table 5.2. Average numbers of leaves per terminal shoot of mandarin seedlings planted at different
elevations.
Altitude
(m ASL)
Average number of
leaves per shoot1
Observations in April 2015
May 2014
April 2015
1500
5.00
3.82
Generally, very poor growth with mostly a single stem.
Powdery mildew present. One plant did not have young
shoots.
1400
5.72
14.19
All plants still alive but two were damaged by cattle. Soft
scales present.
1300
6.74
14.19
One plant had died and three plants damaged by cattle.
1200
6.93
7.62
Three plants did not have young leaves. Soft scales were
common.
1100
4.11
2.00
All plants present but with single stem, and almost no
leaves. Cattle damage and severe powdery mildew present.
1000
8.38
29.68
One plant had died, and four had sparse shoots.
900
8.29
14.19
Severe cattle damage on four plants. Most shoots on two
plants had dried up.
800
10.02
24.84
One plant had died, and 10 other plants did not have young
shoots in April 2015
1
Average of four young shoots per plant comprising 48 samples at each elevation.
5.3.3 Rainfall
Figure 5.11 shows monthly total rainfall at the eight sites along DroopchugangPhuensoomgang. The records indicate that the monsoon season commences in June
and lasts until September. Generally, during the monsoon, the sites at the higher
elevations (≥ 1200 m ASL) received more rainfall compared to the sites at lower
elevation (˂ 1200 m ASL) in each year except for June 2015. Total rainfall during
the months of January, February and May was variable between years but
consistently higher from June to September for all locations (Figure 5.11, Tables 5.3
& 5.4). November and December received the least amount of rainfall.
147
Figure 5.7. Results of analysis of soil samples collected from the eight experimental sites in Tsirang in June 2015.
148
34
34
Leaf
Ambient
32
30
30
28
28
26
26
24
24
22
22
o
20
18
20
May
June
18
34
32
32
30
30
28
28
26
26
24
24
22
22
20
20
o
34
Temperature ( C)
Temperature ( C)
32
18
July
800
August
900
1000
1100
1200
1300
1400
1500
800
18
900
1000
1100
1200
1300
1400
1500
Elevation (m ASL)
Figure 5.8. Comparison of average leaf temperature and ambient temperature at the experimental sites in Tsirang in May, June, July and August 2014.
149
Figure 5.9. Monthly average minimum and maximum ambient temperatures recorded at the
experiment sites along Droopchhugang-Phuensoomgang.
150
Figure 5.10.
Monthly average minimum
Droopchugang−Phuensoomgang in Tsirang.
151
and
maximum
relative
humidity
along
Figure 5.11. Monthly total rainfall (mm) recorded from June 2013 to October 2015 at the sites along
Droopchugang-Phuensoomgang.
152
Table 5.3. Average monthly rainfall (mm) in June, July, August and September 2014–2015 at
elevations ranging from 1500 m to 800 m ASL along Droopchugang-Phuensoomgang in Tsirang.
Elevation (m ASL)
2014
2015
Range
Average
Range
Average
1500
143
*
196–303
254
1400
129–379
290
191–353
275
1300
125–326
269
172–309
242
1200
126–392
285
194–289
249**
1100
117–290
220
207–286
235
1000
99–259
207
207–289
243
900
98–324
237
188–268
228
800
113–298
236
214–272
236
* June only for 1500 m.
** June to August 2015 for 1200 m.
Table 5.4. Average annual rainfall, minimum and maximum ambient temperatures, and minimum
and maximum relative humidity in 2014 at elevations ranging from 800–1500 m ASL along
Droopchugang-Phuensoomgang in Tsirang.
Elevation
ASL)
(m
Rainfall
(mm)
Temperature (°C)
Relative humidity (%)
minimum
maximum
minimum
maximum
800
1118
16.02
28.73
50.59
95.98
900
1128
15.60
29.50
48.80
97.60
1000
1072
15.60
29.20
48.90
96.10
1100
1041
15.53
27.55
53.39
95.55
1200
1461
14.73
24.71
64.18
97.05
1300
1293
13.92
24.15
64.25
98.07
1400
1357
13.47
25.89
55.12
98.10
1500
851*
13.00
24.00
58.60
98.70
* data for July to September 2014 not recorded accurately.
153
5.3.4 Psyllid incidence
The incidence of psyllids recorded on young mandarin plants at different elevations
is shown in Table 5.5. Adult Diaphorina citri were observed at 800 to 1100 m ASL
in different months while adults of Diaphorina communis or Cacopsylla heterogena
were never observed on the young plants at any elevation (Table 5.5A). Immature
stages of Diaphorina citri were also found at 900 and 1000 m ASL while immature
stages of Cacopsylla heterogena within the pouch galls were observed at 1400 m
ASL in April 2015 (Table 5.5B).
154
Table 5.5. Incidence of Diaphorina citri (DC), Diaphorina communis (DCO) and Cacopsylla heterogena (Ca) nymphs and adults on mandarin at different altitudes along
Droopchhugang-Phuensoomgang, Tsirang.
Altitude
(m ASL)
March 2014
DC
DCO
April 2014
May 2014
June 2014
July 2014
August 2014
April 2015
Ca
DC
DCO
Ca
DC
DCO
Ca
DC
DCO
Ca
DC
DCO
Ca
DC
DCO
Ca
DC
DCO
Ca
A. Adult psyllids
1500
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1400
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1300
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1200
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1100
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1000
0
0
0
0
0
0
0
0
0
3
0
0
4
0
0
0
0
0
0
0
0
900
0
0
0
1
0
0
0
0
0
6
0
0
4
0
0
1
0
0
1
0
0
800
0
0
0
0
0
0
0
0
0
6
0
0
5
0
0
5
0
0
5
0
0
B. Psyllid nymphs
1500
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1400
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1300
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1200
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1100
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1000
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
900
0
0
0
0
0
0
0
0
0
0
0
0
4
0
0
0
0
0
0
0
0
800
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
155
5.3.5 Incidence of ‘CLas’
The incidence of ‘CLas’ was assessed from plant tissues sampled at different
elevations from mature mandarin trees from Droopchhugang-Phuensoomgang and
Mendrelgang Gewog and from young mandarin trees from DroopchhugangPhuensoomgang.
Bark and root tissues were sampled from the plants along
Droopchhugang-Phuensoomgang and bark, roots and leaves were collected from
Mendrelgang Gewog.
5.3.5.1 Samples from mature trees
Table 5.6 shows the detection of ‘CLas’ in the bark samples collected from mature
mandarin trees along Droopchhugang-Phuensoomgang. Generally, the majority of
the bark samples collected from 800 to 1100 m ASL produced FAM Ct values >
0.00. At these elevations, the FAM Ct values of samples collected in June 2014 and
March 2016 ranged from 23.13 to 38.55. However, samples collected in May 2015
had a higher range (35.42 to 39.81) of FAM Ct values. In contrast, few samples
collected from 1200 to 1500 m ASL produced FAM Ct > 0.00. The FAM Ct values
of samples above 1200 m ASL that were greater than zero ranged from 26.15 to
38.94. The internal control for all samples worked well as indicated by the overall
CAL Ct value range of 15.80–31.23. The extraction controls, which consisted either
of apple or peach midribs, had FAM Ct values of zero, while the FAM Ct value
range for the positive controls was 16.41–30.52.
The results of qPCR analysis of root samples collected from mature trees along
Droopchhugang-Phuensoomgang are presented in Table 5.7. More than half of the
root samples collected in May 2015 from elevations 1200 to 1500 m ASL produced
FAM Ct values > 0.00 that ranged from 30.64 to 39.18; however, fewer samples
collected at 800 and 900 m ASL had FAM Ct values > 0.00. In contrast, the root
samples collected in March 2016 produced results similar to the bark samples with
majority of the root samples collected from 800–1100 m ASL producing FAM Ct >
0.00, whilst the majority of samples collected from elevations 1200 m ASL and
above had Ct values of zero. The FAM Ct values for the March 2016 samples
156
ranged from 25.12–39.26 and 28.05–39.18 at elevations 800–1100 m and 1200–1500
m ASL, respectively. One root sample from 800 m ASL that had a FAM Ct value of
37.75 produced an amplicon using cPCR. The sequence of this amplicon showed
100% identity with sequences of isolates of ‘CLas’ from Bhutan, China, India, Iran
and Japan. The FAM Ct values for the extraction controls were 0.00 and those for
the positive control ranged from 19.14–30.52. The internal control CAL Ct values
for the root samples ranged from 14.39–34.7.
The ‘CLas’ Ct values for bark, root, and leaf samples from mature mandarin trees in
Mendrelgang Gewog are presented in Table 5.8. All bark samples produced FAM Ct
values of 0.00 except for one sample each at 909 and 1190 m ASL. Similarly, the
leaf samples had FAM Ct values of zero except for the two samples from 986 m
ASL. In contrast, at least one root sample from all but one elevation produced a
FAM Ct value > 0.00. The FAM Ct values ranged from 31.49–39.38. The root
sample with a FAM Ct value of 31.49 was successfully sequenced, and its sequence
showed 100% identity with ‘CLas’ isolates from Bhutan, China, India, Iran and
Japan. The extraction control produced FAM Ct values of zero, and the positive
control FAM Ct values ranged from 30.19 to 30.52. The internal control CAL Ct
values ranged from 14.31 to 22.61.
157
Table 5.6. FAM Ct values (‘CLas’) for bark samples from 10 mature mandarin trees at each elevation along Droopchhugang-Phuensoomgang.
FAM Ct values1
1
2
3
4
5
6
7
8
9
10
EC
PC
CAL Ct range
1500
June 2014
0.00
0.00
0.00
0.00
35.11
34.73
33.93
0.00
0.00
0.00
NA
22.94
16.40–25.30
May 2015
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
30.14
15.91–19.51
36.68
March 2016
0.00
0.00
33.56
0.00
0.00
26.72
0.00
0.00
0.00
0.00
0.00
19.36
17.69–21.11
1400
June 2014
0.00
35.19
35.78
39.05
0.00
0.00
0.00
0.00
0.00
33.39
NA
22.94
15.48–18.77
May 2015
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
30.14
17.04–19.16
March 2016
0.00
0.00
36.65
0.00
28.93
0.00
0.00
0.00
0.00
0.00
0.00
19.53
19.18–26.34
1300
June 2014
0.00
0.00
0.00
34.38
0.00
0.00
0.00
0.00
0.00
0.00
NA
0.00
16.11–19.61
May 2015
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
30.25
16.02–20.77
35.45
March 2016
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
19.53
22.29–24.23
1200
June 2014
0.00
0.00
37.12
0.00
0.00
0.00
0.00
0.00
0.00
28.37
NA
22.94
15.84–17.27
May 2015
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
29.30
21.49–25.69
36.04
March 2016
0.00
30.12
38.94
37.17
0.00
0.00
26.15
0.00
0.00
0.00
0.00
19.32
16.84–20.55
1100
June 2014
32.49
30.36
33.08
33.34
33.27
34.43
33.65
33.42
29.29 32.41
0.00
22.94
17.89–24.98
May 2015
0.00
0.00
0.00
0.00
0.00
0.00*
0.00
29.30
17.60–31.23
36.85
37.00
39.67
35.08
March 2016
30.94
32.99
30.13
30.85
30.92
31.09
30.59
32.15
28.64 25.92
0.00
19.32
17.96–24.24
1000
June 2014
34.42
32.09
30.58
33.08
29.97
30.34
29.55
37.67
31.34 34.06
NA
22.94
15.80–18.98
May 2015
0.00
0.00
0.00
0.00
0.00
29.81
14.88–18.80
37.26
38.37
35.42
35.95
39.07 0.00
March 2016
26.80
27.54
31.92
27.82
27.33
30.81
31.37
27.44
27.69 29.14
0.00
19.14
18.16–23.29
900
June 2014
30.01
31.70
32.1
29.69
0.00
31.18
0.00
33.56
35.35 29.66
NA
22.94
16.96–22.58
May 2015
0.00
0.00
0.00
29.81
14.66–19.23
36.65
38.59
37.38
35.46
36.44
36.56
39.81 0.00
March 2016
32.28
33.16
34.15
32.77
34.59
34.39
32.73
38.55
33.57 31.49
0.00
20.21
22.27–25.69
800
June 2014
28.09
29.72
24.67
31.11
28.4
23.13
31.18
26.94
32.09 24.83
NA
22.94
17.50–26.47
0.00
30.52
13.56–14.93
May 2015
36.82
35.76
34.77
37.55
36.15
38.10
34.60
37.45
37.37 36.78
March 2016
26.64
25.95
27.31
29.05
29.77
28.78
30.64
31.97
31.86 29.48
0.00
16.41
15.82–25.04
1
FAM Ct = ‘CLas’ DNA; samples subjected to cPCR using primers A2/J5 are emboldened; Italicised FAM Ct values indicate samples that are positive by cPCR; EC =
extraction controls (peach or apple midribs); PC = positive control; NA = no analyses; CAL = internal control (plant cytochrome); *the internal control (CAL) for the sample
did not work.
Altitude
(m ASL)
Month & Year
158
Table 5.7. qPCR FAM Ct values (‘CLas’) for roots of from 10 mature mandarin trees at each elevation along Droopchhugang-Phuensoomgang May 2015 and March 2016.
Altitude
(m ASL)
1500
1400
1300
1200
1100
1000
900
800
FAM Ct values1
Month &Year
May 2015
March 2016
May 2015
March 2016
May 2015
March 2016
May 2015
March 2016
May 2015
March 2016
May 2015
March 2016
May 2015
March 2016
May 2015
March 2016
1
2
3
4
5
6
7
8
9
10
EC
PC
CAL Ct range
32.76
0.00
0.00
0.00
34.54
0.00
31.45
0.00
0.00
30.73
0.00
0.00
0.00
35.26
0.00
28.29
39.18
0.00
0.00
30.39
0.00
0.00
0.00
30.92
36.73
36.65
38.79
32.50
37.60
36.70
0.00
31.40
37.49
29.95
0.00
0.00
0.00
0.00
39.13
0.00
0.00
30.90
0.00
31.81
0.00
32.96
38.94
32.04
31.95
0.00
38.94
0.00
36.88
0.00
35.41
0.00
37.94
30.27
0.00
29.68
0.00
32.63
0.00
29.33
31.74
0.00
38.79
29.62
36.27
0.00
31.97
0.00
37.52
38.40
37.88
31.57
0.00
37.11
0.00
30.13
34.76
28.05
0.00
0.00
35.10
0.00
36.07
36.70
0.00
34.53
0.00
31.00
0.00
32.80
0.00
32.53
0.00
0.00
0.00
0.00
37.94
0.00
0.00
29.97
0.00
32.60
39.26
25.12
0.00
0.00
0.00
34.47
30.64
29.77
0.00
0.00
36.02
0.00
0.00
0.00
33.39
0.00
0.00
28.47
0.00*
34.20
0.00
31.71
31.47
0.00
0.00
0.00
37.41
0.00
35.21
0.00
32.45
34.61
0.00
29.31
0.00
38.58
37.75
31.34
36.71
0.00
0.00
0.00
0.00
0.00
34.63
0.00
37.73
31.21
34.97
28.98
0.00
31.87
0.00
28.36
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
30.14
19.36
30.14
19.36
30.25
19.80
29.30
19.32
29.80
19.32
29.81
19.14
30.52
19.67
30.52
20.21
14.79-19.60
16.50-30.94
17.83-21.87
20.48-30.89
17.13-26.12
20.72-24.81
21.11-24.59
20.93-25.51
20.72-26.55
18.75-29.01
16.10-19.19
17.00-27.31
14.47-18.24
18.75-26.52
15.00-34.05**
17.95-20.36
1
FAM Ct = ‘CLas’ DNA; samples subjected to cPCR using primers A2/J5 are embolden; Italicised and underlined FAM Ct values are samples confirmed by sequencing; EC =
extraction controls (peach or apple midribs); PC = positive control; CAL = internal control (plant cytochrome oxidase); *the internal control (CAL) for the sample did not
work; **only one sample with the highest range value.
159
Table 5.8. qPCR ‘CLas’ FAM Ct values for mandarin tissue samples collected from a range of
altitudes in Mendrelgang Gewog, Tsirang Dzongkhag.
FAM Ct values for different tissue types1
Altitude (m ASL)
Bark
root
leaves
1434
0.00 (4)
0.00–35.17 (4)a
0.00 (2)
1376
0.00 (4)
0.00–37.62 (4)a
0.00 (2)
1303
0.00 (4)
0.00–35.98 (4)a
0.00 (2)
1251
0.00 (4)
0.00–39.38 (4)a
0.00 (1)
1190
0.00–39.91 (4)a
0.00–38.25 (4)a
0.00 (2)
1100
0.00 (9)
0.00–34.58 (4)b
0.00 (2)
1065
0.00 (4)
0.00–37.82 (4)c
0.00 (2)
1012
0.00 (4)
986
0.00 (4)
909
0.00–37.63 9 (3)a
EC
0.00
PC
30.19
CAL Ct
value range
14.32–22.61
0.00 (4)
0.00–36.82 (4)a
33.61–34.16 (2)
0.00–38.01 (3)a
0.00 (2)
0.00
0.00
30.52
17.25–22.13
1
0.00 (2)
30.19
14.74–19.34
FAM Ct = ‘CLas’ DNA, FAM Ct for the type of tissues tested and number of sample tested are
given in parentheses; aonly one sample with a Ct > 0.00; btwo samples with FAM Ct values of 31.49
& 34.58 (sample with FAM Ct of 31.49 successfully sequenced); ctwo sample with FAM Ct values of
32.91 & 37.82; EC = extraction controls (rose leaf midribs); PC= positive control; CAL Ct values =
internal control (plant cytochrome oxidase).
160
5.3.5.2 ‘CLas’ detection in samples from young mandarin trees along
Droopchhugang-Phuensoomgang
Results of qPCR analysis of bark samples collected from young mandarin seedlings
planted along Droopchhugang-Phuensoomgang are presented in Table 5.9. At least
four or more seedlings planted at elevations from 800 to 1300 m ASL produced
FAM Ct values > 0.00 from extracts from bark samples collected in 2015 and 2016.
The FAM Ct values for these samples ranged from 22.69–39.69. At least one sample
from 800, 900 and 1000 m ASL was successfully sequenced and shared 100%
identity with ‘CLas’ isolates from Bhutan and other countries in the region. In
contrast, only two samples from 1400 m ASL produced FAM Ct values > 0.00 while
none of the samples from 1500 m ASL produced FAM Ct values > 0.00. The
extraction controls produced FAM Ct values of zero. The FAM Ct value range for
the positive controls was 19.14–30.52 and the internal control CAL Ct range was
14.51–34.70.
The real-time results of the root samples collected from young mandarin trees are
shown in Table 5.10. Almost all root samples collected in May 2015 from 1000 to
1500 m ASL produced FAM Ct values > 0.00 with a range of 30.66–38.08.
However, only two samples collected at 1000 m ASL were positive as determined by
cPCR. Fewer samples from 800 and 900 m ASL collected in May 2015 produced
FAM Ct value > 0.00, and these ranged from 33.44–37.46.
The samples that
produced FAM Ct values of 34.33 and 37.46 from 800 m ASL were successfully
sequenced, and the sequences matched 100% identity with ‘CLas’ isolates from
Bhutan, China, India, Iran and Japan. In contrast, the March 2016 samples collected
from 1100 to 1500 m ASL produced FAM Ct values equal to zero, whereas the
majority of the samples collected from 800 to 1000 m ASL produced FAM Ct values
ranging from 22.36 to 36.81. The extraction controls produced FAM Ct values of
zero. The FAM Ct range for the positive controls was 19.14 to 30.52, and the
internal control CAL Ct value ranged from 14.13 to 32.5.
161
Table 5.9. FAM Ct values (‘CLas’) for bark samples collected from 12 young mandarin seedlings at each elevation planted along Droopchhugang-Phuensoomgang.
Altitude
(m ASL)
1500
1400
1300
1200
1100
1000
900
800
FAM Ct value1
Year &
Month
1
2
3
4
5
May 2015
0.00
0.00
0.00
0.00
March 2016
0.00
0.00
0.00
May 2015
0.00
0.00
March-2016
0.00
May 2015
CAL Ct
range
6
7
8
9
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
38.79
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
37.09
0.00
39.69
March 2016
0.00
0.00
0.00
0.00
May 2015
35.16
0.00
39.70
March 2016
dead
0.00
10
11
12
EC
PC
NS
0.00
0.00
0.00
30.14
16.15-21.18
0.00
NS
0.00
0.00
0.00
19.36
16.26-33.90**
0.00
0.00
37.07
0.00
0.00
0.00
30.25
14.51-19.53
0.00
0.00
0.00
0.00
0.00
0.00
0.00
19.36
17.12-34.70**
0.00
35.91
37.32
0.00
0.00
0.00
35.69
0.00
30.25
16.00-21.96
0.00
0.00
0.00
0.00
0.00
39.18
0.00
0.00
0.00
19.80
18.87-25.49
0.00
35.93
38.18
0.00
36.11
0.00
0.00
0.00
0.00
0.00
29.30
20.07-23.31
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
19.32
17.02-26.14
May 2015
38.88
0.00
36.40
0.00
39.22
0.00*
0.00
0.00
0.00*
36.07
33.19
0.00
0.00
28.93
23.16-25.79
March 2016
dead
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
19.32
17.88-29.43
May 2015
0.00
0.00
0.00
0.00
37.81
0.00
0.00
0.00
31.92
29.49
0.00
29.00
0.00
29.81
14.39-17.14
March 2016
38.19
36.11
25.65
0.00
38.22
36.67
36.59
39.47
20.03
19.14
37.61
23.51
0.00
19.14
15.96-21.73
May 2015
32.44
33.67
32.66
34.35
30.58
34.86
32.88
33.84
0.00
0.00
0.00
0.00
0.00
30.52
14.54-18.12
March 2016
33.87
26.73
24.48
dead
25.29
25.08
27.22
28.60
37.83
0.00
0.00
34.93
0.00
19.14
17.23-29.35
May 2015
28.55
0.00
0.00
0.00
31.09
0.00
0.00
31.00
0.00
0.00
0.00
dead
0.00
30.52
14.52-15.87
March 2016
22.69
38.00
0.00
0.00
22.77
26.55
35.86
24.79
0.00
0.00
0.00
dead
0.00
20.21
16.98-23.07
1
FAM Ct = ‘CLas’ DNA; samples subjected to cPCR using primers A2/J5 are emboldend; Italicised FAM Ct values indicate samples that are positive by cPCR; Italicised
and underlined FAM Ct values are samples confirmed by sequencing; EC= extraction controls (peach or apple midribs); PC= positive control; CAL = internal control (plant
cytochrome); *the internal control (CAL) for the sample did not work; **only one sample with the highest range value; NS= no sample
162
Table 5.10. FAM Ct values (‘CLas’) for root samples collected from 12 young mandarin trees planted along Droopchhugang-Phuensoomgang.
Altitude
(m ASL)
Year
Month
1500
May 2015
1
34.90
2
33.37
3
34.61
4
33.93
5
35.93
6
32.54
7
36.27
8
32.93
9
36.44
10
36.48
11
35.01
12
36.77
EC
0.00
PC
30.14
16.74-23.93
March 2016
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
19.36
16.50-30.94
May 2015
34.16
32.95
31.76
30.06
31.51
35.10
30.66
34.83
35.36
34.94
36.08
0.00
0.00
30.25
16.25-19.26
March 2016
0.00
0.00
0.00
0.00
0.00
0.00
38.74
0.00
0.00
0.00
0.00
0.00
0.00
19.36
20.37-30.89
May 2015
34.62
34.53
37.14
39.05
32.79
34.85
33.71
35.03
36.27
33.04
38.08
34.76
0.00
30.25
15.49-23.01
March 2016
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
19.80
20.72-25.19
May 2015
35.10
31.95
32.49
34.02
32.43
33.22
34.24
32.86
32.91
33.79
33.26
33.84
0.00
29.30
20.10-23.07
March 2016
Dead
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
19.80
20.93-25.86
May 2015
34.92
34.40
33.79
33.82
38.56
33.77
36.13
35.32
33.70
32.85
36.59
32.78
0.00
28.93
22.39-27.18
March 2016
Dead
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
19.32
18.75-29.01
May 2015
35.30
32.09
35.65
34.27
34.91
37.61
0.00
35.55
34.70
30.85
0.00
32.84
0.00
29.81
15.74-18.23
March 2016
0.00
35.50
27.55
36.91
0.00
22.36
24.64
0.00
28.59
28.79
0.00
26.18
0.00
19.14
17-27.31
May 2015
0.00
0.00
0.00
36.06
0.00
0.00
0.00
0.00
0.00
35.25
0.00
33.44
0.00
30.52
16.59-32.5**
March 2016
27.21
30.69
30.20
dead
29.11
27.64
29.09
25.88
0.00
36.81
0.00
0.00
0.00
19.14
18.75-26.38
May 2015
34.33
35.70
36.45
0.00
37.46
0.00
0.00
35.57
0.00
0.00
0.00
dead
0.00
30.52
14.13-19.67
March 2016
27.73
0.00
0.00
0.00
23.41
24.16
0.00
29.27
0.00
0.00
0.00
dead
0.00
20.21
17.95-21.6
1400
1300
1200
1100
1000
900
800
&
FAM Ct value
1
CAL
values
Ct
FAM Ct = ‘CLas’ DNA; samples subjected to cPCR using primers A2/J5 are emboldened; Italicised FAM Ct values indicate samples that are positive by cPCR; Italicised
and underlined FAM Ct values are samples confirmed by sequencing; EC= extraction controls (peach or apple midribs); PC= positive control; CAL = internal control (plant
cytochrome); *the internal control (CAL) for the sample did not work; **only one sample with the highest range value.
163
5.4
Discussion
5.4.1 Growth of mandarin seedlings.
In my study, mandarin seedlings planted, in 2013, in existing orchards along
Droopchhugang-Phuensoomgang at 100 m intervals from 800 to 1000 m ASL grew
better than the seedlings planted at 100 m intervals from 1100 m and 1500 m ASL.
This was in contrast to my hypothesis that rapid transmission of ‘CLas’ from ‘CLas’infected mature trees in the orchards to the seedlings at the lower altitudes would
impede their growth in contrast to plants at the higher elevations where the risk of
infection would be less. More seedlings became infected at the lower altitudes, but,
despite this the plants grew better. Populations of Diaphorina citri at these altitudes
were also noticeably lower than anticipated due to rapid decline and loss of mature
trees, and low incidence of flush growth on mature trees that were still alive. Thus,
factors other than ‘CLas’ influenced the growth of the seedlings. Severe infections
with powdery mildew occurred at 1100 and 1500 m, soil quality varied and was poor
at 1100, 1200 and 1500 m ASL, and damage caused by cattle, goats and wild boars
was frequently observed (Table 5.2 & Figure 5.7).
5.4.2 Relationships
between
ambient
temperatures,
relative
humidity, rainfall, elevation, and incidence of psyllids
Prior to my studies, as noted in the introduction to this chapter, Diaphorina citri has
been recorded in Bhutan at elevations ranging from 200 m ASL in the south to ~1350
m ASL at Richina in the west-central region of Punakha and, as nymphs, at 1380 m
in Dzamling Zor Gewog, Tsirang.
In the current study, Diaphorina citri adults were observed on the young mandarin
plants at 800, 900 and 1100 m ASL. High populations were also observed on young
mandarin trees in a nursery at 1100 m, and ‘CLas’ was detected in the nursery plants
and in psyllids collected from them. With one exception on 19 April 2013 at
Riserboo ‘A’ at 1438 m when four adults were tapped from a mature tree, the psyllid
was never observed in Tsirang at elevations ≥ 1200 m ASL during the study despite
164
ambient temperatures and relative humidity at all elevations in citrus orchards in
Tsirang being favourable (see Liu & Tsai 2000) for its development and survival.
Annual minimum temperature at ≥ 1200 m ASL ranged from 13°C to 14.73°C, and
annual rainfall from 1293 to 1461 mm averaged in 2014 (Table 5.4). It is thus
possible that the adults recorded at Riserboo ‘A’ may have been from breeding
populations in the orchard. It is also possible that they may have flown or were
dispersed by wind from lower elevations, or were introduced on infested plants from
nurseries at lower elevations shortly before they were recorded. Bové (2014) stated,
in reference to HLB in Bhutan, that the psyllid cannot survive at elevations above
1400 m.
Aubert (1987; 1990) commented that Diaphorina citri is a ‘surprisingly enduring
insect’, surviving temperatures of 45°C in arid areas to −7°C in wet subtropical
areas. Lakra et al. (1983) reported that in Hissar, Haryana, India (29.14464°N,
75.7188°E, 215 m ASL), the daily minimum temperatures of below 5°C and daily
maximum of > 45°C, with a relative humidity lower than 40% are lethal to the
psyllid.
Aubert (1987) reported that Diaphorina citri is commonly found from sea level to
1500 m in south-western Arabia. Maximum summer temperatures at 1500 m ranged
from 32–34°C and minimum temperatures in winter were as low as 2.5°C. The
psyllid was absent in orchards above 1700–1800 m where occasional frosts occur
(Aubert 1987). Subsequently, ‘CLas’ and Diaphorina citri were recorded at 1700 m
in Taif (21.2622°N, 40.4090°E) in Saudi Arabia (Bové & Garnier 1984; Bové 2014).
However, Aubert (1987), Bové and Garnier (1984) and Bové (2014) did not cite the
latitudes and longitudes of the locations where the surveys were undertaken, and
orchards viewed in Google Earth suggest that the observations in Taif were at
elevations less than 1500 m ASL, possibly ~1450 m.
Diaphorina citri infestations were not recorded by Aubert (1990) on Réunion Island
(21.12°S, 55.53°E) at elevations of 700–800 m where minimum temperatures were
165
7°C. In peninsular Malaysia, between 3–5°N and 101–103.5°E, the psyllid was
recorded up to 1200 m where minimum temperatures of 14°C occur (Aubert 1990).
In China, the greatest infestations of the psyllid and the greatest incidence of
huanglongbing occurs in the southeast in Guangxi, Guangdong, Fujian, and Taiwan,
with the boundary separating high from low incidence running from 29°N in
Zhejiang to 25°N in Yunnan in a sea-facing arc running slightly inland from the
mountain ranges that separate inland Sichuan, Guizhou, Hunan and Jiangxi from
coastal Guangxi, Guangdong, Fujian and Zhejiang (Yang et al. 2006).
The
elevations of localities between 26.00°N and 29.00°N where Diaphorina citri occurs
in these provinces range from ~500–1200 m (Yang et al. 2006).
Beattie et al. (2010) assessed populations of Diaphorina citri in orchards at
Purworejo (7.7145°S, 110.0096°E, 60 m ASL), Grabag (7.3667°S, 110.3000°E, 640
m ASL) and Ngablak (7.3963°S, 110.3893°E, 1300 m ASL) in Central Java,
Indonesia, over 2–3 years. The insect was common at Purworejo, uncommon at
Grabag and never observed at Ngablak. Online records and unpublished records
from the study (Rachmad Gunadi, Universitas Gadjah Mada) indicate that average
temperatures and annual rainfall at the three elevations are ~26.5°C and 2447 mm,
22.1°C and 3068 mm, and 17.4°C and 2908 mm, respectively, with peak monthly
rainfall of ~470 mm at Purworejo, ~580 mm at Grabag, and ~630 mm at Ngablak in
December and January.
The presence of Diaphorina citri at elevations ranging from 10 m to 880 m ASL in
Puerto Rico (18.2208°N, 066.5901°W) was determined by Jenkins et al. (2015). It
was not recorded at elevations above 600 m ASL. This was attributed to lower
ambient temperatures at higher elevations limiting flush growth on trees and
generations of the psyllid at the higher elevations, and to differences in air pressure,
oxygen levels and short wave radiation, partial pressure and precipitation. They also
hypothesised that slower development of the psyllid at the higher elevations may
have prolonged the risk of attack by predators and parasites (Jenkins et al. 2015).
They did not record ambient temperatures or rainfall, but weather information
166
accessible online indicates that average rainfall ranges from 700–1400 mm near sea
level to 2300–2500 mm from 600–800 m ASL where surveys were undertaken.
Average minimum and maximum temperatures differ by ~3°C and ~4°C repectively.
(http://pr.water.usgs.gov/drought/climate.html; http//www.topuertorico.org/reference
/tempera.shtml).
Populations of Diaphorina citri decline after heavy rainfall.
Aubert (1987)
mentioned 150 mm, and Chavan & Shummanvar (1993) recorded the lowest
population densities near Pune in Maharashtra, India, after monsoon rains that occur
from June to October. Average monthly rainfall recorded from June to September in
Pune by Tinmaker et al. (2010) over four years was 145 mm, ranging from 115 mm
to 187 mm per month. Average rainfall cited by World Weather Online for the same
interval in Pune was 165 mm, ranging from 126 mm to 207 mm per month
(https://www.worldweatheronline.com/pune-weather-averages/maharashtra/in.aspx).
Bové (2014) summarised records for ‘CLas’ and Diaphorina citri in Nepal, noting
that the pathogen and the vector had been recorded in orchards that occur at similar
latitudes (~26° to 27°N) to orchards in Bhutan, at elevations ranging from ~450 m to
~1350 m.
‘Candidatus Liberibacter asiaticus’ titres and the extent of psyllid
infestations were not mentioned.
Garnier & Bové (2002) surveyed orchards in Bhutan from 28 to 29 August 2002.
Most of the trees in the orchards visited were 10–30 years old. The altitude of the
orchards was determined with an altimeter, but the elevations appear, based on my
knowledge and surveys, and cross-checking with Google Earth, in most instances, to
have been overestimated by 70–100 m. ‘Candidatus Liberibacter asiaticus’ was
detected at Rimchhu (27.6675°N, 89.7753°E, 1380 m ASL, cited as 1450 m),
Botakha (27.6401°N, 89.7948°E, 1287–1325 m ASL, cited as 1400 m), Serigang
(27.6399°N, 89.7925°E, 1318 m ASL, cited as 1400 m), Phuntshopelri (27.6128°N,
89.8406°E, 1264 m ASL, cited as 1350 m), Upper Sonagasa (27.6110°N, 89.8467°E,
1239 m ASL, cited as 1350 m ASL) and Lower Sonagasa (27.6093°N, 89.8499°E,
1232 m ASL, cited as 1350 m ASL), and the Renewable Natural Resources Research
167
Center (RNRRC) at Bajo, Wangdue Phodrang (27.4906°N, 89.8999°E, 1225 m, cited
as 1300 m). The presence of Diaphorina citri eggs, nymphs and adults was not
recorded. At Chhuzomsa (27.5053°N, 89.9634°E, 1362 m ASL, cited as 1300 m), to
the east of Wangdue Phodrang, no symptoms of HLB were recorded and Diaphorina
citri was not mentioned. The surveys continued southwards along the right bank of
the Punatsangchhu. At Rurechhu (27.3537°N, 89.9144°E, 1000 m ASL, cited as
1100 m) no evidence of HLB was found and Diaphorina citri was not mentioned: the
orchard is now an electricity substation. At Baychhu (27.2984°N, 89.9674°E, 776 m
ASL, cited as 850 m), ‘CLas’ was detected but Diaphorina citri was not mentioned.
At Kamichhu (27.2706°N, 90.0381°E, 640 m ASL, cited as 700 m) ‘CLas’ and
Diaphorina citri were present.
Bové (2004) reported the outcome of surveys undertaken in Bhutan from 9 to 24
October 2004.
These surveys were undertaken in the Damphu and Tashipang
(Mendrelgang) regions of Tsirang and on the Thimphu (spelt as Timphu) and
Pasakha roads near Phuentsholing. No evidence for HLB was found.
Diaphorina communis was never observed in the seedling mandarin plots along
Droopchhugang-Phuensoomgang during the study. Some adults were observed on
Bergera koenigii in the vicinity of the plots at 800, 900 and 1000 m.
Cacopsylla heterogena was observed on mature trees at 1500 m ASL and on young
trees in an orchard between 1300 m and 1400 m ASL along DroopchhugangPhuensoomgang in 2013. A few pouch galls with nymphs and aged honeydew were
found on mature mandarin trees at 1300 m ASL in 2015. The psyllid was recorded
once in the seedling mandarin plots at 1400 m ASL in 2015. Eggs, nymphs and
adults were observed in a nursery near these plots in 2014. In areas, other than
Droopchhugang-Phuensoomgang, the psyllid was found at elevations ranging from
983–2444 m ASL (Chapter 6). No parasitoids were recorded despite the large
number of nymphs observed during the study.
168
My results and the observations reported by Aubert (1987; 1990), Chavan &
Shummanvar (1983), Beattie et al. (2010) and Jenkins et al. (2015) suggest that
monsoon rains have significant impacts on populations of Diaphorina citri in Tsirang
and that these impacts are likely to be greater as prevailing ambient temperatures
decline with increasing elevation. However, the differences in ambient temperatures
(Figure 5.9, Table 5.4) may not be the sole reason for the abrupt change in the
incidence of the psyllid between 1100 and 1200 m.
The fall in the incidence of Cacopsylla heterogena with declining elevations,
particularly the abrupt change in incidence below 1200 m, is most probably related to
increasing ambient temperatures and falling relative humidity (Figures 5.10 & 5.11,
Table 5.4) with the decline in elevation and increasing risk of egg and nymph
mortality despite protection afforded to these immature stages by the pouch galls in
which they develop.
5.4.3 Prevalence of ‘CLas’ at different elevations
Few mandarin seedlings (up to three plants) representing each seedling consignment
received from the NSC in 2013 and 2014 were maintained in the green house in
NPPC for observations. Pooled samples of these seedlings were tested for ‘CLas’
and one sample (out of three) produced FAM Ct of 34.27 by qPCR in 2014 but did
not yield any amplicons in cPCR. This result, however, cast doubt over the health
status of the seedlings at the NSC. Therefore, more sampling and testing were
conducted on the same plants as well as sampling from the plants at NSC.
Subsequent testing of samples from the same plants maintained at NPPC did not
yield any FAM Ct values.
Further, the PCR results obtained from the young
mandarin seedlings planted along Droopchhugang-Phuensoomgang also indicate that
the plant used in the current study could not have been infected. If seedlings were
infected before planting, then ‘CLas’-positive results would also be obtained from
seedlings planted at the higher elevations as the seedlings planted at each altitude
were selected randomly from the same group of plants.
If the seedlings were
infected before planting, then ‘CLas’ would have been detected in seedlings planted
at all elevations, which was not the case.
169
I hypothesised that leaf temperatures detrimental to ‘CLas’ may influence the
incidence of ‘CLas’ and severity of huanglongbing at higher altitudes in Tsirang, due
to lower evaporative cooling of leaves in relation to higher relative humidity at the
prevailing ambient temperatures. However, the leaf temperatures recorded were ~8
to 19°C below temperatures required in heat treatments to kill ‘CLas’ in plant tissues
(e.g., 40 to 51°C for 30 minutes to 48 hours) (Lin & Lo 1965; Martinez et al. 1971;
Raychaudhuri et al. 1974; Su & Chang 1976; Huang 1978; Lo et al. 1981; Cheema et
al. 1982; Lo 1983; Hoffman et al. 2013). Therefore, leaf temperatures could not
have been the factor limiting the titres of ‘CLas’ and severity of huanglongbing at the
higher altitudes.
Results of qPCR analysis of the root and bark samples collected for two and three
years, respectively, from mature mandarin trees (> 30 years) along DroopchhugangPhuensoomgang showed that ‘CLas’ was more prevalent in plants at the lower
elevations (800–1100 m) than in plants at 1200 m or above (Table 5.6, & 5.7). This
is more evident in the analysis using bark samples (Table 5.6). The low incidence of
‘CLas’ at higher elevations could be due to the absence of Diaphorina citri in these
areas restricting spread of the disease. ‘Candidatus Liberibacter asiaticus’ could
have been introduced in these areas by infected planting material.
Most citrus
orchards in Bhutan were established with material produced without any appropriate
phytosanitary measures. Planting materials used to establish orchards in the 1960s80s were sourced from lower elevations where both the pathogen and Diaphorina
citri were prevalent. Although the presence of ‘CLas’ in Bhutan was only confirmed
in 2002 (Ahlawat 2003; Doe Doe et al. 2003), the disease was present at the lower
elevations much earlier as reported in Lama & Amtya (1991) and Lama & Amatya
(1993). Records indicate that the pathogen was confirmed in neighbouring states of
India and Nepal in the 1960s (Fraser 1966; Nariani & Raychaudhuri 1968; Knorr et
al. 1970; Regmi & Lama 1988), and in Bangladesh in the late 1970s (Catling et al.
1978).
Leaf and bark samples from elevations above 1200 m in Mendrelgang Gewog did not
yield any FAM Ct values, whereas at least one or more root samples produced FAM
170
Ct value > 0.00 with most of them close to 36. A root sample from 1100 m with
FAM Ct value of 31.49 was confirmed by sequencing. The observation of many root
samples from higher elevations producing FAM Ct values > 0.00 compared to bark
(or leaf) both along Droopchhugang-Phuensoomgang as well as in the Mendrelgang
Gewog seem to indicate that ‘CLas’ may be present but is not readily detected in
aerial plant tissues at such elevations. However, there is no evidence to confirm this,
since not a single root sample from the higher elevation could be amplified using
cPCR and sequenced. It is also possible that the root samples may have produced
non-specific amplification. Kunta et al. (2014) obtained ‘CLas’-positive Ct values
ranging from 22.90 to 31.78 in root samples from asymptomatic trees using the 16S
primer-probe of Li et al. (2006) whereas the leaf samples from the same trees did not
produce any positive real time test results. Kunta et al. (2014) concluded that the
16S primer-probe of Li et al. (2006) was not suitable for detection of ‘CLas’ in root
samples based on further evaluations using the LJ900fpr primer-probe on root and
soil samples. Kunta et al. (2014) found that the PCR products that the 16S primerprobe amplified from root and soil samples matched with beta-glucosidase belonging
to several proteobacteria. The primers-probe set used in the current study was also
that of Li et al. (2006). However, further work is required to assess the results of the
root samples in the current study.
Real-time PCR analysis of samples taken from young mandarin seedlings planted at
100 m intervals from 800 m to 1500 m showed a similar trend with a greater
proportion of plants at lower elevations producing FAM Ct values > 0.00 compared
to plants at higher elevations, especially in the bark and root samples collected in
March 2016. Bark samples collected in May 2015 showed a higher number of plants
at lower elevations producing FAM Ct value > 0.00 than the corresponding root
samples.
The overall results indicate that few plants at higher elevations produced FAM Ct
values in the range of 0.00 < FAM Ct < 36 which are deemed as ‘CLas’ positive.
Many samples that produced FAM Ct values between 30 and 40 did not yield any
amplicons when subjected to cPCR with primers A2/J5, although two samples with
171
FAM Ct values of 37.46 and 37.75 from young and mature trees respectively, both
collected from 800 m, were successfully amplified and sequenced.
5.5
Conclusions
My results suggest that factors other than ambient temperature, RH and rainfall limit
the occurrence of Diaphorina citri and, directly or indirectly, the presence of ‘CLas’
at ≥ 1200 m in Tsirang.
UV radiation may be a possible factor limiting the
distribution of Diaphorina citri at or above 1200 m. For UV radiation to have
impacts on tissues, radiation must enter and be absorbed in the tissues, and many
animals and plants are thought to have developed UV shielding mechanisms
(Caldwell et al. 1998). This possibility is supported by occurrence of eggs and
nymphs of Cacopsylla heterogena inside pouch galls in contrast to opposed to the
exposed eggs and nymphs of Diaphorina citri and Diaphorina communis. It is also
supported by the occurrence of an unidentified leaf gall midge (Diptera:
Cecidomyiidae) at elevations ranging from 800 to 1400 m observed during my study
(Figure 5.12) that develops within wart-like, tubular leaf galls formed by adaxial leaf
surfaces rolled inwards along midribs. The formation of pouch and tubular leaf galls
would provide shielding mechanisms against the effects of UV light, high rainfall
and desiccation. Moreover, Bhutan lies within a region of the Earth’s surface where
high UV radiation occurs (Figure 5.13) (Liley & McKenzie 2006), and higher UV
radiation than in Arabia where, as noted above, Diaphorina citri occurs in the
absence of monsoon rains, at slightly higher elevations than in Bhutan. Unidentified
spider mites (Acari: Tetranychidae) were also observed during the study, on abaxial
leaf surfaces (Figure 5.14) of Zanthoxylum sp. leaflets, 27.0610°N, 89.5642°E, 1607
m, near the Chhukha Bridge on the Phuentsholing-Thimphu Highway on 24 April
2013 and mandarin trees at Kamichhu (650 m) on 16 April 1014. Some spider mites
are known to stay on abaxial leaf surfaces in order to avoid UV (Ben-Yakir &
Fereres 2016). UV radiation (UVA & UVB) has been reported to affect herbivores
directly, e.g., by causing high mortality of immature stages of a chrysomelid species,
and reduced the egg laying capacity of the two spotted spider mite (Tetranychus
urticae Koch: Acari: Tetranychidae) (Kuhlmann & Müller 2011). Indirect effects of
172
UV radiation on herbivores is associated with changes in plant morphology,
physiology and photochemistry (Kuhlmann & Müller 2011). Under enhanced UVB
radiation, probing behaviour as well as the nymphal development period,
reproductive and post-reproductive period, and difference in weight after moulting of
the grain aphid (Sitobion avenae: Hemiptera: Aphididae) were negatively affected
(Hu et al. 2013a & b). Hu et al. (2013b) noted differences in response to UV
radiation among the brown and green morphs of the grain aphid. Further, UV
radiation causes changes in plant secondary metabolites—which are known to
contribute to protection against pathogens and insects (Katerova et al. 2012). Salt et
al. (1998) observed progressively low populations of the psyllid, Strophingia ericae
(Curtis) (Hemiptera: Liviidae), and reduced level of the amino acid, isoleucine, in the
heather plants (Calluna vulgaris (L.) Hull) (Ericales: Ericaceae) compared to the
controls over a 27 month period. Solar radiation is said to increase with increase in
altitude. Comparing the total irradiance and UVA irradiance between Jungfraujoch
at 3576 m ASL in Switzerland and Innsbruck at 577 m ASL in Austria, Blumthaler et
al. (1997) noted that under clear sky condition of summer, total irradiance increased
by 8% per 1000 m while UVA irradiance increased by 9%.
Figure 5.12. Wart-like tubular leaf gall of an unidentified cecidomyiid observed between 800 m and
1400 m ASL during the study, and larvae within a gall (Photos: GAC Beattie).
173
Figure 5.13. Map of peak UV indexes using a modified scale to highlight the absolute peaks (Liley &
McKenzie 2006). The location of Bhutan is circled in red and indicated with an arrow.
Figure 5.14. Spider mites on abaxial leaf surfaces of Zanthoxylum sp. (1600 m ASL) and mandarin
(650 m ASL). (Photos: GAC Beattie)
174
5.6
Highlights of the study:
Altitude, ambient temperature and relative humidity did not lead to leaf
temperatures detrimental to ‘CLas’ titres.
Diaphorina citri was more commonly found below 1200 m while Cacopsylla
heterogena was found quite abundantly at higher altitudes.
The distribution of Diaphorina citri in Tsirang at higher altitudes seems to be
limited, possibly due to combination of UV radiation and rainfall, since the
ambient temperatures obtained during the two years of the study period are
within the favourable conditions for Diaphorina citri.
Diaphorina communis was not observed in the experimental plots or on mature
mandarin trees in the study area, although it was observed nearby on Bergera
koenigii.
‘Candidatus Liberibacter asiaticus’ is more prevalent at elevations up to 1100
m. At elevations above 1200 m, ‘CLas’ may have been introduced in planting
materials but remained localised due to absence of Diaphorina citri at these
altitudes. In the absence of a vector, it is possible to grow mandarins and other
Citrus species and hybrids above 1200 m using pathogen-free planting material.
175
Chapter 6: Preliminary investigation into the role of
Cacopsylla heterogena Li in huanglongbing
__________________________________________________________________
6.1
Introduction
As detailed in the literature review, besides Diaphorina citri and Diaphorina
communis, about eight other species of psyllids are reported to feed on rutaceous
plants in Asia (Table 6.1). In Bhutan, four adults of a species of green psyllid were
first recorded in April 2013 in Tsirang, initially in an orchard (27.0082°N,
90.1391°E, 1260 m ASL) in Dekidling Chiwog, Kilkhorthang Gewog, and then on
the same day at Riserboo ‘A’ (Kamigaon) (26.9532°N, 90.1347°E, 1436 m ASL) in
Mendrelgang Gewog when a larger number of adults were tapped from immature
and mature seedling mandarin trees on which nymphs were also present. Adult
females of the green psyllid lay their eggs on young leaves that rapidly fold inwards
longitudinally at 90° along midribs so forming a pouch gall that protects the nymphs
as they develop. High populations of the psyllid cause significant deformation of
flush growth, which is manifested as a twisting and bending of leaves as they grow in
length before hardening (pers. obs.). Since these initial observations, damage caused
by this psyllid has been observed in mandarin orchards in the Dagana, Mongar,
Punakha, and Thimphu Dzongkhags (Beattie, pers. comm.). This psyllid species was
also observed infesting an undescribed wild citrus species (see Chapter 7) growing
near Wengkhar and Korilla in the Mongar Dzongkhag and at Basochhu in the
Wangdue Phrodrang Dzongkhag (Beattie, pers. comm.).
Two other species of
psyllids have subsequently been found causing similar foliage damage on an
undescribed species of Zanthoxylum in various parts of the Tsirang Dzongkhag and
at Baychhu in the Wangdue Phodrang Dzongkhag.
Until recently, the only species of green psyllid on citrus known to acquire and
transmit ‘CLas’ is Cacopsylla citrisuga (Yang & Li) (Cen et al. 2012 a; 2012b). The
high incidence of the psyllids found in Bhutan on mandarin and other citrus relatives
and their possible role in the epidemiology of ‘CLas’ necessitated an investigation of
these species to determine any role they may play in relation to the aetiology of
176
‘CLas’.
Additionally, molecular characterisation of the psyllids is important to
inform the morphological identification of the currently known psyllids on Rutaceae.
Thus, this chapter focuses on the molecular characterisation of the psyllids found on
citrus (mandarin, lime, lemon, orange and wild citrus) and Zanthoxylum in Bhutan,
and the detection of ‘CLas’ within the psyllids and the host plants on which they
were found.
For insects, the mitochondrial 16S region and the COI gene (Simon et al. 1994; Lunt
et al. 1996; Shouche & Patole 2000; Costa et al. 2003) are widely used in
phylogenetic studies and were using in this study. In addition, the nuclear ITS region
has also been employed to infer phylogenetic relationships in closely related groups
of insects (Miller et al. 1996) and was also used in this study.
The specific objectives of this chapter were to:
study the morphological and molecular characteristics of the green psyllids
found on mandarin and wild citrus, and other psyllids found on an
unidentified Zanthoxylum sp., and,
determine the presence of ‘CLas’ within the green psyllids and their host
plants.
177
Table 6.1. Summary of species of psyllids feeding on Rutaceae in Asia1
Species
Host plant
Places
Reference
Cacopsylla (Psylla) citricola (Yang & Li)
Citrus maxima (Burm.) Merr.
China
Yang & Li (1984)
Citrus medica L.
China; India
Yang & Li (1984)
Citrus maxima L.
China
Yang & Li (1984)
Citrus medica L.
India
Beattie (pers. comm.)
Murraya paniculata L.
Japan
Inoue et al. (2006)
Cacopsylla (Psylla) citrisuga (Yang & Li)
Cacopsylla (Psylla) evodiae (Miyataki))
Tetradium glabrifolium (Champ.
ex Benth.) T. G. Hartley
Zanthoxylum beecheyanum var.
alatum (Nakai) Hara
Inoue (2010)
Toddalia asiatica (L.)
Cacopsylla heterogena Li
Citrus sp.
China
Li (2011)
Cacopsylla (Psylla) murrayi (Mathur)
Bergera koenigii L.
Northern
India
Mathur (1975)
Citrus maxima L.
Cacopsylla toddaliae (Yang)
Lahiri & Biswas (1979, 1980)
Citrus × limon (L.) Osbeck
Malaysia
Osman & Lim (1992)
Citrus × aurantium L.
Japan;
Taiwan
Inoue 2010
Toddalia asiatica (L.) Lam.
Diaphorina murrayi Kandasamy
Bergera koenigii (L.)
India
Kandasamy (1986)
Trioza citroimpura Yang & Li
Citrus reticulata Blanco
China
Yang & Li (1984)
1
For complete descriptions, refer to the literature review in Chapter 1.
178
6.2
Materials and methods
6.2.1 Psyllid samples for phylogenetic analyses and morphological
identification
Specimens of green psyllids on citrus occurring at altitudes ranging from 983 to 2444
m ASL and on the Zanthoxylum sp. from altitudes from 1175 to 1264 m ASL were
collected as described in Chapter 2 for phylogenetic analysis (Table 6.2). Nymphs
were included to confirm that the adults and nymphs collected from each host were
of the same species. Specimens were stored in 100% ethanol until DNA extraction.
Representative psyllid specimens were sent to Dr Susan Halbert (Taxonomic
Entomologist, Division of Plant Industry, Florida Department of Agriculture and
Consumer Services) and Luo Xinyu (Department of Entomology, China Agricultural
University) for morphological identification.
Three specimens of Cacopsylla citrisuga from Yunnan, China were received from Dr
Cen Yijing, South China Agricultural University, Guangzhou, Guangdong, China to
compare with the psyllids collected in Bhutan; two specimens were adults collected
in Yunnan, China, and preserved in 100% ethanol, and one specimen was received as
DNA.
6.2.2 Psyllid DNA extraction for phylogenetic studies
Genomic DNA from single psyllid was extracted from specimens stored in 100%
ethanol in the Molecular Laboratory of the School of Science and Health (SSH),
Western Sydney University, Hawkesbury Campus. The extraction method using the
ISOLATE II Genomic DNA Kit (Bioline) was employed but without crushing the
samples Section 4.2.2 (Chapter 4). Subsequent extractions were made using the
REDExtract-N-Amp™ Plant PCR Kit (Sigma-Aldrich) as in Section 3.2.4 (Chapter
3). For specimens from China, DNA was extracted from the two psyllid specimens
using the ISOLATE II Genomic DNA Kit (Bioline). The third specimen from China
was received as a DNA extract.
179
Table 6.2. Host plants and locations where psyllid specimens were collected. Abbreviations used: Bhutan (BT); nymph with round and not black abdomen (RNBA); nymph
with round and black abdomen (RBA); slender nymph (SN). Authorities of plant species are as in Chapter 7.
Sample
Name of samples based on
Location
Host plant
Life stage
GPS coordinates
Elevation
number
results of this study
CHBT.1
Cacopsylla heterogena
Droobchhugang, Tsholingkhar
Gewog, Tsirang, BT
Citrus reticulata
adult
27.0189°N, 90.1275°E
1478
CHBT.2
Cacopsylla heterogena
Droobchhugang, Tsholingkhar
Gewog, Tsirang, BT
Citrus reticulata
adult
27.0189°N, 90.1275°E
1478
CHBT.3
Cacopsylla heterogena
Droobchhugang, Tsholingkhar
Gewog, Tsirang, BT
Citrus reticulata
adult
27.0189°N, 90.1275°E
1478
CHBT.4
Cacopsylla heterogena
Riserboo ‘A’, Mendregang Gewog,
Tsirang, BT
Citrus reticulata
adult
26.9532°N, 90.1347°E
1436
CHBT.6
Cacopsylla heterogena
Riserboo ‘A’, Mendregang Gewog,
Tsirang, BT
Citrus reticulata
adult
26.9532°N, 90.1347°E
1436
CHBT.9
Cacopsylla heterogena
Riserboo ‘A’, Mendregang Gewog,
Tsirang, BT
Citrus reticulata
nymph
26.9532°N, 90.1347°E
1436
CHBT.10
Cacopsylla heterogena
Riserboo ‘A’, Mendregang Gewog,
Tsirang, BT
Citrus reticulata
adult
26.9532°N, 90.1347°E
1436
CHBT.11
Cacopsylla heterogena
Riserboo ‘A’, Mendregang Gewog,
Tsirang, BT
Citrus reticulata
adult
26.9532°N, 90.1347°E
1436
CHBT.12
Cacopsylla heterogena
Riserboo ‘A’, Mendregang Gewog,
Tsirang, BT
Citrus reticulata
adult
26.9532°N, 90.1347°E
1436
CHBT.14
Cacopsylla heterogena
Riserboo ‘A’, Mendregang Gewog,
Tsirang, BT
Citrus reticulata
nymph
26.9532°N, 90.1347°E
1436
(m ASL)
180
Sample
Name of samples based on
number
CHBT.17
results of this study
Cacopsylla heterogena
CHBT.18
Location
Host plant
Life stage
GPS coordinates
Elevation
RDC, Maenchhana, Kilkhorthang
Gewog, Tsirang, BT
Citrus × aurantium
(sweet orange)
nymph
26.9977°N, 90.1250°E
(m ASL)
1531
Cacopsylla heterogena
RDC, Maenchhana, Kilkhorthang
Gewog, Tsirang, BT
Citrus × aurantium
(sweet orange)
nymph
26.9977°N, 90.1250°E
1531
CHBT.20
Cacopsylla heterogena
Patala, Drujegang Gewog, Dagana,
BT
Citrus reticulata
adult
26.9847°N, 90 0180°E
1496
CHBT.21
Cacopsylla heterogena
Patala, Drujegang Gewog, Dagana,
BT
Citrus reticulata
nymph
26.9847°N, 90 0180°E
1496
CHBT.22
Cacopsylla heterogena
Patala, Drujegang Gewog, Dagana,
BT
Citrus reticulata
nymph
26.9847°N, 90 0180°E
1496
CHBT.23
Cacopsylla heterogena
Patala, Drujegang Gewog, Dagana,
BT
Citrus reticulata
nymph
26.9802°N, 90.0129°E
1493
CHBT.24
Cacopsylla heterogena
Patala, Drujegang Gewog, Dagana,
BT
Citrus reticulata
nymph
26.9802°N, 90.0129°E
1493
CHBT.25
Cacopsylla heterogena
Patala, Drujegang Gewog, Dagana,
BT
Citrus reticulata
adult
26.9802°N, 90.0129°E
1493
CHBT.26
Cacopsylla heterogena
Patala, Drujegang Gewog, Dagana,
BT
Citrus reticulata
adult
26.9802°N, 90.0129°E
1493
CHBT.27
Cacopsylla heterogena
Thangna, Drujegang Gewog,
Dagana, BT
Citrus reticulata
nymph
26.9870°N, 90.0326°E
1164
CHBT.28
Cacopsylla heterogena
Thangna, Drujegang Gewog,
Dagana, BT
Citrus reticulata
nymph
26.9870°N, 90.0326°E
1164
CHBT.29
Cacopsylla heterogena
Thangna, Drujegang Gewog,
Dagana, BT
Citrus reticulata
adult
26.9870°N, 90.0326°E
1164
181
Sample
Name of samples based on
Location
Host plant
Life stage
GPS coordinates
Elevation
number
results of this study
CHBT.30
Cacopsylla heterogena
Thangna, Drujegang Gewog,
Dagana, BT
Citrus reticulata
adult
26.9870°N, 90.0326°E
1164
CHBT.31
Cacopsylla heterogena
Thangna, Drujegang Gewog,
Dagana, BT
Citrus reticulata
adult
26.9870°N, 90.0326°E
1164
CHBT.31
Cacopsylla heterogena
Thangna, Drujegang Gewog,
Dagana, BT
Citrus reticulata
adult
26.9870°N, 90.0326°E
1164
CHBT.32
Cacopsylla heterogena
Citrus reticulata
nymph
26.9826°N, 90.0533°E
1035
CHBT.33
Cacopsylla heterogena
Citrus reticulata
nymph
26.9748°N, 90.0550°E
983
CHBT.34
Cacopsylla heterogena
Pangna, Drujegang Gewog,
Dagana, BT
Below Pangna, Drujegang Gewong,
Dagana, BT
Talo, Punakha, BT
Citrus reticulata
nymph
27.5516°N, 89.8238°E
2444
CHBT.35
Cacopsylla heterogena
Talo, Punakha, BT
Citrus reticulata
adult
27.5516°N, 89.8238°E
2444
CHBT.37
Cacopsylla heterogena
Talo-Walakha Road 1, Punakha,
BT
Citrus reticulata
adult
27.5403°N, 89.8521°E
1791
CHBT.39
Cacopsylla heterogena
Mendrelgang - Thimphu-Punakha
Road, BT
Citrus reticulata
nymph
27.5248°N, 89.8323°E
1604
CHBT.41
Cacopsylla heterogena
Mendrelgang - Thimphu-Punakha
Road, BT
Citrus reticulata
nymph
27.5248°N, 89.8323°E
1604
CHBT.42
Cacopsylla heterogena
Khuengkha, Dungna Gewog,
Chukkha, BT
Citrus reticulata
nymph
26.9335°N, 89.4322°E
1090
CHBT.45
Cacopsylla heterogena
Khuengkha, Dungna Gewog,
Chukkha, BT
Citrus × aurantiifolia
nymph
26.9335°N, 89.4322°E
1090
(m ASL)
182
Sample
Name of samples based on
number
CHBT.46
results of this study
Cacopsylla heterogena
CHBT.47
Location
Host plant
Life stage
GPS coordinates
Elevation
Khuengkha, Dungna Gewog,
Chukkha, BT
Citrus × aurantiifolia
nymph
26.9335°N, 89.4322°E
(m ASL)
1090
Cacopsylla heterogena
Khuengkha, Dungna Gewog,
Chukkha, BT
Citrus × aurantiifolia
adult
26.9335°N, 89.4322°E
1090
CHBT.48
Cacopsylla heterogena
Khuengkha, Dungna Gewog,
Chukkha, BT
Citrus × aurantiifolia
adult
26.9335°N, 89.4322°E
1090
CHBT.49
Cacopsylla heterogena
Khuengkha, Dungna Gewog,
Chukkha, BT
Citrus × aurantiifolia
adult
26.9335°N, 89.4322°E
1090
CHBT.50
Cacopsylla heterogena
Near Wengkhar, Mongar, BT
wild native citrus
nymph
27.2515°N, 91.2597°E
2018
CHBT.51
Cacopsylla heterogena
Near Wengkhar, Mongar, BT
wild native citrus
nymph
27.2515°N, 91.2597°E
2018
CCN.1
Cacopsylla citrisuga
Yunnan, China (received as DNA)
lemon
adult
24.0712°N, 97.7999°E,
1200
CCN.2
Cacopsylla citrisuga
Yunnan, China
lemon
adult
24.0712°N, 97.7999°E,
1200
PCOBT.1
Cornopsylla rotundiconis
Pemathang, Gosarling Gewog,
Zanthoxylum sp.
adult
27.2414°N, 90.3581°E
1175
Zanthoxylum sp.
adult
27.2414°N, 90.3581°E
1175
Zanthoxylum sp.
adult
27.2414°N, 90.3581°E
1175
Zanthoxylum sp.
adult
27.2414°N, 90.3581°E
1175
Tsirang, BT
PCOBT.2
Cacopsylla sp.
Pemathang, Gosarling Gewog,
Tsirang, BT
PCOBT.4
Cornopsylla rotundiconis
Pemathang, Gosarling Gewog,
Tsirang, BT
PCOBT.5
Cornopsylla rotundiconis
Pemathang, Gosarling Gewog,
Tsirang, BT
183
Sample
Name of samples based on
number
PCOBT.6
results of this study
Cornopsylla rotundiconis
Location
Host plant
Pemathang, Gosarling Gewog,
Life stage
GPS coordinates
Elevation
Zanthoxylum sp.
adult
27.2414°N, 90.3581°E
(m ASL)
1175
Zanthoxylum sp.
adult
27.2414°N, 90.3581°E
1175
Zanthoxylum sp.
adult
27.2414°N, 90.3581°E
1175
Zanthoxylum sp.
adult
27.2414°N, 90.3581°E
1175
Zanthoxylum sp.
RNBA
27.0120°N, 90.1579°E
1264
Zanthoxylum sp.
RNBA
27.0120°N, 90.1579°E
1264
Zanthoxylum sp.
SN
27.0120°N, 90.1579°E
1264
Zanthoxylum sp.
RBA
27.0120°N, 90.1579°E
1264
Zanthoxylum sp.
RBA
27.0120°N, 90.1579°E
1264
Tsirang, BT
PCOBT.7
Cacopsylla sp.
Pemathang, Gosarling Gewog,
Tsirang, BT
PCOBT.8
Cacopsylla sp.
Pemathang, Gosarling Gewog,
Tsirang, BT
PCOBT.9
Cacopsylla sp.
Pemathang, Gosarling Gewog,
Tsirang, BT
PCOBT.10
Cacopsylla sp.
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
PCOBT.11
Cacopsylla sp.
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
PCOBT.15
Cornopsylla rotundiconis
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
PCOBT.16
Cacopsylla sp.
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
PCOBT.17
Cacopsylla sp.
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
184
Sample
Name of samples based on
number
results of this study
PCOBT.18
Cacopsylla sp.
Location
Host plant
Life stage
GPS coordinates
Elevation
(m ASL)
Dangreyboog, Dunglagang Gewog,
Zanthoxylum sp.
RBA
27.0120°N, 90.1579°E
1264
Zanthoxylum sp.
adult
27.0120°N, 90.1579°E
1264
Zanthoxylum sp.
adult
27.0120°N, 90.1579°E
1264
Zanthoxylum sp.
adult
27.0120°N, 90.1579°E
1264
Zanthoxylum sp.
adult
27.0120°N, 90.1579°E
1264
Tsirang, BT
PCOBT.19
Cacopsylla sp.
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
PCOBT.20
Cacopsylla sp.
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
PCOBT.21
Cacopsylla sp.
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
PCOBT.22
Cornopsylla rotundiconis
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
PCOBT.24
Cornopsylla rotundiconis
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
Zanthoxylum sp.
adult
27.0120°N, 90.1579°E
1264
PCOBT.25
Cacopsylla sp.
Dangreyboog, Dunglagang Gewog,
Zanthoxylum sp.
RNBA
27.0120°N, 90.1579°E
1264
Zanthoxylum sp.
RBA
27.0120°N, 90.1579°E
1264
Tsirang, BT
PCOBT.27
Cacopsylla sp.
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
185
Sample
Name of samples based on
number
PCOBT.28
results of this study
Cornopsylla rotundiconis
Location
Host plant
Dangreyboog, Dunglagang Gewog,
Life stage
GPS coordinates
Elevation
Zanthoxylum sp.
SN
27.0120°N, 90.1579°E
(m ASL)
1264
Zanthoxylum sp.
SN
27.0120°N, 90.1579°E
1264
Zanthoxylum sp.
adult
27.0120°N, 90.1579°E
1264
Zanthoxylum sp.
adult
27.0120°N, 90.1579°E
1264
Zanthoxylum sp.
adult
27.0120°N, 90.1579°E
1264
Zanthoxylum sp.
adult
27.0120°N, 90.1579°E
1264
Zanthoxylum sp.
adult
27.0120°N, 90.1579°E
1264
Tsirang, BT
PCOBT.29
Cornopsylla rotundiconis
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
PCOBT.31
Cornopsylla rotundiconis
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
PCOBT.33
Cornopsylla rotundiconis
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
PCOBT.34
Cacopsylla sp.
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
PCOBT.35
Cacopsylla sp.
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
PCOBT.36
Cacopsylla sp.
Dangreyboog, Dunglagang Gewog,
Tsirang, BT
186
6.2.3 Psyllid DNA amplifications and sequencing
The COI region was amplified similar to methods described in Section 4.2.2
(Chapter 4) using primers, CI-J-1718 (5’-GGA GGA TTT GGA AAT TGA TTA
GTT CC-3’) and C1-N-2191 (5’-CCC GGT AAA ATT AAA ATA TAA ACT TC3’) (Simon et al. 1994), with PCR conditions adapted from Boykin et al. (2012) as in
Section 2.2.2 (Chapter 2). The ITS region was amplified with the universal primers,
ITS1 and ITS4, using a protocol adapted from White et al. (1990). The PCR cycling
parameters were performed with an initial denaturation at 94°C for 90 sec, followed
by 30 cycles of denaturation at 95°C for 50 sec, annealing at 55°C for 70 sec and
extension at 72°C for 90 sec, and a final elongation step at 72°C for 3 min. For the
mitochondrial 16S region, amplification was performed as described in Section 2.2.2
(Chapter 2). Sequencing was performed in both directions using the same primers
sets as described in Section 2.2.3 (Chapter 2).
6.2.4 Phylogenetic relationships of green psyllids on citrus and
Zanthoxylum sp.
For the green psyllids, phylogenetic relationships were inferred through maximum
likelihood (ML) analyses of the mitochondrial 16S and COI genes and the nuclear
ITS region. Bactericera cockerelli (Šulc) (Hemiptera: Sternorrhyncha: Triozidae)
was used as the outgroup in all analyses. Sequences were aligned using Muscle as
enabled in MEGA (Version 6, Tamura et al. 2013). For the COI region, sequences
were translated using the invertebrate mitochondrial code and the resulting protein
alignment checked for stop codons within the sequences. Evolutionary analyses
were conducted in MEGA with 1000 bootstrap replicates. Prior to each analysis, the
appropriate evolutionary model was determined. For the COI gene, the General
Time Reversible model (Nei & Kumar 2000) with a discrete gamma distribution ((6
categories (+G, parameter 0.5542)) and invariable sites was used to model the
evolutionary rate among the sites. For 16S region, the ML tree was obtained using
the Hasegawa-Kishino-Yano model (Hasegawa et al. 1985) with a discrete gamma
distribution (6 categories (+G, parameter 0.4105)). The best evolutionary model was
determined based on the lowest Bayesian Information Criterion (BIC) score
187
determined using MEGA6. For the ITS region, the ML analysis used the Tamura 3parameter model (Tamura 1992) with a discrete gamma distribution (6 categories
(+G, parameter 1.1435)) to model evolutionary rate differences among sites.
6.2.5 Detection of ‘CLas’ in green psyllids and their host plants
6.2.5.1 Development of primers for the wingless gene of the green psyllids
The primers and probe of Manjunath et al. (2008), designed against the wg gene of
Diaphorina citri, failed to amplify this gene from the Cornopsylla and Cacopsylla
species. Therefore, a pair of primers, CaF and CaR, was designed based on the
primers (DCF and DCR) of Manjunath et al. (2008) for Diaphorina citri. A second
set of primers, WGCaF and WGCaR (Figure 6.1), was designed based on the primers
(WGf and WGr) of Li et al. (2008). In both instances, primer design was informed
by an alignment of the sequences of the wg gene of Cacopsylla brunneipennis
(Edwards), Cacopsylla peregrina (Förster), and Cacopsylla pyri (Förster) (Thao et al.
2000). The new primers were checked by conventional PCR using DNA templates
from Cacopsylla heterogena, the undescribed Cacopsylla sp. and Cornopsylla
rotundiconis. The primer pair, WGCaF and WGCaR, which amplified the wg gene
of the Cacopsylla species, was selected for use in real-time PCR. For real-time PCR,
the new primer pair was multiplexed with the primers for ‘CLas’ and with the
TaqMan probes for wg (Li et al. 2008) and for ‘CLas’ (Li et al. 2006). The Taqman
probe for wg was labelled at the 5’ terminal with tetrachlorofluorescein (TET)
reporter dye and the 3’ terminal labelled with Black Hole Quencher (BHQ-1)
(Biosearch technologies). The ‘CLas’ probe was labelled with 6-carboxy-fluorescein
(FAM) and BHQ-1. Primers, WGCaF, and WGCaR, do not amplify the wg gene
from Diaphorina spp.; therefore, when DNA from Diaphorina spp. was amplified,
the primers and probe of Manjunath et al. (2008) were used.
188
Figure 6.1. Sequences of primers and probes used for the amplification of the wingless (wg) gene of
the citrus green psyllids based on the primers (WGf and WGr) designed by Li et al. (2008) and the
sequences from Cacopsylla spp. Primer (WGf & WGr; WGCaF & WGCaR) and probe (WGp)
regions are underlined, and the direction of amplification is indicated using arrows. Bases identical to
Diaphorina citri in the Cacopsylla sequences are represented by dots.
6.2.5.2 Collection of psyllid and plant samples for detection of ‘CLas’ and
detection procedures
The Cacopsylla and Cornopsylla spp. were collected from different altitudes in five
dzongkhags (Table 6.3). Although all three species of green psyllids were observed
in 2013, only samples collected in 2015 and 2016 were used for the detection of
‘CLas’.
Samples of Cornopsylla rotundiconis Lou, Li, Li & Cai (Hemiptera:
Sternorrhyncha: Psyllidae) and the undescribed Cacopsylla sp. were included in this
analysis even though the WGp probe does not bind with DNA from Cornopsylla
rotunidiconis; hence, there is no internal amplification control for this psyllid.
Psyllid DNA was extracted using REDExtract-N-Amp™ Plant PCR Kit (SigmaAldrich) as described in Section 3.2.4 (Chapter 3).
Detection of ‘CLas’ was also performed for host plants collected from locations
where psyllid specimens were collected, especially where the plants appeared
symptomatic. The plant samples included mandarin, sweet orange, lemon, lime and
an unidentified Zanthoxylum sp. Midribs and petioles of leaves were excised and
preserved in 100% ethanol within 1–2 d of collection, transported to Western Sydney
189
University and stored at −20°C until further use. Before DNA extraction, samples
were placed on autoclaved paper towel and air dried for 5 min. Then ~0.20 g
aliquots of tissue were chopped into small pieces (1–2 mm) with disposable blades
and placed in bead beating-tough, 2 mL tubes (MO BIO Laboratories Inc. supplied
by Genworks) each containing a 6.35 mm chrome steel beating bead (Daintree
Scientific). The tubes with the samples and the beating beads were then placed in
liquid nitrogen for 30 sec before placing in a tissue lyser (FastPrep). Leaf tissues
were ground at 4 m/sec for 20 sec. DNA from plant samples were then extracted
using ISOLATE II Plant DNA kit (BIOLINE) following the manufacturer’s
instructions with slight modifications. The pre-lysis incubation was performed at
65°C for 30 min instead of 10 min.
The lysis buffer, PA2, was used for all
extractions.
Procedures for ‘CLas’ detection and result interpretation were performed in the same
manner as described in Section 3.2.5 (Chapter 3) except for the primers and probes
used for amplification. For the psyllid assays, the psyllid sample is deemed to be
‘CLas’ positive if the FAM Ct value is within the range of 0.00 < FAM Ct ≤ 32,
‘CLas’ is not detected in the sample if the FAM Ct is zero, and the result for the
sample is inconclusive if the FAM Ct value is in the range of 32 < FAM Ct ≤ 40. All
assays included non-template (NTC) and positive controls. For the psyllid assays,
samples of Diaphorina citri previously determined to be ‘CLas’-positive were used
as positive controls using the primers and probes for the wg gene designed by
Manjunath et al. (2008) multiplexed with primers and probes for ‘CLas’ designed by
Li et al. (2006).
Similarly for plants, the positive controls comprised samples
previously confirmed as positive.
Additionally, for the plant sample assays,
extraction controls consisting of DNA extracted from apple or peach leaf midribs
were included. For plant assays, the sample is ‘CLas’ positive if the FAM Ct value
is greater than zero but less than or equal to 36. Samples that showed Ct values
greater than zero were further subjected to conventional PCR using primers, A2 & J5
(Hocquellet et al. 1999).
Successful amplifications were sequenced in both
directions at Macrogen using the same primer set.
190
Table 6.3. Locations where Cacopsylla heterogena was collected in 2015 and 2016 for detection of 'CLas'.
Locations*
GPS coordinates
Altitude
Host
(m ASL)
Chhukha
Khuengkha,
Dagana
Menchuna, Drujegang
Patala, Drujegang
Thangna, Drujegang
Punakha
Nobgang
Talo-Walakha Road 1
Talo-Walakha Road 2
Thimphu
Mendrelgang, Thimphu-Punakha Road
Tsirang
Drupchigang
Mendrelgang
RDC, Maenchhana
Riserboo 'A'
Riserboo
Dzamling Zor
26.9335°N, 89.4322°E
1090
mandarin & lime
26.9802°N, 90.0129°E
26.9847°N, 90.0180°E
26.9870°N, 90.0326°E
1493
1496
1164
mandarin
mandarin
mandarin
27.5647°N, 89.8438°E
27.5403°N, 89.8521°E
27.5327°N, 89.8673°E
1985
1791
1419
mandarin
mandarin
mandarin
27.5248° N, 89.8323°E
1604
mandarin
27.0189°N, 90.1275°E
26.9517°N, 90.1041°E
26.9977°N, 90.1250°E
26.9532°N, 90.1347°E
26.9572°N, 89.1380°E
27.0243°N, 90.1198°E
1478
1182
1531
1436
1553
1386
mandarin
mandarin
mandarin, orange, lemon
mandarin
mandarin
mandarin
* Dzongkhag names are emboldened
191
6.3
Results
6.3.1 Morphological descriptions of green psyllids on citrus
A species of green psyllid (Figure 6.2) was recorded on mandarin for the first time in
Bhutan in April 2013 at Riserboo ‘A’, Mendrelgang Gewog, Tsirang Dzongkhag
(26.9532°N, 90.1347°E, 1436 m ASL). The psyllid on the mandarins was identified
as Cacopsylla heterogena Li (Li 1976) by Dr Susan Halbert (Division of Plant
Industry, Florida Department of Agriculture and Consumer Services) and confirmed
by Luo Xinyu (Department of Entomology, China Agricultural University). Later,
psyllids identical to the one on mandarin were observed on lemons, limes, sweet
oranges and wild citrus at altitudes ranging from 983–2444 m ASL (Table 6.2).
6.3.2 Psyllids on Zanthoxylum sp.
Two further species of psyllids were observed on the species of Zanthoxylum (Figure
6.3) around the same time as the green psyllid was found on mandarin. The wings of
the two species on Zanthoxylum appear more orange–yellow compared to the green
psyllid on mandarin. In the field, the sizes of two species on Zanthoxylum appeared
differed: one was observed to be larger than the other, and possessed exceptionally
long antennae. The larger psyllid with long antennae was identified as Cornopsylla
rotundiconis (Luo et al. 2012) (Figure 6.4) and the smaller one is an undescribed
Cacopsylla sp. (Figure 6.5).
192
Figure 6.2. Cacopsylla heterogena: ovipositing female (A); eggs (B); nymphs (C); adult (D).
(Photos: GAC Beattie).
Figure 6.3. Leaves and fruits of the Zanthoxylum sp. from where Cornopsylla rotundiconis and the
undescribed Cacopsylla sp. were collected. (Photos: N. Om).
193
Figure 6.4. Cornopsylla rotundiconis: adult male (A); slender nymph with honeydew (B) (Photos: N.
Om).
Figure 6.5. Unidentified Cacopsylla sp. from Zanthoxylum: young nymph with round and black
abdomen (A); young nymph with round but not black abdomen (B); fifth instar nymph (C); adult
female (D). (Photos: N. Om).
194
6.3.3 Phylogenetic analyses
6.3.3.1 Psyllid sequence analysis
A BLAST search using the COI sequences obtained from Cornopsylla rotundiconis
and the Cacopsylla spp. from the Zanthoxylum sp. showed that they have 84–88%
identity with Cacopsylla fraudatrix Labina & Kuznetsova, Cacopsylla citrisuga,
Cacopsylla myrtilli (Wagner) and Cacopsylla ledi (Flor), while the sequences of
Cacopsylla heterogena have 99% identity with Cacopsylla citrisuga, 85% with
Cacopsylla myrtilli and Cacopsylla ledi and 82–84% with Cacopsylla coccinea
(Kuwayama), Cacopsylla pruni (Scopoli), Cacopsylla picta (Förster), and
Cacopsylla pyricola (Förster). The 16S sequences obtained for the psyllids in the
current study have 90–93% identity with Cacopsylla chinensis (Yang & Li),
Cacopsylla qianli (Yang & Li), Cacopsylla pyrisuga (Förster) and Psylla lanceolata
Yang.
Only a few ITS sequences were obtained from the Bhutanese psyllids on mandarin
and Zanthoxylum. Some of the ITS sequences obtained were found to be ~98%
identical to strains of Cladosporium and Davidiella (Cladosporiaceae: Capnodiales)
but with only ~30% sequence coverage. Attempts at sequencing the ITS region of
psyllids using DNA extracted via a destructive method did not improve the
sequencing results. A BLAST search using data from successful sequencing of the
ITS regions of Cacopsylla heterogena, Cacopsylla citrisuga and the undescribed
Cacopsylla sp. only had similarity to the ITS2 region of Cacopsylla pyricola with
74–76% identity and 50–71% sequence coverage, and ITS2 and 28S of Cacopsylla
fulguralis (Kuwayama) with 73–76% identity and 62–64% sequence coverage. The
ITS sequence from Cornopsyla rotundiconis showed 69–70% identity and 56%
sequence coverage to the ITS2 and 28S region of Cacopsylla pyricola. For the
Cacopsylla citrisuga accessions, one of the extracts made in this study and the DNA
extracted in China produced amplicons for all three regions. For all three regions of
all three psyllid species, the sequences obtained were compared with appropriate
sequences in GenBank (Table 6.4).
195
Table 6.4. List of GenBank accessions of psyllids used for molecular phylogeny.
Species
Genetic regions
COI
Bactericera cockerelli
16S
Beard & Bulman (unpublished)
KF623528
Swisher & Crosslin (2014)
AY971914
Bactericera cockerelli
KM206147
Liu et al. (2006)
Oettl & Schlink (2015)
AB363711
Cacopsylla chinensis
Cacopsylla citrisuga
ITS
KC008074
Bactericera cockerelli
Cacopsylla affinis
References
Lee et al. (2007)
KJ850285
Wang et al. (2015)
Cacopsylla citrisuga
KJ850304
Wang et al. (2015)
Cacopsylla coccinea
KP245955
Que et al. (2016)
Cacopsylla fraudatrix
JX987969
Kuznetsova et al. (2012)
KF305121
Cacopsylla fulguralis
Peccoud et al. (2013)
Cacopsylla ledi
JX987974
Kuznetsova et al. (2012)
Cacopsylla melanoneura
FJ648818
Malagnini et al. (unpublished)
Cacopsylla myrtilli
KR044343
Gwiazdowski et al. (2015)
Cacopsylla myrtilli
JX987981
Kuznetsova et al. (2012).
Cacopsylla myrtilli
KF494331
Nokkala et al. (2013)
Cacopsylla picta
KM206184
Oettl & Schlink (2015)
196
Genetic regions
Species
COI
Cacopsylla pruni
KM206191
Cacopsylla pyricola
JF327670
Cacopsylla pyricola
KP843860
16S
Referemce
ITS
Oettl & Schlink (2015)
JF327721
Kang et al. (2012)
Zohdi & Hossini (unpublished)
Cacopsylla pyrisuga
AB721003
Katoh et al. (2013)
Cacopsylla qianli
AB363717
Lee et al. (2007)
Psylla lanceolata
AB363700
Lee et al. (2007)
197
Table 6.5 shows the sequence analysis for the different genetic regions of the
psyllids. The range of sequence lengths obtained were 447–514, 413–514, and 889–
1079 bp for COI, 16S rDNA and the ITS regions, respectively. Greatest sequence
divergence between the ingroup and the outgroup was obtained for the ITS region
(21.79–37.22%).
Within the Cacopsylla heterogena accessions, the 16S region
showed greater sequence divergence (0.00–1.40) than in the COI (0.00–0.60) and
ITS (0.00–1.00) regions. The sequence divergence between Cacopsylla heterogena
and Cacopsylla citrisuga was higher in the 16S and COI regions than in the ITS
region. For Cacopsylla heterogena, the intraspecific sequence divergence overlaps
that of the interspecific sequence divergence between Cacopsylla heterogena and
Cacopsylla citrisuga for all three genes.
As for Cacopsylla citrisuga, the
intraspecific sequence divergence is lower than the interspecific divergence for the
16S and ITS regions, but overlaps with the interspecific sequence divergence for
COI. For all three genetic regions, the highest sequence divergence was obtained
between Cacopsylla heterogena and Cornopsylla rotundiconis, and the lowest
divergence between Cacopsylla heterogena and Cacopsylla citrisuga. Alignment of
translated sequences of COI region did not show any stop codons within the
sequences. The number of amino acid differences between Cacopsylla heterogena
and Cacopsylla citrisuga for COI was between 0.00–1.00, between Cacopsylla
heterogena and the undescribed Cacopsylla sp. was 2.00–3.00, with nine amino acid
differences occurring between Cornopsylla rotundiconis and Cacopsylla heterogena
(Table 6.5).
198
Table 6.5. Sequence analysis of psyllids.
Analysis
Genetic regions
COI
16S
ITS
Range of sequence length
447–514
413–514
889–1079
Number of variable sites
201
160
426
Number of parsimonious informative sites
163
84
194
18.58–21.94
15.34–19.11
21.79–37.22
Range of sequence divergence within Cacopsylla heterogena (%)
0.00–0.60
0.00–1.40
0.00–1.00
Range of sequence divergence within Cacopsylla citrisuga (%)
0.00–0.66
0.22
0.10
Range of sequence divergence between Cacopsylla heterogena & Cacopsylla citrisuga (%)
0.24–0.88
0.63–1.32
0.20–0.80
Range of sequence divergence between Cacopsylla heterogena &Cacopsylla sp. from Zanthoxylum
(%)
10.74–11.98
5.30–5.70
7.33–10.07
Range of sequence divergence between Cacopsylla heterogena & Cornopsylla rotundiconis (%)
16.50–17.84
9.62–10.83
10.52–12.85
0.00
N/A
N/A
Number of amino acid difference within Cacopsylla citrisuga
0.00–1.00
N/A
N/A
Number of amino acid difference between Cacopsylla heterogena & Cacopsylla citrisuga
0.00–1.00
N/A
N/A
Number of amino acid difference between Cacopsylla heterogena & Cacopsylla sp. from
Zanthoxylum
2.00–3.00
N/A
N/A
9.00
N/A
N/A
Range of sequence divergence between in-group & Bactericera cockerelli (%)
Number of amino acid difference within Cacopsylla heterogena
Number of amino acid difference between Cacopsylla heterogena & Cacopsylla rotundiconis
N/A = no analysis
199
6.3.3.2 Phylogenetic relationships of psyllids derived from the mitochondrial
COI gene
Figure 6.6 shows the phylogenetic relationships of green psyllids based on the COI
gene. The analysis resulted in the following clades.
A clade comprising the Cornopsylla rotundiconis adults and the slender nymphs
(Figure 6.4 & Table 6.1) from the Zanthoxylum sp. (Figure 6.3) that forms a
sister clade to the other clades that comprise the Cacopsylla species.
The Cacopsylla species form the following clades:
o
A weakly supported clade containing Cacopsylla pyricola.
o
A second clade consisting of Cacopsylla affinis (Low), Cacopsylla
melanoneura (Förster), Cacopsylla pruni, Cacopsylla picta, Cacopsylla
coccinea, Cacopsylla fraudatrix, Cacopsylla myrtilli and Cacopsylla ledi.
o
A third clade further separating into three subclades.
The first contains
nymphs that had black (RBA) and non-black abdomens (RNBA) and the
adults of the undescribed Cacopsylla sp. (Figure 6.5 & Table 6.2) collected
from Zanthoxylum. The second subclade comprising the adults and nymphs
of Cacopsylla heterogena (Figure 6.2 & Table 6.2) collected from mandarin
and wild citrus.
The final subclade contains Cacopsylla citrisuga from
Yunnan, China and two GenBank accessions of the same species
corresponding to the haplotypes, C1 and C2, of Wang et al. (2015).
6.3.3.3 Phylogenetic relationships of psyllids derived from the 16S gene
Figure 6.7 shows the phylogenetic relationships of psyllids based on the 16S region.
The followings clades are formed:
A basal clade consisting of Cacopsylla chinensis.
A clade consisting of Psylla lanceolata and Cacopsylla pyrisuga.
A clade comprising accessions Cornopsylla rotundiconis collected from
Zanthoxylum sp. with Cacopsylla qianli as a sister member to the clade.
A clade that formed three subclades with the first consisting of accessions of the
Cacopsylla sp. collected from the Zanthoxylum sp. The other two subclades
were closely related with one consisting of the Cacopsylla heterogena
200
accessions from Bhutan and the other comprising the accessions of Cacopsylla
citrisuga from China.
The nymphs of each species of psyllid grouped within the same clade as that of the
adults.
6.3.3.4 Psyllids phylogeny based on the ITS region
The phylogenetic analysis of the psyllids based on the ITS region is shown in Figure
6.8.
The analysis resulted in a basal clade (A) comprising the accessions of
Cornopsylla rotundiconis and a second clade that further divides into three subclades
(B–D):
B
comprising Cacopsylla fulguralis and Cacopylla pyricola;
C
containing
the
unidentified
Cacopsylla
species
from
the
Zanthoxylum sp. from Bhutan; and
D
a poorly resolved clade containing Cacopsylla citrisuga and
Cacopsylla heterogena.
The analysis was repeated with a reduced data set comprising only the
accessions of Cacopsylla citrisuga and Cacopsylla heterogena (Figure 6.9)
with an accession of the undescribed Cacopsylla sp. used as the outgroup. In
this analysis, the sequences from Cacopsylla citrisuga grouped within the
sequences from Cacopsylla heterogena.
201
Figure 6.6. Phylogenetic analysis based on the COI gene of psyllids derived using maximum
likelihood based on the General Time Reversible model (Nei & Kumar 2000). A discrete gamma
distribution was used to model evolutionary rate differences among sites (6 categories (+G, parameter
= 0.5479)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I],
55.5942% sites). The tree with the highest log likelihood (-3047.7307) is shown. The percentage of
trees in which the associated taxa clustered together is shown next to the branches. Accession
numbers are in parentheses following each taxon.
202
Figure 6.7. Phylogenetic analysis of the 16S rDNA region derived using maximum likelihood based
on the Hasegawa-Kishino-Yano model of evolution (Hasegawa et al. 1985). A discrete gamma
distribution was used to model evolutionary rate differences among sites (6 categories (+G, parameter
0.4105)). The tree with the highest log likelihood (-1859.4981) is shown. The percentage of trees in
which the associated taxa clustered together is shown next to the branches. Accessions obtained from
GenBank are shown with their accession numbers in parentheses.
203
Figure 6.8. Phylogenetic analysis based on the ITS region of psyllids inferred by maximum
likelihood based on the Tamura 3-parameter model (Tamura 1992). A discrete gamma distribution
was used to model evolutionary rate differences among sites (6 categories (+G, parameter 1.1435)).
The tree with the highest log likelihood (-4233.2235) is shown. The percentage of trees in which the
associated taxa clustered together is shown next to the branches. GenBank accession numbers are
given in parentheses after the taxon.
Figure 6.9. Phylogenetic analysis of a reduced data set of the ITS region of the Cacopsylla species
using the unidentified Cacopsylla species as the outgroup. The analysis used ML based on the
Tamura 3-parameter model of evolution (Tamura 1992). The tree with the highest log likelihood (2042.2051) is shown. The percentage of trees in which the associated taxa clustered together is shown
next to the branches.
204
6.3.4 Detection of ‘CLas’ in green psyllids and host plants
6.3.4.1 Primer development
The wingless (wg) gene primers (DCF & DCR) and TaqMan probe designed by
Manjunath et al. (2008) for Diaphorina citri failed to amplify the wg gene of the
green psyllids from mandarin. The new primers, CaF and CaR, based on this region
by aligning the sequences of wg genes of Cacopsylla brunneipennis, Cacopsylla
peregrina, and Cacopsylla pyri (Thao et al. 2000) also failed to produce an amplicon
for Cacopsylla heterogena and Cacopsylla sp. from Zanthoxylum. Another set of
primers (WGCaF & WGCaR) based on different positions within the wingless gene
of Diaphorina citri used by Li et al. (2008) were then designed. This primer set
amplified DNA from both Cacopsylla heterogena and Cacopsylla sp. but not
Cornopsylla rotunidconis by conventional PCR.
Similarly, when used in
conjunction with the TaqMan probe designed by Li et al. (2008), the wg gene was
also amplified by real-time PCR using DNA from the Cacopsylla species but not
from Cornopsylla rotunidconis.
6.3.4.2 Detection of ‘CLas’ in Cacopsylla heterogena and host plants
A total of 66 and 141 psyllid DNA extracts comprising of 262 and 447 psyllids
collected in 2015 and 2016, respectively, were tested for ‘CLas’ (Table 6.6). Of the
extracts made from individuals collected in 2015, only two samples of adults showed
a FAM (the fluorophore for ‘CLas’) Ct value in the range of 0.00 < FAM Ct ≤ 32;
the remainder were HLB negative. For insects collected in 2016, only four samples
of adults had FAM Ct values within the range of 0.00 < FAM Ct ≤ 32. The lowest Ct
values obtained were 32.98 and 30.13 for collections made in 2015 and 2016,
respectively. The Ct range for ‘CLas’ (FAM) positive controls was 19.82–20.30 for
2015 and 17.43–21.34 for the 2016 samples. The Ct range for the internal control of
the psyllid gene (TET) was 20.53–23.91 and 20.66–22.72 for 2015 and 2016
samples, respectively. Among the samples that showed FAM Ct values in the range
of 0.00 < FAM Ct ≤ 32, one sample from a composite of five adults collected in 2016
(FAM Ct = 30.13) was amplified and successfully sequenced with the primers A2/J5.
205
A BLAST search using this sequence showed 99% identity and sequence coverage
with ‘CLas’ isolates from China, India and Iran among others.
Table 6.7 shows the results of PCR tests for ‘CLas’ detection in plant species and
varieties on which Cacopsylla heterogena was collected. The host plants tested
included mandarin, sweet orange, lemon and lime.
Seventeen of the 24 plant
samples tested showed FAM Ct values in the range of 0.00 < FAM Ct ≤ 36, i.e., they
were HLB positive. The FAM Ct range for the 17 samples was 17.45–35.06. The
lowest FAM Ct (17.45) was obtained from a mandarin sample collected at 933 m
ASL in the Samtse Dzongkhag. The highest FAM Ct value was obtained from one
of samples collected from Talo at 2444 m ASL, and is the only sample that showed a
FAM Ct value above 36. The positive controls showed a FAM Ct range of 18.89–
21.55 while all extraction controls had FAM Ct values equal to zero. The CAL Ct
value (plant internal control) ranged between 19.70−32.76.
6.2.1.1 Detection of ‘CLas’ in Zanthoxylum sp. and its psyllids
A total of 30 extracts made from either single individuals or composites (5
individuals) of the undescribed Cacopsylla sp. and 20 extracts from either 1 or 5
adults were used.
Extracts of nymphs of Cornopsylla rotundiconis were also
included that consisted of composites of five nymphs per extract. Results of samples
obtained from a Zanthoxylum sp. that exhibited blotchy mottled symptoms and its
psyllids tested for ‘CLas’ are shown in Table 6.8. The Zanthoxylum sp. samples
show a FAM Ct range of 20.98–23.18 with a corresponding CAL Ct range of 16.76–
21.83. These same samples were subjected to cPCR similar to other samples with
FAM Ct ≤ 36 but did not produce an amplicon. All samples of the undescribed
Cacopsylla sp. and Cornopsylla rotundiconis adults and nymphs resulted in FAM Ct
values equal to zero. The wg primer and probe sets designed for Cacopsylla species
do not amplify the DNA of Cornopsylla rotundiconis; hence, internal control Ct
values are not available.
206
Table 6.6. Screening of Cacopsylla heterogena collected in 2015 and 2016 in Bhutan for the
presence of ‘CLas’ by real-time PCR.
Parameters
Year of collection
2015
2016
Total number of nymphs included in the test1
123
262
Total number of adults included in the test2
139
185
Total number of psyllids tested
262
447
3
32.98–38.47
30.13–39.84*
0
0
2†
4*
19.82–20.30
17.43–21.34
Range of TET Ct for all samples
20.22–26.25
20.16–34.48**
Range of TET5 Ct for positive control4
20.53–23.91
20.66–22.72
0
1
Range of FAM Ct > 0
Number of nymph samples with FAM3 Ct values in the
range of 0.00 < FAM Ct ≤ 32
3
Number of adult samples with FAM Ct values in the
range of 0.00 < FAM Ct ≤ 32
Range of FAM3 Ct for positive control4
5
Confirmation through sequencing6
1
21 extractions made with five nymphs per extractions, two extractions made with 6 and 12 nymphs
per extraction, and 7 extracts with three nymphs in 2015. For 2016, five nymphs per extraction were
used. All nymphs were mostly 4–5th instars;
2
Eighteen extracts with single adults per extraction; 28 extracts with 5 psyllids per extraction in
2015. In 2016, forty five extracts with 5 adults per extract & 55 extracts with single adults were
made;
3
'CLas' DNA ;
4
Range taken from different test runs;
5
Psyllid DNA (internal control);
6
Samples with FAM Ct values in the range of 30 ≤ FAM Ct ≤ 36 were subjected to conventional PCR
with A2/J5 primers (Hocquellet et al. 1997) and successful amplifications sequenced;
† Ct values obtained for extracts made from 5 adults per extraction;
* Samples from Talo-Walakha Road 2 (Table 6.3);
** 1 nymph sample (composite of five 4-5th instar nymphs), from RDC Maenchhana, Tsirang, with a
‘CLas’ Ct value of 34.48.
207
Table 6.7. Detection of 'CLas' in host plants of Cacopsylla heterogena.
Locations1, 2
GPS coordinates
Altitude
Host plant3
Ct value range
(m ASL)
FAM
CAL
Chhukha
Khuengkha
26.9335°N, 89.4322°E
1090
mandarin (1)
33.53
27.74
26.9335°N, 89.4322°E
1090
lime (1)
22.01
26.16
Menchhuna, Drujegang
26.9802°N, 90.0129°E
1493
mandarin (2)
32.37–33.87
22.72–23.34
Patala, Drujegang
26.9847°N, 90.0180°E
1496
mandarin (2)
0.00–35.06
24.72–28.48
below Pangna, Drujegang
26.9748°N, 90.0550°E
983
mandarin (1)
20.26
26.18
27.5516°N, 89.8238°E
2444
mandarin (5)4
31.56–37.87
21.31–32.76
27.5327°N, 89.8673°E
1419
mandarin (5)
4
17.54–23.96
20.50–24.26
26.9032°N, 89.2911°E
933
mandarin (1)
17.45
23.04
RDC, Maenchhana
26.9977°N, 90.1250°E
1531
lemon (5)5
0.00–25.10
23.32–26.33
RDC, Maenchhana
26.9977°N, 90.1250°E
1531
sweet orange (1)
0.00
23.66
Positive controls
18.89–21.55
19.70–22.72
Extraction controls (peach)
0.00
24.84–27.11
Dagana
Punakha
Talo
Talo-Walakha Road 2
Samste
Tsirang
1
District names are emboldened: 2 Cacopsylla heterogena nymphs/adults from Pangna, Talo and Samste were not evaluated for ‘CLas’: they were too few or, for
nymphs, too small (first and second instars). Adults from Talo-Walakha Road 2 were ‘CLas’ +ve. Nymphs/adults from the other locations were ‘CLas’ –ve;
3
Number of samples (petioles or midribs) tested are given in parentheses; 4 All samples showed Ct values > 0.00; 5 One out of five samples showed a Ct value > 0.00
208
Table 6.8. Detection of 'CLas' in Zanthoxylum sp., Cacopsylla sp. and Cornopsylla rotundiconis.
Sample type1
Ct value
FAM
CAL
20.98 & 23.18
16.76 & 21.83
25.77
21.49
Plant
1.
Zanthoxylum sp.
Positive control
Psyllids
Number of extracts based on:
single adults
5 adults
5 nymphs
Ct value
FAM
TET2
15
15
0
0.00
N/A
11
9
5
0.00
N/A
Positive control3
21.86
N/A
Positive control4
30.19
22.32
2.
Cacopsylla sp.
3.
Cornopsylla rotundiconis
1
Zanthoxylum mid-rib samples were collected from Dangreyboog, Dunglagang Gewog, Tsirang in July 2014, and psyllid samples were collected
in May 2014 and July 2015;
2
WGCaF and WGCaR primers with WGp TaqMan probe of Li et al. (2008);
3
Diaphorina citri sequenced extract;
4
Cacopsylla heterogena extract that was sequenced (Table 6.7).
N/A = not applicable
209
6.4
Discussion
6.4.1 Phylogeny of psyllids
The mitochondrial COI and the 16S rDNA genes and the ITS region of the nuclear
DNA were used to determine the phylogenetic relationships of species of green
psyllids recorded on Rutaceae in Bhutan. In the molecular phylogeny of closely
related individuals, it is important to have a lower sequence divergence within
species compared to that between species to delimit the species (Meyer & Paulay
2005; Roe & Sperling 2007), and many studies have shown this (Tang et al. 1996).
In the current study, pairwise sequence divergence comparisons (Table 6.5) show
that the sequence divergence in the 16S and ITS regions within Cacopsylla citrisuga
is lower than the sequence divergences between Cacopsylla heterogena and
Cacopsylla citrisuga.
However, for the COI gene, the intra-specific sequence
divergence overlaps that of the inter-specific divergence between Cacopsylla
citrisuga and Cacopsylla heterogena.
Similarly, the intra-specific sequence
divergences of Cacopsylla heterogena overlap the range of inter-specific sequence
divergences between Cacopsylla citrisuga and Cacopsylla heterogena in all three of
the genetic regions examined. Intra-specific variation is said to vary from species to
species ranging from no variation in some species to significant variation in others
(Tang et al. 1996). The overlap of intra- and interspecific sequence divergence has
been observed in the ITS region of the species complex of the black fly Simulium
damnosum Theobold (Diptera: Simuliidae) (Tang et al. 1996), in mitochondrial and
nuclear genes in the families of Coleoptera, Diptera, Hemiptera, Hymenotera,
Isoptera, Lepidotera and Phthiraptera (Cognato 2006), in the COI gene of the
whitefly Bemisia tabaci (Gennadius) (Hemiptera: Sternorrhyncha: Aleyrodidae)
(Boykin et al. 2007) and in the COI-COII region of 23 species of Lepidoptera and
Diptera (Roe & Sperling 2007). Therefore, such an observation in the current data is
not unusual.
The results of the phylogenetic analyses show that the green psyllids collected from
different locations in Bhutan (Table 6.2) could be placed in one of three taxa. In the
analyses of all three genetic regions, Cornopsylla rotundiconis from the species of
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Zanthoxylum always formed a clade that was sister to the clades containing the
undescribed Cacopsylla sp. also from Zanthoxylum and Cacopsylla heterogena
found on Citrus spp. In addition, all Bhutanese accessions of Cacopsylla grouped
separately from all other Cacopsylla spp. whose sequence data have been placed in
GenBank. Analyses of the two mitochondrial genes seem to indicate that Cacopsylla
citrisuga and Cacopsylla heterogena are separate species, as separate clades
containing these two species were obtained. However, in the analysis of the ITS
region (Figures 6.8 & 6.9), Cacopsylla citrisuga grouped together with Cacopsylla
heterogena within one clade. Hence, it is possible that these two taxa represent
closely related species. However, both the genetic distances and the phylogenetic
analysis of the ITS region call into question the separation of these two species.
The incongruence in the analyses of the mitochondrial and the nuclear genes may be
due to at least two possibilities. Firstly, it is possible that Cacopsylla citrisuga and
Cacopsylla heterogena may either belong to one species or to a species complex. A
species complex is a group of ‘closely related isolates whose individual members
may represent more than one species’ (Fegan & Prior 2005). Species complexes are
known to exist in several insect species including true fruit fly (Diptera: Tephritidae)
species such as Bactrocera dorsalis (Hendel) (Augustinos et al. 2014). Augustinos
et al. (2014) raised uncertainties about the actual number of species recognised in
this species complex. Similarly, Schutze et al. (2015) argued that three Bactrocera
species namely, Bactrocera dorsalis, Bactrocera invadens Drew, Tsuruta & White
and Bactrocera papayae Drew & Hancock are the same species. Species complex
problems are reported in the hermit beetles wherein the species, Osmoderma eremita
(Scopoli) (Cleoptera: Scarabaeidae), includes at least two or more species (Audisio et
al. 2009). Both Cacopsylla citrisuga and Cacopsylla heterogena are described from
Yunnan Province in China. Cacopsylla citrisuga (syn. Psylla citrisuga) was first
recorded in 1981 from Baoshan (25.1138°N, 99.1618°E, 1680 m ASL), Tengchong
(25.0219°N, 98.4911°E, 1650 m ASL) and Ruili (24.0138°N, 97.8519°E, 750 m
ASL) in western Yunnan (Yang & Li 1984). Cacopsylla heterogena was recorded in
1990 and described in 2011 from Wulongxiang (24.6119°N, 104.2921°E, 952 m
ASL) in eastern Yunnan (Li 2011). Cacopsylla citrisuga is reported on pumelo
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(cited as Citrus grandis) and citron (Citrus medica) (Yang & Li 1984), while Li
(2011) found Cacopsylla heterogena on the same host plants (cited as ‘Citrus sp.
(Rutaceae). In Bhutan, Cacopsylla heterogena was first observed in 2013 at 1260 m
ASL and, since then, has been observed at altitudes ranging from 983–2444 m ASL
(Table 6.2) in five dzongkhags. The other dzongkhags have not been surveyed yet,
but it will not be surprising if this insect has a wider distribution. Cacopsylla
heterogena in Bhutan is found on mandarin, lemon, lime, and sweet orange (pers.
observation), and wild citrus (Beattie, pers. comm.), nominally a hybrid of Citrus
cavaleriei (see Chapter 7). Hence, both in Bhutan and Yunnan, the two species are
found in the same regions and on the same range of host plants. In addition to
occurring in similar locations, according to Li (2011) Cacopsylla heterogena is also
morphologically similar to Cacopsylla citrisuga except that Cacopsylla heterogena
has shorter antennae.
Molecular study of Cacopsylla citrisuga is limited to that of Wang et al. (2015) who
reported the existence of two haplotypes of this species in Yunnan, one from Ruili in
the west and the other from near Shiping (23.7073°N, 102.4944°E, 1444 m ASL) to
the east. The two haplotypes are separated with moderate bootstrap support (63%) in
an analysis of the COI region (Wang et al. 2015). Data from both haplotypes are
shown in Figure 6.6 of my study, and show that there is at least a third haplotype in
Yunnan. Hence, there is genetic variation within the COI gene from Cacopsylla
citrisuga, and it is possible that the variation found in Cacopsylla heterogena forms
part of this variation. As for Cacopsylla heterogena, there is no other record of
molecular work, and my study is the first study to determine its relationship with
Cacopsylla citrisuga and other psyllid species.
A second explanation for the observed difference in the present data could be
introgressive hybridisation. Introgression through hybridisation is defined as the
introduction of genes from one species into another resulting in hybrid offspring that
interbreed with one of the parental species (Baum & Smith 2013). Introgression of
genes (either in mtDNA or nuclear genome) has been observed in many animal
species ranging from salamanders (Amphibia: Salamandridae) (Canestrelli et al.
212
2014) and cichlid fishes (Perciformes: Cichlidae) (Nevado et al. 2011) to Drosophila
species (Diptera: Drosophilidae) (Herrig et al. 2014) and mosquitoes (Diptera:
Culicidae) (Crawford et al. 2015).
Therefore, it is possible that if Cacopsylla
citrisuga and Cacopsylla heterogena are, indeed, different species, the incongruence
in the analysis of the nuclear and mitochondrial genes could be due to hybridisation
between the species.
As mentioned earlier, both Cacopsylla heterogena and
Cacopsylla citrisuga occur in Yunnan. Therefore, it is possible that the place where
the Cacopsylla citrisuga accessions used in the current study were obtained come
from a contact zone where hybridisation between the two species could occur.
Genes of Cacopsylla heterogena could have introgressed into Cacopsylla citrisuga
leading to individuals with shared similarity in their nuclear genomes but with
differing mitochondrial genes.
Mixed populations of different psyllid species are known. Cacopsylla citricola often
occurs as mixed populations with Cacopsylla citrisuga on pumelo and citron in
China (Yang & Li 1984). This apparently occurs despite competition for the same
ecological niche for oviposition and development of nymphs.
Hence, mixed
populations of Cacopsylla heterogena and Cacopsylla citrisuga could also occur.
Therefore, based on the results of the current study, the status of whether Cacopsylla
heterogena and Cacopyslla citrisuga are the same species, part of a species complex
or different species cannot be established. There is limited sequence data available
for Cacopsylla citrisuga and no sequences of the 16S and ITS regions of Cacopsylla
citrisuga are available in GenBank. Further morphological work combined with
molecular analyses using additional mitochondrial and nuclear markers and including
samples of both Cacopsylla heterogena and Cacopsylla citrisuga from China and
elsewhere are needed to compare with the Bhutanese accessions of Cacopsylla
heterogena to elucidate the relationship between the two psyllids.
213
6.4.2 Detection of ‘CLas’ in Cacopsylla heterogena and host plants
A total of 207 psyllid DNA samples sourced from 709 psyllids collected in 2015–
2016 were tested for the presence of ‘CLas’. None of the DNA samples from 123
nymphs and 139 adults collected in 2015 showed FAM Ct values in the range of 0.00
< FAM Ct ≤ 32. Thus, ‘CLas’ was not detected in these psyllids. In 2016, the
pathogen was not in DNA samples from 262 nymphs but four samples out of 185
adults produced FAM Ct values in the range of 0.00 < FAM Ct ≤ 32. One of these
samples, with a FAM Ct value of 30.13, was able to produce an amplicon by cPCR
using the primers, A2 and J5 (Hocquellet et al. 1999).
This amplicon was
successfully sequenced and the data matched that from ‘CLas’ isolates from Bhutan,
China, India and Iran with 99% identity and sequence coverage. The DNA was from
five Cacopsylla heterogena adults collected on a seedling mandarin tree at 1419 m
ASL on the Talo-Walakha Road 2 (Table 6.2) in the Punakha Dzongkhag. These
results indicate that Cacopsylla heterogena nymphs cannot acquire ‘CLas’ but
Cacopsylla heterogena adults may occasionally acquire the pathogen under some
field conditions.
Of the 24 plant samples tested, 20 showed FAM Ct values in the range of 0.00 <
FAM Ct ≤ 36. The lowest values were obtained from mandarin collected from a
location at 933 m ASL (Table 6.6), and the extracts from leaf midribs and petioles
produced FAM Ct values ranging from 17.54 to 23.96 (Table 6.6). These positive
samples were from trees (Table 6.2) representative of mandarin trees at all of the
locations from which Cacopsylla heterogena was recorded and collected during the
study. This suggests that ‘CLas’ may be present in most mature mandarin trees
irrespective of their elevation above sea level. However, titres of the pathogen may
be low, as trees with symptoms of the disease are uncommon above 1200 m.
Nevertheless, my results suggest very low acquisition rates of ‘CLas’ by Cacopsylla
heterogena, zero for nymphs and negligible for adults (~2%). This suggests that
Cacopsylla heterogena may not transmit ‘CLas’.
In contrast to my results for
Cacopsylla heterogena, Cen et al. (2012a) detected ‘CLas’ in 41% of the late instar
nymphs of Cacopsylla citrisuga using nested PCR. The trees where nymphs were
214
collected were also ‘CLas’ positive (Cen et al. 2012a). In 1992 in India, less than 1%
of the Diaphorina citri tested were positive for ‘CLas’ (Bové et al. 1993); however,
this may be a reflection of the assay method used. More recently, 8.6% and 9.7% of
field-collected Diaphorina citri tested were positive for ‘CLas’ (Manjunath et al.
2008; Halbert et al. 2012). However, infection rates for this insect are usually higher
with 38–100% of psyllids being HLB-positive in surveys in Florida (Coy & Stelinski
2015). In addition, ‘CLas’ was detected in about 40–75% of a psyllid population
confined to infected plants for five to 12 weeks under laboratory conditions (Hung et
al. 2004; Pelz-Stelinski et al. 2010). The figures given above for Diaphorina citri are
based on much larger sample sizes, and titres of ‘CLas’ in mandarin trees in relation
to titres within Cacopsylla heterogena needs to be examined more thoroughly. Razi
et al. (2014) assayed single individuals of Diaphorina citri in Pakistan and found
substantial seasonal variation in HLB-positive psyllids with infection rates varying
from 0–25% in different months. Therefore, in addition to larger sample sizes,
seasonal effects need to be taken into account in future surveys. Further studies are
needed to determine whether Cacopsylla heterogena can transmit ‘CLas’, although
my results indicate that the psyllid does not play a major role in the spread of HLB in
Bhutan.
6.4.3 Detection of ‘CLas’ in Zanthoxylum sp., Cacopsylla sp. and
Cornopsylla rotundiconis
The Zanthoxylum sp. from which most of the samples of the undescribed Cacopsylla
sp. and Cornopsylla rotundiconis were collected exhibited strong mottling symptom
on the leaflets of one branch. The two samples of Zanthoxylum sp. examined did not
produce any amplicon when subjected to conventional PCR although the FAM Ct
values were lower than the ‘CLas’ positive control (Table 6.8). This suggests that
the FAM Ct value obtained may be a false positive.
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6.5
Highlights of the study:
Three species of green psyllids occur in Bhutan, one on citrus and two on
Zanthoxylum sp.
The green psyllids occurring on mandarin, wild citrus (Chapter 7), lemons,
limes and oranges in Bhutan belong to one species, Cacopsylla heterogena.
The two psyllids collected from the Zanthoxylum sp. are confirmed as
Cornopsylla rotundiconis, and the other is an undescribed species of
Cacopsylla.
The taxonomic separation of the Cacopsylla citrisuga accessions from
Yunnan, China and Cacopsylla heterogena from Bhutan needs to be
confirmed to determine whether they are the same or distinct species.
Cacopsylla heterogena can acquire ‘CLas’ under field conditions.
There was no evidence of ‘CLas’ in Cornopsylla rotundiconis and Cacopsylla
sp., the green psyllid species recorded on Zanthoxylum sp.
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Chapter 7:
Molecular and morphological
characterisation of Murraya species and wild citrus
taxa from Bhutan and their relevance to
huanglongbing
____________________________________________________________________
7.1
Introduction
As described in the comprehensive list given in Chapter 1 of this thesis, a range of
rutaceous plants has been reported to be hosts of ‘Candidatus Liberbacter asiaticus’
(‘CLas’). All Citrus species and cultivars are regarded as being susceptible to the
pathogen although the severity of infections and manifestations of symptoms vary
(Halbert & Manjunath 2004; Gottwald et al. 2007; Beattie & Barkley 2009;
Ramadugu et al. 2016). Among citrus relatives, species of Murraya (sensu stricto)
and curry leaf, Bergera koenigii (L.), are of particular concern as potential alternative
hosts of the bacterium. Murraya paniculata (syn. Murraya exotica sensu Mabberley
2016) is a common and widely grown ornamental in temperate, subtropical and
tropical regions of the world (the taxonomy of Murraya species is discussed below
and in the discussion), and curry leaf occurs naturally in South Asia and is commonly
cultivated in Asia and elsewhere for the culinary use of its leaves. Both species may
be found in proximity to citrus and are hosts of Diaphorina citri, the principal, and
most widely distributed, vector of ‘CLas’ (Aubert 2009; Hall et al. 2013). Early
studies of Diaphorina citri reported the psyllid on Bergera koenigii (cited as
Murraya koenigii) and Citrus species and hybrids in India (Lal 1917; 1918; Fletcher
1917; 1919; Husain & Nath 1924, 1927; Mathur 1975) and on Citrus (Kuwayama
1908) and Murraya paniculata in Taiwan (Maki 1915; Kuwayama 1931). Husain &
Nath (1927) were the first to describe the serious impact of the psyllid on citrus
production. They attributed impacts of the psyllid (small dry, insipid fruit, twig and
branch dieback, and death of trees) to ‘some poison’ injected by the psyllid during
feeding. The symptoms they described are now regarded as the first description of
symptoms of huanglongbing (Beattie et al. 2009). The black psyllid, Diaphorina
communis, has commonly been reported on Bergera koenigii and also on Murraya
paniculata (Mathur 1975). Past studies have demonstrated that Murraya paniculata
217
can harbour ‘CLas’ and serve as an inoculum reservoir (Deng et al. 2007; Zhou et al.
2007; Lopes et al. 2010; Walter et al. 2012b; Ramadugu et al. 2016). Zhou et al.
(2007) detected ‘CLas’ in Murraya paniculata transmitted through dodder (Cuscuta
pentagona Engelm. (Solanales: Convolvulaceae), while Deng et al. (2007), Walter
(2012a; 2012b), and Ramadugu et al. (2016) detected low-titres ‘CLas’ in Murraya
paniculata as a result of field transmission by Diaphorina citri. Lopes et al. (2010)
detected ‘CLas’ and ‘Candidatus Liberibacter americanus’ (‘CLam’) in Murraya
paniculata growing in urban areas (Lopes et al. 2010). Walter et al. (2012a & b)
hypothesised that low titres of ‘CLas’ in Murraya paniculata and in Diaphorina citri
reared from Murraya paniculata could be due to a resistance trait in Murraya
paniculata against the bacterium. Ramadugu et al. (2016) also considered Murraya
paniculata to be resistant to ‘CLas’ with infections not persisting. Thus, infections in
Murraya paniculata are transient.
The systematics of Murraya exotica and Murraya paniculata has been debated since
Linnaeus with the plants being described as Chalcas paniculata in 1767 and
Murraya exotica in 1771 (Linnaeus 1767; 1771).
They have been regarded as
separate species by some authors (e.g., Stone 1985; Jones 1995; Huang 1997) and as
synonyms by others (e.g., Tanaka 1929; Swingle & Reece 1967; Mabberley 1998).
Recent morphological and molecular evidence (Nguyen 2011) suggests that plants
classified as Murraya paniculata by Swingle & Reece (1967) belong to four distinct
species: Murraya paniculata (syn. Murraya exotica), the widely cultivated form
known as orange jasmine, orange jessamine and mock orange; Murraya elongata A.
DC. ex Hook. f. (Hooker 1875), a taxon found on mainland Asia in India, across
Indochina to the southern provinces of China, and on the Andaman Islands and the
island of Langkawi in the Indian Ocean; Murraya sumatrana Roxburgh (Roxburgh
1832), with a west Malesian distribution; and Murraya heptaphylla Spanoghe
(Spanoghe 1841) found in eastern Malesia and Australasia.
Another species,
Murraya gleniei Thwaites ex Oliv. (Oliver 1861), occurs in Sri Lanka (Swingle &
Reece 1967; Mabberley 2016) and one other, Murraya alata Drake, with winged
rachises occurs in southern China (Guangdong, Guangxi and Hainan) and Indochina
(Drake 1892; Swingle & Reece 1967; Zhang et al. 2008). Nguyen (2011) included
218
six Murraya accessions from Brazil, three from California and two from Florida in
his study. All of these accessions were Murraya paniculata (orange jasmine) which,
despite studies reported by Damsteegt et al. (2010) and Lee et al. (2011) appears to
be the only species of Murraya in the Americas aside from a University of
California, Riverside accession of ‘Murraya ovatifoliolata var. ovatifoliolata’ from
Woongarra (24.9016°S, 152.4232°E, 20 m ASL), Queensland, Australia
In the early 1990s, Grierson (1991) recorded Murraya paniculata and Bergera
koenigii (as Murraya koenigii) in the southern and eastern regions of Bhutan; no
other records of Murraya species in Bhutan exist. However, in April 2014, during
the course of this study, plants resembling Murraya elongata, a species described
from Myanmar, were found in a citrus orchard at Baychhu (27.2975°N, 89.9669°E,
784 m ASL) and on the roadside of the Rinchending–Pasakha Highway at Reldri
(26.8400°N, 89.4045°E, 400 m ASL) in Chukhha Dzongkhag.
Adults of both Diaphorina citri and Diaphorina communis were observed on the
Murraya plants in Baychhu. In April 2015, eggs and young nymphs of one or both
psyllid species were observed on the plants in Baychhu. Sleeve cages were placed
over the infested branches. All adult psyllids that emerged from nymphs after about
21 d were Diaphorina citri males and females. This represents the first record of
Diaphorina citri on a species of Murraya native to the region where the psyllid
evolved. Murraya paniculata (orange jasmine), on which Diaphorina citri was
recorded in India in 1975 (Cheema & Kapur 1975), was introduced to the Indian
Subcontinent from China before the 1800s (Edwards & Lindley 1819–1820;
Roxburgh 1832).
In October 2015, a wild citrus taxon growing near Wengkhar (27.2515°N,
91.2597°E, 2018 m ASL) in the Mongar Dzongkhag was observed to be infested
with what was assumed to be nymphs of Cacopsylla heterogena (Beattie, pers.
comm.) (Figure 7.1). Leaf damage resembling that caused by Cacopsylla heterogena
on mandarins in Tsirang was also evident (Figure 7.1). Wild plants identical to that
at Wengkhar were also observed in Basochhu in April 2016. The plants at Basochhu
219
had foliar damage caused by Cacopsylla heterogena (Beattie, pers. comm.) (Figure
7.2). Morphologically, the foliage and the fruit of the wild citrus appeared to be
similar to Ichang papeda (Citrus cavaleriei H. Léveillé ex Cavalerie syn. Citrus
ichangensis Swingle), but its foliage can be confused with foliage of Citrus latipes
(Swingle) Tanaka as both species have large petioles. Swingle and Reece (1967)
described the leaves of ‘Citrus ichangensis’ as narrow, 4–6 times longer than wide
with large, broadly-winged petioles and those of Citrus latipes as ‘leaf blades more
variable in size and shape and with the tips subacute or even bluntly rounded, not
apiculate or subcaudate with blunt points as in Citrus ichangensis’.
Striking
differences between ‘Citrus ichangensis’ and Citrus latipes are observed in the
flowers and the fruits. The flowers are borne singly in the leaf axils in ‘Citrus
ichangensis’ while in Citrus latipes, flowers are borne in ‘small axillary racemes
with 5–7 flowers’. The fruit peel of Citrus latipes is much thicker (5–6 mm) than
that of ‘Citrus ichangensis’ (2–4 mm) (Swingle & Reece 1967). Grierson (1991)
recorded Citrus latipes being present in Trongsa Dzongkhag in Bhutan.
They
described the leaves as ovate, 40–70 × 15–25 mm, acuminate, base rounded, margin
minutely crenate-serrate, glabrous; petiole obovate, often larger than the lamina, 35–
70 × 20–30 mm, the flowers as solitary or in short racemes, 4-merous, and the fruit
as subglobose, ca. 50 mm diameter, peel somewhat thick, segments ca. 9, seeds
numerous. Penjor et al. (2014) reported that a wild citrus accession from Mongar
was identical to Citrus latipes based on the matK gene but referred to the accession
as an ‘Ichang relative’ based on the similar morphological characters between the
wild citrus and ‘Citrus ichangensis’. In Bhutan, no information on the Murraya
plants mentioned above existed at the start of this study other than the records by
Grierson (1991). Grierson recorded one species of Murraya (sensu stricto) referring
to it as Murraya paniculata (syn. Murraya exotica sensu Mabberley (2016)) and
describing it as an evergreen shrub or tree to 6 m, leaves 120–150 mm, 3–7-foliolate;
leaflets ovate, 50–100 × 20–50 mm, bluntly acuminate, base ± asymmetrically
cuneate, margin entire, glabrous, inflorescence up to 20-flowered, berry ovate, ca. 8
× 5 mm.
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Figure 7.1 Nymph of Cacopsylla heterogena on wild citrus at Wengkhar (top); leaf distortion caused
by Cacopsylla heterogena on wild citrus at Wengkhar (bottom) (Photos: GAC Beattie).
221
Figure 7.2. Foliage of wild citrus at Basochhu (Photos: GAC Beattie).
222
In order to confirm the presence of Murraya elongata in Bhutan and to resolve the
identity of the wild citrus plant, the first part of this chapter focuses on the molecular
and morphological identification of the Murraya and wild citrus taxa found in
Bhutan. Moreover, as outlined above, as Murraya paniculata is a transient host of
‘CLas’ and the host status of the other putative species of Murraya or wild citrus is
unknown, the latter part of this chapter focuses on detection of ‘CLas’ in these
accessions.
Many molecular markers have been developed for phylogenetic studies.
More
commonly, the chloroplast genome (cpDNA) (Clegg & Zurawski 1992; Morton et al.
2003; Shaw et al. 2005; Bayer et al. 2009; Lu et al. 2011; Carbonell-Caballero et al.
2015) and the nuclear ITS regions (Sang et al. 1995; Kyndt et al. 2010; Li et al.
2010; Kumar et al. 2013; Yamaji et al. 2013; Hynniewta et al. 2014; Sun et al. 2015)
of plants have been used. For the current study, five and four cpDNA markers for
Murraya and the wild citrus, respectively, together with the nuclear ITS region were
used to investigate the phylogenetic relationships among Murraya accessions and
wild citrus accessions.
The specific objectives of this chapter were to:
determine the phylogenetic relationships of species of Murraya occurring in
Bhutan with those proposed by Nguyen (2011), and assess their
morphological differences;
determine the phylogenetic relationships of the wild citrus species; and
test the Murraya species and wild citrus for ‘CLas’.
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7.2
Materials and methods
7.2.1 Collection of Murraya accessions
The plant samples used in the current study included nine accessions of Murraya
from Bhutan and six from Yingde, Guangdong, China. The samples of Murraya
from Bhutan were collected from: (1) a citrus orchard at Baychhu, Wangdue
Phodrang Dzongkhag; (2) from the roadside of the Rinchending–Pasakha Highway
at Reldri near Phuentsholing, Chukhha Dzongkhag; and (3) from Zangtopelri Park in
Phuentsholing, Chukhha Dzongkhag. GPS coordinates of the localities are given in
Table 7.1. Voucher specimens of all plants were collected, pressed and mounted on
herbarium sheets. Fresh leaflets of plants corresponding to the pressed specimens
were used for DNA extractions. DNA extractions were performed within 3−5 d of
collection. Specimens from Yingde, China, were obtained as DNA for molecular
analysis (Table 7.1).
7.2.1 Collection of the wild citrus
Young leaves of the wild citrus species were collected for molecular studies from
near Wengkhar, Mongar Dzongkhag in October 2015 by George Beattie, and then in
April 2016 from Korilla also in the Mongar Dzongkhag by Tshering Penjor of the
Research and Development Centre (RDC), Wengkhar.
Leaves of seedlings
cultivated from wild plants at Basochhu were obtained from RDC Maenchhana,
Tsirang, in April 2016. Leaf samples were collected and preserved in 100% ethanol
until being used for DNA extraction. GPS coordinates of the localities are given in
Table 7.1.
7.2.2 DNA extractions from Murraya
DNA extractions from plants were made at the Plant Pathology Laboratory of the
National Plant Protection Centre (NPPC) in Bhutan. Extraction methods similar to
Section 3.2.3 of Chapter 3 were used except whole leaf samples were used for the
extraction of plant genomic DNA. For detection of ‘CLas’ from leaf samples, only
mid ribs were used by excising the mid ribs with disposable razor blades.
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Table 7.1. A list of Murraya samples collected from Bhutan and China and citrus samples from Bhutan used for molecular analysis in the current study.
Abbreviations used: Bhutan (BT); Murraya (M); Phuentsholing (P/ling); RDC (Research & Development Centre); Wangdue (Wangdue Phodrang).
Sample name
Location
GPS coordinates
Elevation
(m ASL)
Species (based on the results of this
study)
MBT.1
Baychhu, Wangdue, BT
27.2975ºN, 89.9669ºE
784
Murraya elongata
MBT.2
Baychhu, Wangdue, BT
27.2975ºN, 89.9669ºE
784
Murraya elongata
MBT.3
Baychhu, Wangdue, BT
27.2975ºN, 89.9669ºE
784
Murraya elongata
MBT.4
Baychhu, Wangdue, BT
27.2975ºN, 89.9669ºE
784
Murraya elongata
MBT.5
Baychhu, Wangdue, BT
27.2975ºN, 89.9669ºE
784
Murraya elongata
MBT.6
Reldri, P/ling, BT
26.8400ºN, 89.4045ºE
400
Murraya elongata
MBT.7
Reldri, P/ling, BT
26.8400ºN, 89.4045ºE
400
Murraya elongata
MBT.8
Reldri, P/ling, BT
26.8400ºN, 89.4045ºE
400
Murraya elongata
MBT.9
Zangtopelri Park, P/ling, BT
26.8619ºN, 89.3827ºE
200
Murraya paniculata
MCN.1
Yingde, China
24.2922°N, 113.3452°E
62
Murraya elongata
MCN.2
Yingde, China
24.2922°N, 113.3452°E
62
Murraya elongata
MCN.3
Yingde, China
24.2922°N, 113.3452°E
62
Murraya elongata
MCN.4
Yingde, China
24.2922°N, 113.3452°E
62
Murraya elongata
MCN.5
Yingde, China
24.2922°N, 113.3452°E
62
Murraya elongata
MCN.6
Yingde, China
24.2922°N, 113.3452°E
62
Murraya elongata
NCBT.1
near Wengkhar, Mongar, BT
27.2516°N, 91.2597°E
2018
unresolved
NCBT.3
Rimchu, Punakha, BT
27.6677°N, 89.7667°E
1520
unresolved
NCBT.5
Korilla, Mongar, BT
27.3108°N, 91.2961°E
2100
unresolved
NCBT.7
RDC, Maenchhana, Tsirang, BT
26.9964°N, 90.1224°E
1502
unresolved
NCBT.9
Basochhu, Wangdue, BT
27.3556°N, 89.8871°E
1929
unresolved
225
7.2.3 DNA extraction from the wild citrus
For the wild citrus specimens, DNA was extracted from specimens preserved in
100% ethanol in the Postharvest Molecular Laboratory, Hawkesbury Campus,
Western Sydney University.
For extraction of plant genomic DNA, leaf tissue
(~0.02 g) was washed in 2% bleach with Triton X-100 (five drops of Triton X-100
per 5 mL of 2% bleach) for 1 min with gentle vortexing, and rinsed three times with
Milli Q water. For detection of ‘CLas’ only midribs (~0.20 g) were used. Leaf
samples were cut into small pieces (1–2 mm) and placed in bead beating-tough, 2 mL
tubes (MO BIO Laboratories Inc. supplied by Genworks) each containing a 6.35 mm
chrome steel beating bead (Daintree Scientific). The tubes with the samples and the
beating beads were then placed in liquid nitrogen for 30 secs before placing in a
tissue lyser (FastPrep). Leaf tissues were ground using the tissue lyser at 4 m/sec for
20 sec. DNA was then extracted using either the DNeasy Plant Mini Kit (Qiagen) or
the ISOLATE II Plant DNA Kit (Bioline).
7.2.4 Plant genomic DNA amplification
Table 7.2 shows the primers used for amplification of the five chloroplast DNA
(cpDNA) regions and the internal transcribed (ITS) region of the nuclear ribosomal
DNA. For all regions, PCR reactions were performed using 2 µL of template DNA
in 20–25 µL reaction volumes following the GoTaq® Flexi DNA polymerase
(Promega) protocol, with final concentrations of 2.5 mM MgCl2, 0.24 mM dNTP
mix (Bioline), 0.2 µg/µL BSA, 0.4 µM of each primer and 1.25 U of GoTaq Flexi
DNA polymerase.
For amplification of regions of Murraya and wild citrus
specimens, the same primer sets were used except for the ITS regions.
conditions for each genetic region were as follows.
226
PCR
Table 7.2. List of primers used for the molecular analysis of Murraya and wild Citrus specimens from Bhutan.
Target region
Primer name (forward
& reverse)
Primer sequence (5’- 3’)
Reference
ITS
ITS1
ITS4
TCC GTA GGT GAA CCT GCG G
TCC TCC GCT TAT TGA TAT GC
White et al. (1990)
White et al. (1990)
ITS
AB101
AB102
ACGAATTCATGGTCCGGTGAAGTGTTC G
TAGAATTCCCCGGTTCGCTCGCCGTTA C
Robinson et al. (2001)
Robinson et al. (2001)
matK-5’trnK spacer
trnK-3914F*
matk5’R
TGG GTT GCT AAC TCA ATG G
GCA TAA ATA TAY TCC YGA AAR ATA AGT GG
Johnson & Soltis (1994)
Shaw et al. (2005)
psbM-trnDGUCspacer
trnDGUCR
GGG ATT GTA GYT CAA TTG GT
Shaw et al. (2005)
psbMF
AGC AAT AAA TGC RAG AAT ATT TAC TTC CAT
Shaw et al. (2005)
trnTUGUR
AGG TTA GAG CAT CGC ATT TG
Shaw et al. (2005)
rps4R2
CTG TNA GWC CRT AAT GAA AAC G
Shaw et al. (2005)
ycf6R
GCC CAA GCR AGA CTT ACT ATA TCC AT
Shaw et al. (2015)
CCA GTT CRA ATC YGG GTG
Shaw et al. (2005)
CGA AAT CGG TAG ACG CTA CG
ATT TGA ACT GGT GAC ACG AG
Taberlet et al. (1991)
Taberlet et al. (1991)
rpS4-trnT spacer
trnCGCA-ycf6
GCA
trnC
F
C
F
*renamed as matK6 in Shaw et al. (2005).
trnL-trnL-trnF
227
7.2.4.1 ITS region
For the Murraya specimens, this region was amplified with primers ITS1 and ITS4
using a protocol adapted from White et al. (1990). The PCR cycling parameters
were performed with an initial denaturation at 94°C for 90 sec, followed by 30 cycles
of denaturation at 95°C for 50 sec, annealing at 55°C for 70 sec and extension at
72°C for 90 sec, and a final elongation step at 72°C for 3 min.
For the wild citrus specimens, the ITS region was amplified using primers AB101
and AB102 (Robinson et al. 2001; Li et al. 2010). PCR conditions for this primer
pair were performed with an initial denaturation at 96°C for 2 min, followed by 30
cycles of denaturation at 96°C for 1 min, annealing at 60°C for 1 min and extension
at 72°C for 2 min, and a final elongation at 72°C for 10 min (Li et al. 2010).
7.2.4.2 matK-5’trnK spacer
This non-coding intron was amplified using primers, trnK-3914F (Johnson & Soltis
1994) (also referred to as matK6 by Shaw et al. (2005)) and matK5’R (Shaw et al.
2005). Reactions were performed following the protocol of Shaw et al. (2005):
80°C for 5 min, and 35 cycles of 95°C for 3 min, 50°C for 1 min, 72°C for 2 min
followed by a final extension step at 72°C for 5 min.
7.2.4.3 psbM-trnDGUC spacer
Primers psbMF and trnDGUCR (Shaw et al. 2005) were used to amplify this region.
Amplification parameters were: 94°C for 5 min followed by 35 cycles of 94°C at 1
min, 55°C for 1 min, 72°C for 3.5 min, and a final step of 72°C for 5 min (Nguyen
2011).
7.2.4.4 rpS4-trnT spacer
This region was amplified with primers trnTUGU and rpS4 (Shaw et al. 2005) with
PCR conditions of 92°C for 3 min, followed by 30 cycles of 92°C for 1 min, 55°C
for 1 min, 72°C for 3 min, and a final elongation at 72°C for 7 min (Nguyen 2011).
228
7.2.4.5 trnCGCA-ycf6 region
PCR conditions were set with an initial denaturation step at 80°C for 5 min, then 35
cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 3.5 min, and a final elongation at
72°C for 5 min using primers ycf6R and trnCGCA (Shaw et al. 2005).
7.2.4.6 trnL-trnL-trnF region
This region comprises the trnL intron and the trnL-trnF spacer (Figure 7.3) and will
be referred as trnL-trnL-trnF following Shaw et al. (2005).
The region was
amplified using primers c and f (Taberlet et al. 1991) with PCR conditions as in
Taberlet et al. (1991) and Nguyen (2011): initial denaturation at 94°C for 5 min, then
30 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 2 min, followed by elongation
step of 72°C for 5 min.
Figure 7.3. The trnL-trnL-trnF region showing the positions and directions of primers (Taberlet et al.
1991).
229
7.2.5 ‘CLas’ detection, gel electrophoresis and sequencing
Detection of ‘CLas’ from both Murraya and wild citrus accessions were performed
as described in Chapter 3 using both real-time PCR (qPCR) and conventional PCR
(cPCR). Post PCR processing of samples (gel electrophoresis and sequencing) used
the same methods detailed in Section 2.2.3 (Chapter 2).
7.2.6 Phylogenetic nomenclature for Murraya
Since the study of Murraya by Nguyen (2011), further analysis of the literature has
suggested names for the taxa identified that should take precedence over those used
by Nguyen (2011). These names will be used in further publications of the data
derived in Nguyen’s study and will be used in this thesis; the two sets of names are
given in Table 7.3.
Table 7.3. Proposed names for various taxa of Murraya.
Nguyen (2011)
Current study
Murraya exotica
Murraya paniculata
Murraya paniculata
Murraya sumatrana
Murraya asiatica
Murraya elongata
Murraya × omphalocarpa
Murraya × omphalocarpa
Murraya ovatifoliolata
Murraya heptaphylla var. ovatifoliolata
Murraya ovatifoliolata var. zollingeri
Murraya heptaphylla var. heptaphylla
230
7.2.7 Phylogenetic relationships of Murraya spp.
Sequence assembly was performed as described in Section 2.2.4 of Chapter 2.
Sequences of other accessions were obtained either from previous studies and/or
from GenBank and are presented in Table 7.4 for Murraya accessions and Tables 7.5
& 7.6 for the chloroplastal and ITS regions of the citrus accessions.
Phylogenetic relationships among the Murraya accessions from Bhutan and of those
from other parts of the world were determined based on five cpDNA regions and the
ITS region. Aligned sequence datasets were subjected to phylogenetic analyse by
maximum likelihood (ML) in MEGA6.
Then ML analyses were performed
separately on each cpDNA sequence set and the ITS region, and then for
combination of all cpDNA and ITS region with 1000 bootstrap replicates. Before
analysis, the appropriate evolutionary model was determined for each gene using
MEGA 6 with the best model being determined based on the lowest Bayesian
Information Criterion (BIC) score. For each of the cpDNA, the Tamura 3-parameter
model (Tamura 1992) was used. For the ITS region and the combined cpDNA data
sets, the Tamura 3-parameter model with a discrete gamma distribution (5 categories
(+G, parameter = 0.2202) for ITS and (5 categories (+G, parameter = 0.1000) for the
combined cpDNA were used to model the evolutionary rate differences among sites.
For each gene, initial tree(s) for the heuristic search were obtained by applying the
Neighbor-Joining method to a matrix of pairwise distances estimated using the
maximum composite likelihood approach, and then selecting the topology with
superior log likelihood value. The combined cpDNA set was also subjected to
maximum parsimony (MP) analysis using MEGA6. The MP tree was obtained using
the subtree-pruning-regrafting algorithm (Nei & Kumar 2000) with search level 1 in
which the initial trees were obtained by the random addition of sequences (10
replicates).
231
Table 7.4. Sequences of Murraya and Bergera accessions used for molecular analyses in this study.
Accession
Location
Species
Reference
2Mu.p. ANSW
Richmond, NSW, Australia
Murraya paniculata
Nguyen (2011)
4Mu.p. ANSW
Richmond, NSW, Australia
Murraya paniculata
Nguyen (2011)
8–ANSW
Royal Botanic Garden, Sydney, NSW, Australia
Murraya paniculata
Nguyen (2011)
13Mu.p. AQ
Brisbane, QLD, Australia
Murraya paniculata
Nguyen (2011)
22Mu.p.IWJ,
Bogor Botanic Garden, WJ, Indonesia
Murraya sumatrana
Nguyen (2011)
23Me.c. IWJ
Bogor Botanic Garden, WJ, Indonesia
Merrillia caloxylon (Ridl.) Swingle
Nguyen (2011)
24Mu.p. IP
Bogor Botanic Garden (from Pegunungan Cycloop, Papua), Indonesia
Murraya × cycloopensis
Nguyen (2011)
25Mu.p.IWJ
Bogor Botanic Garden, WJ, Indonesia (from Merubetiri
Murraya sumatrana
Nguyen (2011)
National Park, EJ)
28Mu.e.IWJ
Bogor Botanic Garden, WJ, Indonesia
Murraya paniculata
Nguyen (2011)
30Mu.p. IL
Bogor Botanic Garden, WJ, Indonesia
Murraya sumatrana
Nguyen (2011)
34Mu.p.IEJ
Purwodadi Botanic Garden, EJ, Indonesia
Murraya sumatrana
Nguyen (2011)
38Mu.p.IEJ
Purwodadi Botanic Gardens, EJ, Indonesia
Murraya sumatrana
Nguyen (2011)
42–IUCR
Bayan, Purworejo, CJ (from UCR), Indonesia
Murraya paniculata
Nguyen (2011)
44Mu.e.ICJ
Bayan, Purworejo, CJ, Indonesis
Murraya paniculata
Nguyen (2011)
45Mu.p. ICJ
Bayan, Purworejo, CJ, Indonesia
Murraya sumatrana
Nguyen (2011)
46Mu.p. ICJ
Yogyakata, CJ, Indonesia
Murraya sumatrana
Nguyen (2011)
48Mu.p. ICJ
Universitas Gadjah Mada, Yogyakata, CJ, Indonesia
Murraya sumatrana
Nguyen (2011)
232
Accession
Location
Species
Reference
54Mu.ov.AQ
Bundaberg, QLD, Australia
Murraya heptaphylla var. ovatifoliolata
Nguyen (2011)
58Mu.p.VTG
Chau Than, Tien Giang, Việt Nam
Murraya paniculata
Nguyen (2011)
61–VCP
Cuc Phuong National Park, Ninh Binh, Việt Nam
Murraya elongata
Nguyen (2011)
65–UCR
University of California, Riverside, USA
Murraya paniculata
Nguyen (2011)
66–Mu.sp.AQ
Woongarra, QLD (ex University of California,
Riverside), Australia
Haddon Head Beach, Blue Mud Bay, NT, Australia
Murraya heptaphylla var. ovatifoliolata
Nguyen (2011)
Murraya heptaphylla var. ovatifoliolata
Nguyen (2011)
71Mu.ov. ANT
Gove, NT, Australia
Murraya heptaphylla var. ovatifoliolata
Nguyen (2011)
72Mu.p. AQ
Mt Carbine, QLD, Australia
Murraya heptaphylla var. ovatifoliolata
Nguyen (2011)
73Mu.p. AQ
Cooktown-Mt Webb National Park, QLD, Australia
Murraya heptaphylla var. ovatifoliolata
Nguyen (2011)
74Mu.ov.AQ
Battle Camp, QLD, Australia
Murraya heptaphylla var. ovatifoliolata
Nguyen (2011)
75Mu.p.AQ
Cairns, QLD, Australia
Murraya heptaphylla var. ovatifoliolata
Nguyen (2011)
76Mu.p.PI
Guangxi via South China Botanical Gardens, China
Murraya elongata
Nguyen (2011)
77Mu.p.VTG
Cai Lay, Tien Giang, Việt Nam
Murraya paniculata
Nguyen (2011)
84VDL
Buon Ma Thuot, Dac Lac, Việt Nam
Murraya paniculata
Nguyen (2011)
86Mu.p.VTH
Dong Ve, Thanh Hoa, Việt Nam
Murraya paniculata
Nguyen (2011)
88Mu.p.VCP
Cuc Phuong National Park, Ninh Binh, Việt Nam
Murraya elongata
Nguyen (2011)
91Mu.om.T
Orchid Island, Taiwan
Murraya × omphalocarpa (Hayata)
Nguyen (2011)
92Mu.omT
Orchid Island, Taiwan
Murraya × omphalocarpa
Nguyen (2011)
93Mu.omT
Orchid Island, Taiwan
Murraya × omphalocarpa
Nguyen (2011)
94Mu.p.CYD
Pipashan, Yingde County, GD, China
Murraya elongata
Nguyen (2011
95Mu.p.CYD
Pipashan, Yingde County, GD, China
Murraya elongata
Nguyen (2011)
69Mu.p.ANT
233
Accession
Location
Species
Reference
96Mu.p.CYD
Pipashan, Yingde County, GD, China
Murraya elongata
Nguyen (2011)
97CYD
Hengshitang, Yingde County, GD, China
Murraya paniculata
Nguyen (2011)
98Be.kw. CGX
Guangxi via South China Botanical, Gardens in
Bergera kwangsiensis (C.C. Huang)
Nguyen (2011)
Bergera microphylla (Merr. & Chun)
Nguyen (2011)
Murraya paniculata
Nguyen (2011)
Guangzhou, GD, China
99Be.mic. CH
Bawangling, Hainan, via South China Botanical
Gardens in Guangzhou, GD, China
100M.sp. CH
Bawangling, Hainan, via South China Botanical
Gardens in Guangzhou, GD, China
101Mu.p.CGD
South China Agricultural University, GD, China
Murraya paniculata
Nguyen (2011)
102Mu.p.BSP
Capão Bonito, SP Brazil
Murraya paniculata
Nguyen (2011)
106M.p.BSP
Araraquara, SP, Brazil
Murraya paniculata
Nguyen (2011)
108M.p. ANSW
Richmond, NSW, Australia
Murraya paniculata
Nguyen (2011)
111UFBG
Fairchild Botanic Garden, Florida, USA
Murraya paniculata
Nguyen (2011)
113Mu.z.INTT
Kupang, NTT, Indonesia
Murraya heptaphylla var. heptaphylla Spanoghe
Nguyen (2011)
114Mu.z.INTT
Kupang, NTT, Indonesia
Murraya heptaphylla var. heptaphylla
Nguyen (2011)
115Mu.p.AQ
Tondoon Botanic Gardens, QLD, Australia
Murraya heptaphylla var. ovatifoliolata
Nguyen (2011)
EF126637
–
Bergera koenigii (Murraya koenigii)
Bayer et al. (2009)
EF138843
–
Bergera koenigii (Murraya koenigii)
Bayer et al. (2009)
JX144255
–
Murraya alata Drake
Mou et al. (unpublished)
JX144256
–
Murraya paniculata
Mou et al. (unpublished)
234
Table 7.5. GenBank accessions of citrus and hybrids used for molecular analyses of chloroplast regions.
Taxa cited in GenBank
Names based on Mabberley (1997, 1998,
2004, 2008) and Zhang et al. (2008)
Accession numbers for the specific genetic region
trnL-trnL-trnF
rpS4-trnT
Matk-5’trnK
psbM-trnD
References
Atalantia monophylla
Atalantia monophylla (Roxb.) A. DC.
EF126636
EF134633
EF138841
EF164813
Bayer et al. (2009)
Citrus aurantiifolia
Citrus × aurantiifolia (Christm.) Swingle
EF126645
EF134643
EF138851
EF164823
Bayer et al. (2009)
Citrus aurantiifolia
Citrus × aurantiifolia (Christm.) Swingle
KJ865401
KJ865401
KJ865401
KJ865401
Su et al. (2014)
Citrus aurantium
Citrus × aurantium L.
EF126647
EF134644
EF138853
EF164824
Bayer et al. (2009)
Citrus bergamia
Citrus × limon (L.) Osbeck
EF126648
EF134646
EF138854
EF164826
Bayer et al. (2009)
Citrus celebica
Citrus hystrix DC.
EF126649
EF134647
EF138855
EF164827
Bayer et al. (2009)
Citrus gracilis
Citrus wintersii Mabb.
EF126650
EF134648
EF138856
EF164828
Bayer et al. (2009)
Citrus halimii
Citrus halimii Stone
EF126651
EF134649
EF138857
EF164829
Bayer et al. (2009)
Citrus hindsii
Citrus japonica Thunb.
JX144237
–
–
–
Mou et al. (unpublished)
Citrus hystrix DC.
Citrus hystrix DC.
EF126652
EF134650
EF138858
EF164830
Bayer et al. (2009)
Citrus ichangensis
Citrus cavaleriei H. Léveillé ex Cavalerie
EF126653
EF134651
EF138859
EF164831
Bayer et al. (2009)
Citrus indica Tanaka
Citrus indica Tanaka
EF126654
EF134652
EF138860
EF164832
Bayer et al. (2009)
Citrus jambhiri
Citrus × taitensis Risso
EF126655
EF134653
EF138861
EF164833
Bayer et al. (2009)
Citrus junos
Citrus × junos Siebold ex Tanaka
EF126656
EF134654
EF138862
EF164834
Bayer et al. (2009)
Citrus latifolia
Citrus × latifolia (Tanaka ex Yu. Tanaka)
EF126657
EF134655
EF138863
EF164835
Bayer et al. (2009)
Tanaka
235
Taxa cited in GenBank
Names based on Mabberley (1997, 1998,
2004, 2008) and Zhang et al. (2008)
Accession numbers for the specific genetic region
trnL-trnL-trnF
rpS4-trnT
Matk-5’trnK
psbM-trnD
References
Citrus latipes
Citrus latipes (Swingle) Tanaka
EF126658
EF134656
EF138864
EF164836
Bayer et al. (2009)
Citrus × limonia
Citrus × limon (L.) Osbeck
–
–
EF138866
–
Bayer et al. (2009)
Citrus limon
Citrus limon (L.) Osbeck
EF126659
–
EF138865
EF164837
Bayer et al. (2009)
Citrus macroptera
Citrus hystrix DC.
EF126661
EF134659
EF138867
EF164839
Bayer et al. (2009)
Citrus maxima
Citrus maxima (Burm.) Merr.
EF126664
EF134661
EF138869
EF164842
Bayer et al. (2009)
Citrus medica
Citrus medica L.
EF126665
EF134663
EF138871
EF164843
Bayer et al. (2009)
Citrus medica
Citrus medica L.
–
–
–
EU369571
Li et al. (2007b)
Citrus myrtifolia
EF126666
EF134664
EF138872
EF164844
Bayer et al. (2009)
Citrus obovoidea Yu. Tanaka
Citrus × aurantium var.
myrtifolia Ker Gawler
Citrus maxima (Burm.) Merr.
EF126667
EF134665
EF138873
EF164845
Bayer et al. (2009)
Citrus platymamma
Citrus reticulata Blanco
KR259987
KR259987
KR259987
KR259987
Lee et al. (2015)
Citrus reticulata
Citrus reticulata Blanco
EF126671
EF134669
EF138877
EF164850
Bayer et al. (2009)
Citrus reticulata
Citrus reticulata Blanco
–
EF134670
–
–
Bayer et al. (2009)
Citrus reticulata
Citrus reticulata Blanco
EU369542
–
–
–
Li et al. (2007b)
Citrus sinensis
Citrus × aurantium L. ‘Sweet orange’
DQ864733
DQ864733
DQ864733
DQ864733
Bausher et al. (2006)
Citrus sinensis
Citrus × aurantium L.
–
EF134671
–
EF164851
Bayer et al. (2009)
Citrus sp. amboinensis
Citrus 'amboinensis'
–
EF134642
–
–
Bayer et al. (2009)
Citrus tachibana
Citrus reticulata Blanco
EF126673
EF134672
EF138880
EF164852
Bayer et al. (2009)
Citrus × paradisi
Citrus × aurantium L. “Grapefruit’
EF126668
EF134668
EF138876
EF164847
Bayer et al. (2009)
236
Taxa cited in GenBank
Names based on Mabberley (1997, 1998,
2004, 2008) and Zhang et al. (2008)
Accession numbers for the specific genetic region
trnL-trnL-trnF
rpS4-trnT
Matk-5’trnK
psbM-trnD
References
Citrus × paradisi
Citrus × aurantium L. “Grapefruit’
EF126668
EF134668
EF138876
EF164847
Bayer et al. (2009)
Clymenia polyandra
Citrus polyandra Tanaka
AY295281
–
–
–
Morton et al. (2003)
Fortunella hindsii
Citrus japonica Thunb.
EU369558
–
–
–
Li et al (2007b)
Fortunella japonica
Citrus japonica Thunb.
EF126679
EF134680
EF138888
EF164860
Bayer et al. (2009)
Fortunella margarita
Citrus japonica Thunb.
–
–
–
EF164861
Bayer et al. (2009)
Fortunella polyandra
Citrus japonica
–
EF134682
EF138890
EF164862
Bayer et al. (2009)
Microcitrus australasica
Citrus australasica F. Mueller
EF126686
EF134690
EF138898
EF164870
Bayer et al. (2009)
Microcitrus australasica
Citrus australasica F. Mueller
EU369567
–
–
–
Li et al. (2007b)
Microcitrus australis
Citrus australis (Mudie)
EF126687
EF134691
EF138899
EF164871
Bayer et al. (2009)
Microcitrus inodora
Citrus indora F.M. Bailey
EF126688
–
–
–
Bayer et al. (2009)
Microcitrus papuana
Citrus wintersii Mabb.
EF126689
EF134694
EF138902
EF164874
Bayer et al. (2009)
Poncirus trifoliata
Citrus trifoliata L.
–
EF134706
EF138914
EF164886
Bayer et al. (2009)
Poncirus trifoliata
Citrus trifoliata L.
EU369562
–
–
–
Li et al (2007b)
× Citrofortunella mitis
Citrus × microcarpa Bunge
–
–
EF138868
–
Bayer et al. (2009)
× Citrofortunella sp.
–
EF126641
EF134639
–
EF164819
Bayer et al. (2009)
237
Table 7.6. GenBank accessions of citrus and hybrids used for molecular phylogeny based on the ITS region.
Taxa cited in GenBank
Names based on Mabberley (1997, 1998, 2004, 2008)
and Zhang et al. (2008)
Accession number
References
Atalantia monophylla
Atalantia monophylla (Roxb.) A. DC.
GQ225867
Kumar et al. (2013)
Citrus clementina
Citrus × aurantium L.
XM 006423861
Jenkins et al. (unpublished)
Citrus deliciosa
Citrus × aurantium L.
AB456093
Yamaji et al. (2013)
Citrus hindsii
Citrus japonica Thunb.
JN681163
Amar et al. (unpublished)
Citrus hindsii
Citrus japonica Thunb.
KP093211
Liu et al. (2015)
Citrus hindsii
Citrus japonica Thunb.
KP093210
Liu et al. (2015)
Citrus hindsii
Citrus japonica Thunb.
JX144194
Mou et al. (unpublished)
Citrus hystrix
Citrus hystrix DC.
FJ641961
Kyndt et al. (2010)
Citrus ichangensis
Citrus cavaleriei H. Léveillé ex Cavalerie
JQ990182
Sun et al. (2015)
Citrus indica
Citrus indica Tanaka
GQ225847
Kumar et al. (2013)
Citrus japonica
Citrus japonica Thunb.
JX144195
Mou et al. (unpublished)
Citrus japonica
Citrus japonica Thunb.
HQ893878
Wang et al. (2012).
Citrus japonica
Citrus japonica Thunb.
FJ641924
Kyndt et al. (2010).
Citrus junos
Citrus × junos Siebold Tanaka
AB456114
Yamaji et al. (2013)
Citrus junos
Citrus × junos Siebold Tanaka
AB456113
Yamaji et al. (2013)
Citrus kinokuni
Citrus japonica Thunb.
AB456098
Yamaji et al. (2013)
Citrus latipes
Citrus latipes (Swingle) Tanaka
GQ225851
Kumar et al. (2013)
Citrus macroptera
Citrus hystrix DC.
GQ225852
Kumar et al. (2013)
238
Taxa cited in GenBank
Names based on Mabberley (1997, 1998, 2004, 2008)
and Zhang et al. (2008)
Accession number
References
Citrus maxima
Citrus maxima (Burm.) Merr.
JQ990179
Sun et al. (2015)
Citrus maxima
Citrus maxima (Burm.) Merr.
FJ641949
Kyndt et al. 2010
Citrus maxima
Citrus maxima (Burm.) Merr.
JN681155
Amar et al. (unpublished)
Citrus madurensis
Citrus japonica Thunb.
KP093204
Liu et al. (2015)
Citrus medica
Citrus medica L
JQ990164
Sun et al. (2015)
Citrus medica
Citrus medica L.
JQ990164
Sun et al. (2015)
Citrus reticulata
Citrus reticulata Blanco
FJ641934
Kyndt et al. (2010)
Citrus reticulata
Citrus reticulata Blanco
FJ641939
Kyndt et al. (2010
Citrus reticulata
Citrus reticulata Blanco
GQ225853
Kumar et al. (2013)
Citrus reticulata
Citrus reticulata Blanco
HQ893877
Wang et al. (2012)
Citrus sinensis
Citrus × aurantium L.
JN681150
Amar et al. (unpublished)
Citrus tachibana
Citrus reticulata Blanco
JQ990178
Sun et al. (2015)
Citrus trifoliata
Citrus trifoliata L.
HQ893876
Wang et al. (2012)
Citrus × tangelo
Citrus × aurantium L
JN661211
Amar et al. (unpublished)
Fortunella polyandra
Citrus japonica
FJ434160
Kyndt et al. 2010
Eremocitrus glauca
Citrus glauca (Lindley) Burkill
AB456049
Yamaji et al. (2013)
Eremocitrus glauca
Citrus glauca (Lindley) Burkill
FJ434161
Kyndt et al. (2010)
Fortunella japonica
Citrus japonica Thunb.
AB456108
Yamaji et al. (2013)
Fortunella japonica
Citrus japonica Thunb.
AB456110
Yamaji et al. (2013)
239
Taxa cited in GenBank
Names based on Mabberley (1997, 1998, 2004, 2008)
and Zhang et al. (2008)
Accession number
References
Fortunella japonica
Citrus japonica Thunb.
AB456051
Yamaji et al. (2013)
Microcitrus australasica
Citrus australasica F. Mueller
AB457061
Yamaji et al. (2013)
Poncirus trifoliata
Citrus trifoliata L.
AB456050
Yamaji et al. (2013)
Poncirus trifoliata
Citrus trifoliata L.
FJ606752
Daniel & Knoess (unpublished)
240
7.2.8 Morphology of Murraya
Two data sets were used for analysis of the morphology of accessions of Murraya:
(A) a set containing accessions of Murraya elongata; and (B) data for Murraya
elongata, Murraya sumatrana, Murraya heptaphylla and Murraya paniculata. The
data for these analyses were obtained from this study and from the data set taken
from the study of Ngyuen (2011). Data Set A comprises 71 leaves from 8 plants of
Murraya elongata. Data set B consists of the data from Set A plus 155 leaves from
19 plants of Murraya paniculata, 41 leaves from 5 plants of Murraya sumatrana, 39
leaves of Murraya heptaphylla var. ovatifoliolata and 15 leaves from Murraya
heptaphylla var. heptaphylla. For the Bhutanese accessions, leaf assessments were
based on a maximum of ten basal and terminal leaflets per plant. Pressed specimens
of three plants from each location were used. The following morphological features
of the leaves were measured:
BL: length basal leaflet;
BW: width of basal leaflet;
BACU: length of acuminate portion of basal leaflet;
BAN: angle of base of basal leaflet;
BRLW: ratio of the length and width of basal leaflets;
BRACU: ratio of the length of the acuminate tip to the length of basal leaflets;
TL: length of terminal leaflet;
TW: width of terminal leaflet;
TACU: length of acuminate portion of terminal leaflet;
TAN: angle of base of terminal leaflet;
TRLW: ratio of the length and width of terminal leaflets; and
TRACU: ratio of the length of the acuminate tip to the length of terminal
leaflets.
Variation among the accessions of Murraya was examined using ANOVA and
principal components analysis (PCA). For Data Set A, before being subjected to
ANOVA, the data relating to the variables above were checked for homogeneity of
the variances using Levene’s test; heteroscedasticity data were transformed to
241
logarithms. The transformed data were also used for PCA; however, the derived
variables (BRACU, TRACU, BRLW & TRLW) were excluded from this analysis.
For Data Set B, the data were again checked for homogeneity of variances with
respect to the taxon to which they were assigned (e.g., Murraya elongata or Murraya
paniculata) before being subjected to PCA; all data were transformed to logarithms.
ANOVA and PCA were performed using Statistica (V12, StatSoft Inc.). Fruits of the
Murraya accessions from Bhutan were also included in the morphological
description but not assessed statistically.
7.2.9 Phylogenetic relationships of wild citrus
Phylogenetic relationships of wild citrus accessions collected in Bhutan were
analysed in similar manner as described for the Murraya accessions; ML analyses
were conducted on each cpDNA and the ITS region. Sequences of the cpDNA were
concatenated using Geneious, and ML analysis was performed on the concatenated
sequences to infer the phylogenetic relationships.
Before performing the ML
analysis, the evolutionary model for each gene was performed. For the combined
cpDNA, the Tamura 3-parameter model (Tamura 1992) was determined with a
discrete gamma distribution (6 categories (+G, parameter 0.1000) with invariable (I)
sites, and for the ITS region, the Tamura 3-parameter model (6 categories (+G,
parameter 0.1908) was used to model the evolutionary rates among the sites. The
cpDNA was also analysed through MP. The MP analysis was obtained by the
subtree-pruning regrafting algorithm (Nei & Kumar 2000) with search level 1 in
which the initial trees were obtained by the random addition of sequences (10
replicates). All analyses were performed with 1000 bootstrap replicates in MEGA6.
Atalantia monophylla was used as outgroup for all phylogenetic analyses. Names of
taxa in all phylogenetic analyses are based on Mabberley (1997; 1998; 2004; 2008)
and Zhang et al. (2008) as detailed in Tables 7.5 and 7.6.
242
7.3 Results
7.3.1 Sequence analysis of the Murraya accessions
Table 7.7 shows the sequence analyses of the individual genes.
The longest
sequence length obtained in this study was for the psbM-trnDGUC region (1142–1166
bp). However, the length was trimmed to match the length of sequences from
Nguyen (2011) which provided many of the other sequences used for comparison
with those obtained in the current study. Among the cpDNA regions, psbM-trnDGUC
had the highest number of variable sites (54), but the trnL-trnL-trnF region had the
highest number of parsimoniously informative sites (29). The rpS4-trnT region of
the cpDNA showed the least number of variable (20) and parsimoniously informative
sites (8). Compared to any of the cpDNA regions, the ITS region, with a sequence
length range of 608–686 bp, provided the highest number of variable (93) and
parsimoniously informative sites (36).
7.3.2 Phylogenetic relationships of Murraya
Phylogenetic analysis of different cpDNA regions yielded congruous results but with
some genetic regions providing better resolution than others. To illustrate this, the
phylogenetic trees from three of the cpDNA regions that gave a high (trnL-trnLtrnF), medium (matK) and poor (trnT) resolution of the taxa are presented together
with the results from the ITS region and an analysis based on the combination of all
five cpDNA regions with the ITS region.
7.3.2.1 trnL-trnL-trnF region
Phylogenetic analysis of the trnL-trnL-trnF region provided a better resolved tree
than the other regions of the cpDNA (Figure 7.4). Six distinct clades were formed
including two separate clades of Murraya elongata: one clade of Murraya elongata
comprising accessions from Yingde, China, and the other clade including accessions
from Bhutan and Guangxi, China. All Murraya paniculata accessions clustered into
one clade together with accession MBT.9 from Bhutan. Accessions of Murraya
heptaphylla var. heptaphylla, Murraya heptaphylla var. ovatifoliolata and the hybrid
243
Murraya × ompalocarpa formed one clade with the two latter taxa forming distinct
subclades. Murraya alata formed a single member clade and Merrillia caloxylon
was a sister taxon to all Murraya accessions.
244
Table 7.7. Sequence analysis of the cpDNA and the ITS region of the Murraya accessions.
Genetic regions
Analysis
ITS
matK-5'trnK
psbM-trnDGUC
rpS4-trnT
trnCGCA-ycf6
trnL-trnL-trnF
Combined
chloroplast
Range of length of sequences from the current study
608–
686
793–819
1142–1166*
546–567
723–760
947–1001
3956–4165**
Number of variable sites of the aligned sequences
93
42
54
20
38
42
241
Number of parsimony informative sites
36
20
17
8
19
29
114
0–10
0–4
0–4
0–2
0–3
0–3
0–4
Range of pairwise sequence divergence among all
taxa
* trimmed to the length of sequences obtained by Nguyen (2011)
** for accessions with sequences for all 5 cpDNA.
245
Figure 7.4. Phylogenetic analysis of the trnL-trnL-trnF region using maximum likelihood and the
Tamura 3-parameter model of evolution (Tamura 1992). The tree with the highest log likelihood (1596.8238) is shown, and the percentage of trees in which associated taxa cluster together is shown
next to the branches. Accessions obtained from the current study are shown within boxes.
Abbreviations used: MBT accessions from Bhutan; MCN, accessions from China.
246
7.3.2.2 matK-5' trnK region
The analysis of the matK-5' trnK region produced a tree (Figure 7.5) that shows little
resolution with many accessions forming a large polytomy comprising accessions of:
Murraya heptaphylla var. ovatifoliolata, Murraya heptaphylla var. heptaphylla, and
Murraya × omphalocarpa along with the accession of Merrillia caloxylon and
Murraya elongata accessions from Yingde, China. Accessions of Murraya elongata
from Bhutan and Guangxi group together in a single clade as do the accessions of
Murraya sumatrana and Murraya paniculata.
7.3.2.3 rpS4-trnT region
Results obtained from the analysis of the rpS4-trnT region are shown in Figure 7.6.
This region shows low resolution with the accessions of Murraya elongata and
Merrillia caloxylon forming a large, basal polytomy. Only two distinct clades are
formed. One clade comprises Murraya accessions of Murraya paniculata that split
into two subclades. The other clade comprises accessions of Murraya sumatrana,
Murraya heptaphylla var. ovatifoliolata, Murraya heptaphylla var. heptaphylla,
Murraya × omphalocarpa and Murraya × cycloopensis.
7.3.2.4 ITS region
Phylogenetic analysis of the ITS region of the Murraya species using Merrillia
caloxylon and Bergera microphylla as outgroups resulted in five clades (Figure 7.7).
One clade consists of all Murraya elongata accessions from Bhutan and from China.
Accessions from Yingde form a subclade nested within this main Murraya elongata
clade. Accessions of Murraya sumatrana form a second clade. A third clade
comprising of all accessions of Murraya paniculata with the accession from Bhutan
(MBT.9) and the accession from Buon Ma Thuot, Việt Nam (80Mu.p.VDL) being
sister to the rest. Murraya alata forms fourth, single member clade. The last clade
consists of accessions of Murraya heptaphylla var. ovatifoliolata and Murraya
heptaphylla var. heptaphylla.
247
Figure 7.5. Evolutionary history inferred by using the maximum likelihood method based on the
Tamura 3-parameter model (Tamura 1992) for the matK-5' trnK region. The tree with the highest log
likelihood (-1408.8929) is shown. Accessions obtained from this study are shown in boxes.
Accessions obtained from GenBank are shown with accession numbers in parentheses. Abbreviation
used: MBT Murraya accessions from Bhutan; MCN Murraya accessions from China.
248
Figure 7.6. Molecular phylogenetic analysis inferred by using maximum likelihood based on the
Tamura 3-parameter model (Tamura 1992) for the rps4-trnT region. The tree with the highest log
likelihood (-907.8983) is shown. The percentage of trees in which associated taxa cluster together is
shown next to the branches. Accessions obtained in this study are shown within boxes.
Abbreviations used: MBT accessions from Bhutan; MCN Accessions from China.
249
Figure 7.7. Phylogenetic relationships of the ITS region inferred using the maximum likelihood
method based on the Tamura 3-parameter with a discrete gamma distribution evolutionary model of
rate differences among sites (5 categories (+G, parameter 0.2202)) (Tamura 1992). The tree with the
highest log likelihood (-1479.1368) is shown. The percentage of trees in which the associated taxa
cluster together is shown next to the branches. Accessions obtained from this study are shown within
boxes. Abbreviations used: MBT accessions from Bhutan; MCN Accessions from China.
250
7.3.2.5 Phylogenetic relationships of Murraya inferred from combination of
five cpDNA and ITS region
Figure 7.8 shows the results of the phylogenetic analysis from the combination of
five cpDNA and the ITS region. The analysis of the concatenated sequences resulted
in better resolution than any analysis of the single genes and the results are
summarised below:
All accessions of Murraya elongata from Bhutan and China form one clade
with strong bootstrap support. Two subclades are found within this clade: one
comprising the accessions from Yingde, China, and the other of the accessions
from Guangxi, China together with the accessions from Bhutan. Accession 88
Mu.p VCP from Cuc Phuong does not group within the Murraya elongata
clade.
All accessions of Murraya paniculata form one clade and the accession from
Bhutan is sister to the rest.
All Murraya sumatrana accessions cluster together.
Murraya heptaphylla var. ovatifoliolata and Murraya heptaphylla var.
heptaphylla form one clade with each variety forming a distinct subclade.
Merrillia caloxylon is sister to all Murraya accessions.
This same data set was analysed using maximum parsimony (Figure 7.9). This
method produced similar tree as with the maximum likelihood. However, accession
88 Mu. p VCP from Cuc Phuong forms a sister clade to the Murraya elongata clade.
251
Figure 7.8. Phylogenetic relationships inferred from the combination of five cpDNA regions and the
ITS region of Murraya accessions from Bhutan using the maximum likelihood based on the Tamura
3-parameter model of evolution (Tamura 1992) with a discrete gamma distribution (5 categories (+G,
parameter 0.1000)). The tree with the highest log likelihood (-8468.8153) is shown. The percentage
of trees in which the associated taxa cluster together is shown next to the branches. Accessions
obtained from this study are shown within boxes. Abbreviation used: MBT accessions from Bhutan;
MCN accessions from China.
252
Figure 7.9. Phylogenetic relationships inferred from the combination of five cpDNA regions and the
ITS region of Murraya accessions from Bhutan using maximum parsimony (Felsenstein 1985). Tree
#1 out of 5 most parsimonious trees (length = 248) is shown. The percentage of replicate trees in
which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the
branches. The MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm (Nei &
Kumar 2000) with search level 1 in which the initial trees were obtained by the random addition of
sequences (10 replicates). Branches with bootstrap values less than 50% were collapsed together.
253
7.3.3 Morphology of Murraya accessions from Bhutan
In Baychhu, Murraya elongata was observed on rocky and dry areas mostly on the
edge of orchards. A few plants were observed within the orchard but were usually
cut back at least three to four times a year along with weeds. The weeds grew back
more quickly taking over the whole ground and hiding any plants underneath. In
Reldri, Murraya elongata was observed on sloping area beside the road; this location
was not as dry as that at Baychhu.
The flowers on the plants both at Reldri and Baychhu were borne terminally with 5
white petals. The leaves of these plants had five to seven leaflets most of which had
acuminate tips; the petioles and petiolules were often glabrous (Figures 7.10).
Mature fruits observed at Baychhu were reddish orange in colour, ~10 mm long and
8 mm wide and almost globular. The fruit (previously undescribed) contained two
seeds that were hairy (Figure 7.11).
ANOVA of the leaves of accessions of Murraya elongata from Bhutan (Data Set A,
Figure 7.12), China and Việt Nam showed significant variation in the parameters
measured (Table 7.8). More variation was found in the basal leaflets as opposed to
the terminal leaflets with the length and width of the leaflets and their ratio being
more variable than the other characteristics assessed. PCA of this data resulted in the
first two factors only having eigenvalues greater than 1.0; hence, these two factors
only were used to examine any grouping of the data (Kaiser 1960). The plot of the
factor coordinates (Figure 7.12) shows that the widths and lengths of both the basal
and terminal leaflets are strongly correlated with Factor 1 whereas the basal angles of
the basal and terminal leaflets were strongly correlated with Factor 2. However,
despite the variation in leaf morphology among the plants from the different
locations being found by ANOVA, no obvious separation of the plants was observed
in the PCA analysis (Figure 7.13).
The data for the proposed four species of Murraya (Ngyuen 2011) (Data Set B) are
shown in Figures 7.14 and 7.15. In this analysis, the correlations of the variables
with the factors were similar to those for Data Set A. The logarithms of the lengths
254
and widths of the basal and terminal leaflets were again strongly correlated with
Factor 1 and the basal angles of the terminal and basal leaflets with Factor 2; again
only two factors had eigenvalues greater than 1.0 and were used to examine the
variation among leaves from the four species. The plots of the case variables show
three distinct groups of plants although with some overlap between each group.
Firstly, a group consisting of accessions of Murraya paniculata including the
accession of this species from Bhutan (upper right). Secondly, a group containing
accessions of the two varieties of Murraya heptaphylla (lower right). Lastly, a group
containing accessions of Murraya elongata from Bhutan and China, together with
accessions of Murraya sumatrana from Indonesia (centre left).
Figure 7.10. Leaves, leaflets and flowers of Bhutanese accessions of Murraya elongata from
Baychhu (A & B) and leaves, leaflets and an inflorescence of Murraya elongata from Reldri,
Phuentsholing (C & D) (Photos: N. Om & GAC Beattie).
255
Figure 7.11. Fruit (A & B) and seeds (C) of Murraya elongata from Baychhu (Photo: GAC Beattie).
256
Figure 7.12. Plot of factor coordinates for Data Set A for the first two principal components. As the
analysis is based on correlation, the largest variable-factor correlation is equal to 1.0; therefore, the
coordinates are shown within a unit circle. BL: length basal leaflet; BW: width of basal leaflet;
BACU: length of acuminate portion of basal leaflet; BAN: angle of base of basal leaflet; TL: length
terminal leaflet; TW: width of terminal leaflet; TACU: length of acuminate portion of terminal leaflet;
TAN: angle of base of terminal leaflet.
257
Table 7.8. ANOVA of the morphology of leaves taken from accessions of Murraya elongata from Bhutan, China and Việt Nam. BL: length basal leaflet; BW: width of
basal leaflet; BACU: length of acuminate portion of basal leaflet; BAN: angle of base of basal leaflet; BRLW: ratio of the length and width of basal leaflets; BRACU: ratio of
the length of the acuminate tip to the length of basal leaflets; TL: length terminal leaflet; TW: width of terminal leaflet; TACU: length of acuminate portion of terminal leaflet;
TAN: angle of base of terminal leaflet; TRLW: ratio of the length and width of terminal leaflets; and TRACU: ratio of the length of the acuminate tip to the length of terminal
leaflets.
Feature Origin
n
mean
Se
Fisher's LSD
ANOVA
F
BL
BW
BRLW
BACU
Cuc Phuong
Yingde
Baychhu
Reldri
Cuc Phuong
Yingde
Baychhu
Reldri
Cuc Phuong
Yingde
Baychhu
Reldri
Cuc Phuong
Yingde
Baychhu
Reldri
9
6
28
28
9
6
28
28
9
6
28
28
9
6
28
28
61.12
34.97
40.93
33.27
28.76
18.58
21.78
19.21
2.13
1.93
1.9
1.72
4.93
2.35
4.92
3.84
2.71
1.95
1.64
1.75
1.02
1.7
0.92
0.72
0.06
0.12
0.06
0.05
0.54
0.42
0.36
0.49
a
bc
b
c
a
bc
b
c
a
abc
b
c
a
b
a
ab
258
P
Levene's test
F
P
24.4
< 0.001
0.97
0.41
12.66
< 0.001
1.64
0.19
6.35
< 0.001
0.91
0.44
3.16
0.03
2.15
0.1
Feature
Origin
n
Mean
se
Fisher's LSD
ANOVA
F
BRACU
Cuc Phuong
Yingde
Baychhu
Reldri
LogBAN Cuc Phuong
Yingde
Baychu
Reldri
TL
Cuc Phuong
Yingde
Baychhu
Reldri
TW
Cuc Phuong
Yingde
Baychhu
Reldri
TRLW
Cuc Phuong
Yingde
Baychhu
Reldri
9
6
28
28
9
6
28
28
9
6
28
28
9
6
28
28
9
6
28
28
0.08
0.07
0.12
0.11
77.33
88
102.57
103.04
82.55
53.31
65.45
58.25
31.86
24.8
29.53
24.71
2.6
2.18
2.27
2.39
0.01
0.01
0.01
0.01
2.46
2.77
2.78
2.17
3.69
1.89
2.44
2.07
1.47
1.86
1.65
0.97
0.08
0.1
0.05
0.06
bc
c
a
ab
c
b
a
a
a
c
b
a
a
bc
ab
c
a
b
b
b
259
P
Levene's test
F
P
3.69
0.016
2.74
0.5
15.41
< 0.001
1.51
0.22
12.04
< 0.001
1.84
0.15
4.24
0.008
1.66
0.18
3.96
0.012
1.32
0.28
Feature Origin
TACU
Cuc Phuong
Yingde
Baychhu
Reldri
TRACU Cuc Phuong
Yingde
Baychhu
Reldri
TAN
Cuc Phuong
Yingde
Baychhu
Reldri
n
Mean
9
6
28
28
9
6
28
28
9
6
28
28
8.1
6.06
7.87
6.7
0.1
0.11
0.12
0.11
64.33
67.83
82.32
72.86
se
Fisher's
LSD
0.87
0.73
0.5
0.63
0.01
0.01
0.01
0.01
3.32
0.83
1.79
1.79
c
bc
a
b
260
ANOVA
F
P
Levene's test
F
P
1.35
0.27
1.72
0.17
0.86
0.46
2.35
0.08
11.68
< 0.001
2.33
0.082
Figure 7.13. Plot of the coordinates for each observation (case) using the first two factors from
principal components analysis of leaves from accessions of Murraya elongata from four locations.
261
Figure 7.14. Plot of factor coordinates for Data Set B for the first two principal components. As the
analysis is based on correlation, the largest variable-factor correlation is equal to 1.0; therefore, the
coordinates are shown within a unit circle. BL: length basal leaflet; BW: width of basal leaflet;
BACU: length of acuminate portion of basal leaflet; BAN: angle of base of basal leaflet; TL: length
terminal leaflet; TW: width of terminal leaflet; TACU: length of acuminate portion of terminal leaflet;
TAN: angle of base of terminal leaflet.
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Figure 7.15. Plot of the coordinates for each observation (case) using the first two factors from principal components for all observations (leaves) from accessions of
Murraya elongata, Murraya paniculata, Murraya sumatrana and Murraya heptaphylla.
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7.3.4 Morphological description of wild citrus accessions
The wild citrus accessions have two distinct morphologies. Firstly, the leaves and
fruits of the wild citrus accessions, NCBT.1, NCBT.5, NCBT.7 and NCBT.9,
resemble the description of Citrus cavaleriei (Citrus ichangensis) given by Swingle
& Reece (1967) in that they have large, winged petioles with apiculate leaf tips
(Figures 7.16A & 7.17A). The fruits (Figure 7.16B) have large, thick seeds as in
Citrus cavaleriei. However, based on the observations of accession NCBT.9, the
flowers are different from either of Citrus latipes or Citrus cavaleriei in that they are
mostly borne singly (Figure 7.17B) between the nodes and not in the leaf axils.
Secondly, the morphology of NCBT.3 was different from the accessions above with
respect to leaf and fruit shapes. The leaves are ovate and do not have large, winged
petioles. The fruit have a mammillated apex (Figure 7.18).
7.3.5 Sequence analysis of wild citrus
Longest sequence length was found for the psbM–trnDGUC region (935–1163 bp)
while the rpS4–trnT region had the shortest length (517–562 bp) (Table 7.9). Among
the four cpDNA regions, the psbM–trnDGUC region had the highest number of
variable and parsimoniously informative sites (106 & 20). The rpS4–trnT region had
the least number of variable and informative sites. Compared to the combined
sequence of the chloroplast regions, the ITS region provided a higher number of
parsimoniously informative sites (50) (Table 7.9).
Pairwise sequence divergence showed that the within in-group sequence divergence
was highest in the ITS regions (0.00–5.58%) followed by psbM–trnD and matK–
5’trnK. The ITS region also showed high sequence divergence between the ingroups and the outgroup (Atalantia monophylla).
Five chloroplast genes and one nuclear gene were amplified from the wild citrus
samples collected from Bhutan. A BLAST search using sequences of different
cpDNA of the Bhutanese wild citrus accessions resulted in 99–100% identity to
citrus hybrids such as Citrus reticulata (‘platymamma’), sweet orange (Citrus ×
264
aurantium) (‘sinensis’) (DQ864733) and Citrus × aurantiifolia (KJ865401).
A
BLAST search using the ITS sequence resulted in 99% identity to Citrus reticulata,
Citrus × aurantium (‘clementina’), varieties of Citrus japonica (‘hindsii’, ‘japonica’,
and ‘madurensis’), and others.
For the trnCGCA–ycf6 region, a BLAST search
yielded only three similar sequences, these belonging to Citrus × aurantium
(‘platymamma’), sweet orange (Citrus × aurantium), and Citrus × aurantiifolia and,
hence, this region was excluded from any further analysis.
Figure 7.16. Accession NCBT.1 showing leaves with large winged petioles (A), whole and crosssectioned fruit (B) and leaf distortion caused by Cacopsylla heterogena (C). (Photos: GAC Beattie).
265
Figure 7.17. Accession NCBT.9 showing leaves with large winged petioles (A), flowers that are
mostly borne singly (B) and Cacopsylla heterogena damage on leaves (C). (Photo: GAC Beattie).
266
Figure 7.18. Accession NCBT.3 showing leaves without large petioles (A), a cross section of a fruit
(B); side view of a fruit (C) and the top view of a fruit (D). (Photos: T. Penjor)
267
Table 7.9. Sequence analysis of wild citrus accessions.
Analysis
matK-5'trnK
psbM-trnDGUC
trnL-trnL-trnF
rpS4-trnT
Combined cpDNA
ITS
Range of sequence length of NCBTs
774 – 822
935 – 1163
941 – 1014
517 – 562
3159 – 3167
658 – 808
Range of sequence length of GenBank
accessions
772 – 871
1176 –1256
937 – 1063
526 – 616
3159 – 3223
564 –740
Number of variable sites
45
106
50
30
235
146
Number of informative sites
19
20
19
5
48
50
Range of sequence divergence within ingroups (%)
0.00 – 2.23
0.00 – 2.64
0.00 – 1.43
0.00 – 1.36
0.00 – 1.99
0.00 – 5.58
Range of sequence divergence between ingroups and Atalantia monophylla (%)
2.46 – 3.69
1.91 – 3.20
1.10 – 2.09
1.55 – 2.33
1.77 – 2.97
4.11 – 7.18
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7.3.6 Phylogenetic relationships of wild citrus based on four cpDNA
regions
The phylogenetic analysis of the wild citrus sequences used a data set comprising:
the Bhutanese accessions; accessions taken from GenBank that share 99% similarity
(the highest similarity in the BLAST search) with the Bhutanese accessions; and
other accessions that the BLAST search and other sources suggested may be related
to the Bhutanese accessions. Phylogenetic analysis of the individual chloroplast
genes resulted in trees with low resolution in which the wild citrus accessions from
Bhutan always grouped with Citrus latipes and hybrids of Citrus reticulata but not to
Citrus reticulata itself. A similar result was obtained when the four cpDNA were
combined. Figure 7.19 shows the phylogenetic analysis based on combinations of
the four chloroplast genes with the tree produced by ML. The tree consists of the
eight clades listed below with species names being taken from GenBank:
1.
Citrus reticulata together with Citrus taitensis (‘jhambiri’) and Citrus × junos;
2.
accessions found in Clade ‘R’ of Bayer et al. (2009)—Australasian species
together with Citrus medica and Citrus indica, the latter both native to India;
3-5. Citrus cavalieri (‘ichangensis’), Citrus (Poncirus) trifoliata and Citrus hystrix
(‘macroptera’) each forming single member clades;
6.
two species of Citrus japonica (Fortunella) together with Citrus halimii;
7.
two accessions of Citrus hystrix, two accessions of Citrus aurantiifolia, and ×
Citrofortunella sp. – corresponding to the ‘lime clade’ of Bayer et al. (2009);
and
8.
a large, poorly resolved polytomy containing the Bhutanese wild citrus
accessions (NCBT.1, NCBT.3, NCBT.5, NCBT.7, and NCBT.9), Citrus latipes
and hybrids of Citrus reticulata with Citrus maxima and Citrus × aurantium as
sister clade.
This data set was also subjected to parsimony analysis (Figure 7.20). The tree
produced using this method showed more resolution, and the accessions in Clade 8
again grouped together. However, the Bhutanese accessions again formed part of a
basal polytomy.
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Figure 7.19. Phylogenetic relationships of wild citrus from Bhutan based on combinations of four
chloroplast genes (matK-5’trnK, psbM-trnDGUC, trnL-trnL-trnF, rpS4-trnT) inferred using maximum
likelihood based on the Tamura 3-parameter model of evolution (Tamura 1992) with discrete a
gamma distribution among sites (6 categories (+G, parameter 0.1000)) with invariable sites (([+I],
66% sites). The tree with the highest log likelihood (-6901.6987) is shown. The percentage of trees
in which the associated taxa clustered together is shown next to the branches and branches with
bootstrap values of less than 50% were collapsed together. Most GenBank sequences were obtained
from Bayer et al. (2009). Sequences obtained from different studies are shown by letters in
parentheses. Sequences obtained from this study are: NCBT.1, NCBT.3, NCBT.5, NCBT.7, and
NCBT.9.
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Figure 7.20. Phylogenetic relationships of wild citrus from Bhutan based on combinations of four
chloroplast genes (matK-5’trnK, psbM-trnDGUC, trnL-trnL-trnF, rpS4-trnT) inferred using parsimony.
Tree #1 out of the 2 most parsimonious trees (length = 319) is shown. The percentage of replicate
trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) (Felsenstein
1985) are shown next to the branches. The MP tree was obtained using the Subtree-PruningRegrafting algorithm (Nei & Kumar 2000) with search level 1 in which the initial trees were obtained
by the random addition of sequences (10 replicates). Branches with bootstrap values of less than 50%
were collapsed together. Most GenBank sequences were obtained from Bayer et al. (2009).
Sequences obtained from different studies are shown by letters in parentheses. Sequences obtained
from this study are: NCBT.1, NCBT.3, NCBT.5, NCBT.7 and NCBT.9.
271
7.3.7 Phylogenetic relationships of wild citrus based on the nuclear
ITS regions
Figure 7.21 shows the phylogenetic relationships of the citrus accessions based on
the ITS region, with the data set again based on accessions closely related to the
Bhutanese accessions identified through a BLAST search. The analysis yielded a
poorly resolved tree consisting of a large polytomy in which the following groups
can be found and to which the Australasian accessions were sister:
Clade 1: Citrus maxima, Citrus reticulata and hybrids of Citrus reticulata;
Clade 2: accessions of Citrus trifoliata;
Clade 3: the Bhutanese accessions, NCBT.1, NCBT. 5, NCBT.7 and NCBT.9;
and
Clade 4: the Bhutanese wild citrus accession NCBT.3 grouped together with
Citrus hystrix (including ‘macroptera’), Citrus latipes, Citrus indica, and
further accessions of Citrus maxima.
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Figure 7.21. Phylogenetic analysis of citrus accessions based on the ITS region using maximum
likelihood based on the Tamura 3-parameter model (Tamura 1992). A discrete gamma distribution
was used to model evolutionary rate differences among sites (6 categories (+G, parameter 0.1908)).
The tree with the highest log likelihood (-2390.5207) is shown. Accession numbers are given in
parentheses next to the taxon. Accessions obtained in this study: NCBT.1, NCBT.3, NCBT.5,
NCBT.7, and NCBT.9. Branches with bootstrap values of less than 50% were collapsed together.
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7.3.8 Detection of ‘CLas’ in Murraya and wild citrus
Table 7.10 shows the results of real-time PCR conducted to test the Murraya
elongata and Murraya paniculata and wild citrus accessions for infection by ‘CLas’.
The Murraya elongata accessions produced FAM Ct values ranging from 32.15–
36.17 while the Murraya paniculata accession, MBT. 9, collected in Bhutan
produced a FAM Ct value of 0.00. The internal control (CAL) Ct values for all the
test samples ranged from 13.22 to 22.06.
The positive control FAM Ct was
19.26−25.77 with CAL Ct value range of 19.78−21.49. The extraction control which
is a mandarin sample obtained from the horticulture block in Hawkesbury Campus
produced a FAM Ct value of 37.55 with CAL Ct value of 20.20. Selected samples of
Murraya elongata and wild citrus were subjected to cPCR but did not result in the
production of amplicons.
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Table 7.10. Detection of 'CLas' in the accessions of Murraya species and wild citrus accession
NCBT.9.
Sample name
Species
Ct values
FAM
CAL
MBT.1
Murraya elongata
32.75
13.98
MBT.2
Murraya elongata
36.17
15.87
MBT.3
Murraya elongata
34.69
14.94
MBT.4
Murraya elongata
32.15
13.22
MBT.5
Murraya elongata
33.16
13.96
MBT.6
Murraya elongata
34.96
20.92
MBT.7
Murraya elongata
34.71
20.02
MBT.8
Murraya elongata
35.69
22.06
MBT.9
Murraya paniculata
0.00
20.35
NCBT. 9a
wild citrus
34.96
20.47
NCBT. 9b
wild citrus
35.85
20.38
NCBT. 9c
wild citrus
34.55
19.70
NCBT. 9d
wild citrus
35.95
20.19
NCBT. 9e
wild citrus
35.63
20.32
Positive control*
Citrus reticulata
19.26-25.77
19.78-21.49
Extraction control**
Citrus reticulata
37.55
20.20
NCBT 9a to 9e= samples taken from the same plant in Bhutan but DNA extracted in WSU.
*Positive control Ct values from two assays.
**included during extraction of NCBT.9 samples and collected from horticulture block in
Hawkesbury campus, Western Sydney University.
Samples subjected to cPCR are emboldened
.
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7.4 Discussion
7.4.1 Phylogenetic relationships and morphology of Murraya from
Bhutan
In inferring plant systematics, non-coding regions of the cpDNA have been used to
study phylogenetic relationships below the family level due to their fast evolving
nature and fewer functional constraints as compared to coding regions (Gielly &
Taberlet 1994; Shaw et al. 2005). Similarly, ITS regions are well suited for the study
of species and closely related genera (Baldwin et al. 1995; Soltis & Soltis 1998). In
the current study, five cpDNA and the ITS region of the rDNA were used to assess
the phylogenetic relationships species of Murraya occurring in Bhutan. Variable
results were obtained from the phylogenetic analysis of the cpDNA with some taxa
better resolved with some genetic regions than others.
In this study, the trnL-trnL-trnF region provided a better resolution of taxa than the
other regions of the cpDNA used (Figure 7.4), as it contained the highest number of
parsimoniously informative sites among the cpDNA (Table 7.7). All cpDNA regions
except the rps4-trnT region resolved Murraya elongata into two clades with the
accessions from Bhutan and Guangxi, China in one clade and the accessions from
Yingde, China into another clade (Figure 7.6).
The rpS4-trnT region failed to
resolve Murraya elongata accessions in any groups. In contrast, the ITS region
showed better resolution, with all Murraya elongata accessions falling within one
clade (Figure 7.7).
Further, the remaining accessions of Murraya sumatrana,
Murraya paniculata, and Murraya heptaphylla accessions grouped into separate
distinct clades.
For the combination of the cpDNA and the ITS region analysed using ML, all
accessions of Murraya paniculata, Murraya sumatrana, Murraya heptaphylla and
Murraya elongata were resolved except for the placement of Murraya elongata
accession 88Mu.p.VCP from Cuc Phoung, Việt Nam (Figure 7.8). Better resolution
was achieved when the same data set was analysed using MP with the Cuc Phoung
accession being sister to the rest of the Murraya elongata accessions (Figure 7.9).
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Morphological data analyses show that in spite of variation in leaf morphology
among the Murraya elongata plants from different locations as shown by ANOVA
(Table 7.8), no actual separation between the accessions were obtained with the PCA
(Figure 7.13). In particular, there appears to be no morphological separation of
accessions from the two clades of Murraya elongata identified in the phylogenetic
analysis, and the variation in leaf shape within a plant appears to be as great as the
variation between plants; this suggests that they form a single group. However, the
sampling is only from 71 leaflets from 5 plants and needs to be extended. The
comparison of leaf morphology between Murraya paniculata, Murraya heptaphylla,
Murraya elongata, and Murraya sumatrana resulted in three separate groups: one
group containing Murraya paniculata, the second containing Murraya heptaphylla
and the third group comprising of Murraya elongata and Murraya sumatrana (Figure
7.15). This indicates that leaves of Murraya elongata and Murraya sumatrana are
morphologically similar.
Duminil & Di Michele (2009), in their review of comparing molecular and
morphological markers for plant species delimitation, noted certain incongruence of
species identification based on the two methods. They (2009) concluded that higher
species delimitation associated with molecular markers mostly corresponded to
cryptic species wherein morphologically similar individuals were separated as
different species molecularly. In contrast, higher species delimitations based on
morphology corresponded to species varying morphologically due to their adaptation
to local environment (phenotypic plasticity) (Duminil & Di Michele 2009). Cryptic
species have been reported in liverworts (Marchantiophyta: Conocephalaceae),
cellular slime moulds (paraplyletic Prostista: Dictyosteliidae), ferns (Pteridophyta:
Pteridaceae) (Odrzykoski & Szweykowski 1991), Drapa species (Brassicaeae) in the
artic (Grundt et al. 2006), Mitella (Saxifragaceae) (Okuyama & Kato 2009), and in
orchids (Orchidaceae) (Efimov 2013). Shneyer and Kotseruba (2015) noted that
cryptic species are rarely described or named after their discovery, as it is
complicated to do so according to all rules. Based on a comparison of leaf shape and
molecular phylogenetics, Murraya sumatrana and Murraya elongata may represent
cryptic species. This similarity in leaf shape also poses a problem if taxa can only be
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sampled when in their vegetative phase. However, the shape of the fruit of the two
taxa appear different with plants of Murraya elongata from Bhutan and China having
globose fruit and seeds (Figures 7.11 & 7.22) and Murraya sumatrana producing
long, ellipsoid fruit with mammiform apex and spindle-shaped seeds (Figure 7.23).
The formation of globose fruit by Murraya elongata needs to be confirmed, but both
fruit shape and place of origin appear to separate the two taxa.
Results of the current studies also provide support for the findings of Nguyen (2011)
wherein both molecular and morphological studies showed distinct separation
between accessions that appear to originate from mainland Asia (Murraya elongata
(Bhutan, Việt Nam and China), from southeast China (Murraya paniculata) and
those that have a Malesian or Australasian origin (Murraya sumatrana and Murraya
heptaphylla). Therefore, using both morphology and molecular phylogenetics, the
accessions of Murraya from Bhutan could be placed within the taxa proposed by
Nguyen (2011). The current study provides further evidence to support the argument
for the division of taxa regarded as orange jasmine (Murraya paniculata, sensu
Mabberley (2016) into four species. Two of the four species occur in Bhutan:
Murraya paniculata and Murraya elongata. Murraya paniculata is a widely grown
ornamental known as ‘orange jasmine’, and its occurrence in Bhutan is not surprising
especially in towns near Bhutan–Indian border. Murraya paniculata was recorded as
being introduced to India from China and grown in gardens (Edwards & Lindley
1819-1820; Roxburgh 1832). Roxburgh (1832) also observed its occurrence in the
wild in the mountains of the Cicars (present day northeast coast of Andhra Pradesh)
(Edwards & Lindley 1819–1920). Hooker (1875) described Murraya elongata A.
DC. (Figure 7.24) (from Taong–dong, Myanmar (Burma) but noted it was very
different to the forms of ‘Murraya exotica’ also occurring in the region. Murraya
elongata appears to be associated with hilly, mountainous, seasonally-dry woodlands
associated with limestone and granite areas (Kong et al. 1986; Huang 1997) from
Pakistan through India, Myanmar, Thailand, peninsular Malaysia, Laos, Việt Nam to
southern China (Hooker 1875; Kong et al. 1986; Huang 1997; Nguyen 2011).
Therefore, its discovery at Baychhu and Reldri in Bhutan is not surprising. The
discovery adds to our knowledge of biodiversity in Bhutan and Asia.
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Figure 7.22. Fruit of Murraya elongata from Yingde, China (Photo: GAC Beattie). Coin
=28.65 mm diameter.
Figure 7.23. Fruit and seeds of Murraya sumatrana from Bogor Botanic Garden,
Indonesia (Photo: Nguyen Huy Chung).
279
.
Figure 7.24. Murraya elongata A. DC. ex Hook. Wallich Herbarium type specimen.
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7.4.2 Phylogenetic relationships of wild citrus from Bhutan
In angiosperms, the cpDNA is maternally inherited and the nuclear ITS region is
biparently inherited. This provides the possibility of examining relationships due to
hybridization (Doebley 1992; Olmstead & Palmer 1994). Baldwin et al. (1995)
stated that lineages of hybrid populations do not form monophyletic groups, but such
individuals will group with non-hybrid parents possessing the orthologous nuclear
ribosomal DNA. In the current study, wild citrus accessions from Bhutan were
evaluated to determine their phylogenetic relationships based on a combination of
four cpDNA genes and their ITS region.
In the cpDNA analysis, the Bhutanese wild citrus accessions clustered with Citrus
latipes, Citrus maxima and hybrid accessions and appears to be more distantly
related to Citrus cavaleriei, Citrus medica and Citrus reticulata. The presence of
Citrus latipes and Citrus maxima in the same cluster as the wild citrus accessions
from Bhutan seem to indicate a possible maternal relationship of these two species
with the wild citrus accessions. This is further supported by the clustering of hybrid
accessions that have Citrus maxima as one of the ancestral parents: Citrus ×
aurantium (Citrus maxima and Citrus reticulata); Citrus × aurantium (‘paradisi’)
(backcross between Citrus maxima and Citrus × aurantium); C.× aurantium
(‘sinensis’) (Citrus reticulata and Citrus maxima), Citrus maxima (‘obovoidea’) (a
probable hybrid between Citrus maxima and Citrus reticulata (Hodgson 1967)), and
Citrus × limon [(Citrus medica (citron) × Citrus × aurantium)]. Based on the
cpDNA analysis of hybrids involving either Citrus medica or Citrus maxima, Citrus
maxima has always been proposed as the maternal parent, while Citrus medica has
been proposed as the paternal parent (Nicolosi et al. 2000; Barkley et al. 2006; Li et
al. 2010; Carbonell-Caballero et al. 2015). Citrus × aurantium (‘platymamma’) and
Citrus × aurantium (‘sinensis’) clustered together indicating their close relatedness.
Lee et al. (2015) characterised the whole chloroplast genome of Citrus × aurantium
(‘platymamma’) and found it closest to Citrus × aurantium (‘sinensis’) (Bausher et
al. 2006). Penjor et al. (2014), based on the coding region of matK, showed that the
wild citrus accession that they collected from Mongar, Bhutan is closer to Khasi
281
papeda which is Citrus latipes. This is consistent with the cpDNA results obtained in
the current study, where the wild citrus accessions grouped in same cluster as Citrus
latipes. The current study used four cpDNA (matK-5’trnK intron, psbM-trnD, trnLtrnL-trnF, rpS4-trnT).
The ITS analysis resulted in a poorly resolved tree. One accession of Citrus maxima
taken from GenBank (JQ990179) clustered with the mandarins in this analysis. This
could be a misidentification or a hybrid of Citrus reticulata, as other accessions of
Citrus maxima are in the cluster that contains Citrus medica, Citrus indica, Citrus
hystrix, and Citrus latipes together with NCBT.3 (Clade 4). Kumar et al (2012) also
found Citrus latipes grouping with Citrus hystrix using ITS sequences. With the
exception of accession NCBT.3, the wild citrus accessions cluster together with weak
bootstrap support and appeared to be equally related to Citrus reticulata and its
hybrids, as to Citrus cavaleriei, Citrus latipes, Citrus maxima and others.
In
contrast, accession NCBT.3 clustered in a subgroup with accessions of Citrus latipes,
Citrus hystrix, Citrus indica, Citrus medica and Citrus maxima indicating its closer
relationship to these accessions than to the other Bhutanese accessions.
The exact nature of the Bhutanese accessions is problematic.
Based on their
morphology and molecular biology, these accessions clearly separate into two
paternal groups: Clade 3 in Figure 7.21 comprising accessions NCBT.1, 5, 7 and 9,
and Clade 4 in Figure 7.21 comprising accession NCBT.3. The sequencing results
suggest that the accessions from the two groups have the same maternal parent. With
respect to the chloroplast sequences (Figures 7.19 & 7.20), the clade to which all
Bhutanese accessions belong contains two true species, Citrus latipes and Citrus
maxima, and all accessions are closer to these two species than to Citrus cavaleriei.
With respect to their nuclear genomes, the results from sequencing the ITS region
suggest that the accessions in Clade 3 in Figure 7.21 are closer to Citrus cavaleriei
and a number of other types than to Citrus latipes and Citrus maxima. In contrast,
the ITS regions of NCBT.3 is more similar to Citrus latipes and other members of
Clade 4 in Figure 7.21 than to Citrus cavaleriei. Therefore, NCBT.3 has a different
paternal parent from the rest. These results also suggest that Bhutan accessions in
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either group are not true Citrus species, with the molecular data suggesting that
accessions NCBT 1, 5, 7 and 9 in Clade 3 in Figure 7.21 represent a hybrid taxon and
that NCBT.3 could be Citrus latipes.
However, the morphology of accession NCBT.3 strongly suggests that it is not
Citrus latipes. Firstly, the leaves of Citrus latipes have winged petioles whereas the
petioles NCBT.3 are not winged.
Secondly, the fruit of Citrus latipes are not
mammillate whereas those borne by NCBT.3 are mammillate.
There is some
resemblance with Citrus hystrix (‘macroptera’), as the petioles of certain accessions
are not winged and the fruit are mammillate. It also resembles Citrus medica in
terms of both having wingless petioles and fruits with mammillated apex.
Concerning the other species in Clade 4 in Figure 7.21, the leaves do not resemble
Citrus maxima, as the petioles of this species have small wings and the fruit are large
and not mammillate. The leaves of Citrus hystrix have winged petioles and the fruit
colliculate. Citrus indica has leaves without winged petioles but the fruit are not
smooth and round in shape. In addition, the fruit and leaves of NCBT.3 do not
resemble those of Citrus cavaleriei, as the leaves of the latter have winged petioles
and the fruit are ellipsoid with a surface that is colliculate. The morphology of
NCBT.3, together with its molecular biology, suggests that it is a hybrid with
possibly Citrus latipes as the maternal parent and Citrus hystrix (‘macroptera’) or
Citrus medica as the male parent. Citrus medica is reported to be growing in the
same location from where accession NCBT.3 was obtained (Penjor, pers. comm.).
My NCBT.1, 5, 7 and 9 accessions had winged petioles and the ellipsoid fruit with
colliculate surfaces resembling leaves and fruit of Citrus cavaleriei as illustrated by
Penjor et al. (2014) and Swingle & Reece (1967). The leaves, but not the fruit, of my
NCBT.1, NCBT.5, NCBT.7 and NCBT.9 accessions also resembled description of
Citrus latipes (Swingle & Reece 1967; Grierson 1991). The flowers of plants at
Basochhu were mostly borne singly (Figure 7.17B) between nodes and not in the leaf
axils. Swingle & Reece (1967) noted that the flowers of ‘Citrus ichangensis’ are
borne singly in the axils of the leaves and those of Citrus latipes are, at least
sometimes, borne in small axillary racemes with 5–7 flowers. Grierson (1991)
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described the flowers of Citrus latipes as solitary or in short racemes, and Zhang et
al. (2009) described the flowers of Citrus cavaleriei as flowers solitary or to 9 in
fascicles (Zhang et al. 2009). These descriptions are not consistent.
Based on morphology and results for matK, Penjor et al. (2014) concluded that their
accession B07001 from near Wengkhar, Mongar, was a Citrus cavaleriei-relative.
However, the matK results also grouped the accession with Citrus latipes. My
NCBT.1, NCBT.5, NCBT. 7 and NCBT.9 accessions from Wengkhar and Basochhu,
and accession B07001 of the Citrus cavaleriei-relative studied by Penjor et al.
(2014), were all collected at elevations ranging from 1900 to 2100 m ASL. At these
elevations temperatures in winter drop below freezing and snowfalls can occur
(Penjor et al. 2014). Swingle & Reece (1967) regarded Citrus cavaleriei as the most
cold-tolerant species of Citrus. Grierson (1991) in Grierson & Long (1991) ‘Flora of
Bhutan’ mentions Citrus latipes occurring between 1920 m and 2100 m ASL in
Bhutan. He did not mention Citrus cavaleriei (Citrus ichangensis). Swingle &
Reece (1967) noted that Citrus latipes occurs in the Khasi Hills in north-eastern
India, and in northern Myanmar, between 500–1830 m ASL. Thus, my NCBT.1,
NCBT.5 and NCBT.9 accessions appear to represent a hybrid of cross between
Citrus latipes as the maternal parent and Citrus cavaleriei as the paternal parent. My
results, and those of Penjor et al. (2014), suggest that one or more hybrids may be
included in descriptions of Citrus cavaleriei by Swingle & Reece (1967) and Zhang
et al. (2009).
At the moment, the taxonomy of the wild citrus accessions in the current study must
remain unresolved. Citrus taxonomy and phylogeny has always been complicated
and confusing due to high rate of hybridisation and apomixis coupled with long
history of cultivation (Mabberley 1997; Nicolosi et al. 2000; Barkley et al. 2006).
Although, the ITS region has been used for phylogenetic studies at lower taxonomic
levels, sometimes they have reduced utility due to high sequence divergence, wide
variation in sequence length, paralogy problems or lack of resolving power (Li et al.
2007b). However, such observations with the ITS region could rather be due to the
nature and number of samples used. There is a need to sequence a greater number of
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genes from a greater range of possible parents and for further molecular techniques
to be employed to deduce their lineage.
7.4.3 Detection of ‘CLas’ in Murraya and wild citrus
All samples of Murraya elongata produced FAM Ct values ranging from 32.15 to
36.17 except for one that produced FAM Ct value equal to zero. Samples of the wild
citrus produced FAM Ct values of 34.55 to 35. 95. The FAM Ct values of both
Murraya elongata and wild citrus accession NCBT.9 that produced FAM Ct > 0.00
were within the FAM Ct value cut off for ‘CLas’ positive. However, no amplicons
could be obtained with the selected samples of both Murraya elongata and wild
citrus samples when subjected to cPCR with primers A2/J5. Based on the Ct values,
the samples of Murraya elongata and wild citrus can be interpreted as ‘CLas’
positive especially for those samples with FAM Ct values in the range of 0.00 <
FAM Ct < 36. Walter et al. (2012a) did not detect ‘CLas’ using the 16S r DNA
primers-probe set of Li et al. (2006) in samples of Murraya paniculata regarded as
‘CLas’ negative using the LJ900 primers targeting the tandem repeats of the
hyvI/hyvII of the bacterium. Walter et al. (2012a), however, noted that the bacterial
titre was generally lower in Murraya paniculata samples than in citrus, and
mentioned that one of the ‘LJ900-positive’ samples was amplified with a Ct value of
38.2 using the 16S primers. Similar results were obtained in another study by Walter
et al. (2012b), where only three of the 19 samples of Murraya paniculata produced
‘CLas’ Ct values of 37.43–40.58 using the 16S primers compared to all 19 samples
producing ‘CLas’ Ct values that ranged from 26.55-37.50 using the LJ900 primers.
Walter et al. (2012b) commented that the discrepancies in the results between the
two primers could be due to higher sensitivity of the LJ900 primers method resulting
from generating higher copies of the hyvI/hyvII compared to the 16S gene. Lopes et
al. (2010) also observed lower titres of ‘CLas’ in Murraya paniculata (syn. Murraya
exotica) than in citrus. Therefore, it is possible that the Murraya elongata samples in
the current study may have been infected with ‘CLas’ but titres of the pathogen may
have been too low to be detected by cPCR as shown by the FAM Ct range of 32.15
to 36.17. This suggests that Murraya elongata may be a poor and transient host of
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‘CLas’ and, therefore, of inconsequential importance in the spread of HLB. If it is
the original host of Diaphorina citri it is unlikely to have been plant species from
which the psyllid acquired ‘Candidatus Liberibacter asiaticus’.
7.5 Highlights of the study:
Murraya elongata from Bhutan, and Guangdong and Guangxi in China, form
a monophyletic group based on the combination of five cpDNA and the ITS
region. Both morphological and molecular data show that Murraya elongata
accessions from mainland Asia including the Bhutanese accessions are
different from Murraya sumatrana and Murraya heptaphylla of Malesian or
Australasian origin.
Murraya paniculata from Bhutan grouped with other accessions of this taxon
from other parts of the world.
The phylogeny of the wild citrus based on the four cpDNA and ITS analysis
is not resolved. The current molecular results and the morphologies of the
citrus accessions indicate that accessions NCBT.1, NCBT.5, NCBT.7 and
NCBT.9 represent a hybrid of Citrus latipes and Citrus cavaleriei while
accession NCBT.3, which is morphologically different from the other
accessions may be a hybrid of Citrus latipes on the maternal side and Citrus
hystrix or Citrus medica on the paternal side.
Murraya elongata is of inconsequential importance in the spread of HLB.
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Chapter 8: General Discussion
____________________________________________________________________
The specific objectives of my research were to determine:
the phylogenetic relationships between Diaphorina communis and Diaphorina
citri;
whether Diaphorina communis can acquire ‘Candidatus Liberibacter asiaticus’
from mandarin and transmit it from mandarin to mandarin and from mandarin
to curry leaf or vice versa, and to study the host preferences of Diaphorina
communis;
the molecular and morphological characteristics of the parasitoid of
Diaphorina communis, and to determine the phylogenetic relationships
between the parasitoids collected from Diaphorina communis and Diaphorina
citri;
the effect of altitude, ambient temperature and relative humidity (RH) on leaf
temperature, and determine the incidences and types of psyllids, and
prevalence of ‘CLas’ at different elevations;
the morphological and molecular characteristics of the green psyllids found on
mandarin and wild citrus, and other psyllids found on an unidentified
Zanthoxylum sp., and to determine the presence of ‘CLas’ within the green
psyllids and their host plants; and,
the phylogenetic and morphological relationships of species of Murraya
occurring in Bhutan with those proposed by Nguyen (2011), to determine the
phylogenetic relationship of the wild citrus species found in Bhutan in relation
to other Citrus species and hybrids, and whether the Murraya species and wild
citrus found in Bhutan can be hosts for ‘CLas’;
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8.1
Key findings and their significance
8.1.1 Phylogenetic relationships of Diaphorina communis and
Diaphorina citri
I studied the phylogenetic relationship between Diaphorina communis and
Diaphorina citri based on the COI and 16S regions. My results showed two distinct
clades, one comprising of Diaphorina communis and the other of Diaphorina citri;
variation within each clade exists. This was the first molecular study characterising
Diaphorina communis. My molecular data indicate that the COI and the 16S regions
can be used to distinguish Diaphorina citri from Diaphorina communis.
This
knowledge is useful for making decisions on psyllid management, particularly if
dealing with immature psyllid stages where morphological identification is difficult.
8.1.2 ‘Candidatus Liberibacter asiaticus’ and Diaphorina communis
Diaphorina communis adults can acquire ‘CLas’ from infected mandarin but at a low
rate as evidenced by the low numbers of adults becoming infected at the end of the
acquisition and transmission interval. There is no evidence of curry leaf being a host
of ‘CLas’, and there is no evidence for transmission of ‘CLas’ by Diaphorina
communis from mandarin to curry leaf or mandarin and vice versa. This conclusion
is based on the results of a combination of qPCR and sequencing. Even though the
results of some of the qPCR analyses showed the Ct values for ‘CLas’ to be within
the range of > 0 ≤ 36, a range in plant assays that is deemed as positive for ‘CLas’,
these results could not be confirmed by sequencing and were deemed inconclusive.
I observed adult Diaphorina communis on mandarin, Murraya elongata, curry leaf
and Zanthoxylum sp., and the psyllid could possibly be feeding on these plants in the
field. However, I found that, given the choice, Diaphorina communis prefers to feed
on curry leaf and exclusively develops on this species. Diaphorina communis was
first observed on curry leaf and described in India (Mathur 1935; 1975), and the
results of my studies appear to indicate that curry leaf is the original host of
Diaphorina communis.
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It has been shown that ‘CLas’ multiplies faster in Diaphorina citri that fed on
‘CLas’-infected plants as nymphs than in psyllids that fed as adults, and successful
transmission of ‘CLas’ to healthy host plants occurred only from those psyllids that
had acquired the pathogen as nymphs (Inoue et al. 2009; Pelz-Stelinski et al. 2010;
Ammar et al. 2016). Considering that nymphs of Diaphorina communis cannot
survive on mandarin, and adults may require longer acquisition and multiplication
periods, the role of Diaphorina communis as vector of ‘CLas’ is concluded to be
negligible.
8.1.3 Comparison of parasitoids of Diaphorina citri and Diaphorina
communis
When I started my studies, the only information available on Diaphorina communis
were the biological descriptions by Mathur (1935; 1975), and that the psyllid
harboured the HLB pathogen (Donovan et al. 2012a; 2012b); knowledge on its role
in the epidemiology of HLB was non-existent. Assuming that the psyllid could
transmit ‘CLas’ and assuming that there would be a need to manage the psyllid, part
of my studies was to investigate the existence of natural enemies of the psyllid. The
parasitoid that I reared from nymphs of Diaphorina communis collected from
Bergera koenigii is a new species of Tamarixia based on molecular phylogenetic
analyses that I conducted and on the morphological descriptions by Zoya Yefremova
(Department of Zoology, The Steinhardt Museum of Natural History) and Ekaterina
Yegorenkova (Department of Geography and Ecology, Ulyanovsk State Pedagogical
University). The new species is named as Tamarixia drukyulensis sp. n.
8.1.4 Incidence of psyllids and ‘CLas’ at different elevations
Prior to my studies, citrus plants grown at altitudes of 1200 m and above in Tsirang
grow well compared to citrus grown in areas below 1200 m, and Diaphorina citri
was generally not observed above this elevation, with the exception of Diaphorina
citri nymphs recorded in the Dzamling Zor Gewog (1380 m ASL) of Tsirang
Dzongkhag by NPPC-ACIAR team in 2009. I hypothesised that the low incidence
and severity of ‘CLas’ and Diaphorina citri contributed to better growth of citrus at
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higher altitudes, and that rapid transmission of ‘CLas’ would impede their growth at
the lower altitudes. I also hypothesised that the low incidence and severity of both
the pathogen and the psyllid was due to higher leaf temperatures as a result of higher
relative humidity and prevailing ambient temperatures at these elevations.
My
investigations involving eight sites ranging from 800 m to 1500 m ASL along
Droopchhugang-Phuensoomgang in Tsirang revealed that:
mandarin seedlings planted along Droopchhugang-Phuensoomgang grew better
at 800–1000 m ASL than seedlings planted at 1100 and 1500 m ASL based on
the number of leaves on terminal shoots
leaf temperatures were lower than the mean maximum ambient temperatures at
all locations.
ambient temperatures and relative humidities at all elevations were within the
the favourable range for development and survival of Diaphorina citri.
in Tsirang, Diaphorina citri was found from 800 to 1100 m ASL on mandarin
(the result does not imply that Diaphorina citri did not occur below 800 m
because this was not assessed during the study).
more plants (both mandarin seedlings and mature trees) at the lower altitude were
infected with ‘CLas’ compared to plants at higher elevations. Based on the
results of bark samples, ~40–80% infection occurred in the mandarin seedlings
planted at the beginning of the study in 2013 at lower elevations of 800 to 1000
m ASL compared to 0% infection at ≥ 1100 m ASL; 100% infection in mature
mandarin trees at 800–1100 m compared to 0–20% ≥ 1200 m ASL.
These findings indicate that leaf temperature is not a limiting factor for both
Diaphorina citri and ‘CLas’ incidences in Tsirang. The results of my studies and the
observations reported in earlier studies (Aubert 1987 & 1990; Chavan &
Shummanvar 1983; Beattie et al. 2010; Jenkins et al. 2015) regarding the effect of
temperature, humidity and elevation on Diaphorina citri suggest that interactions
between monsoon rainfalls and temperature are more likely to impact Diaphorina
citri and may limit the distribution at higher altitude as the ambient temperature
decreases with increasing elevations. However, the results obtained in the current
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study revealing an abrupt change in the incidence of Diaphorina citri between sites
at 1100 m and 1200 m suggest that ambient temperature may not be solely
responsible for this. The annual average ambient temperatures at 1200 m are higher
than at 1100 m by ~1°C and 3°C for the annual minimum and maximum
temperatures respectively (Table 5.4 of Chapter 5).
My investigations also looked at the incidence of Diaphorina communis and
Cacopsylla heterogena along the study sites. The results showed that Diaphorina
communis never occurred on mandarin at the observed elevations, although adults
and nymphs of Diaphorina communis were observed on Bergera koenigii at 800, 900
and 1000 m.
This observation of Diaphorina communis on Bergera koenigii
corroborates the findings of the host preference test in Chapter 3, and seems to
suggest that the distribution of Diaphorina communis may be restricted by the
distribution of Bergera koenigii. Cacopsylla heterogena, as identified in Chapter 6,
was found from 1300 m to 1500 m ASL in the experimental plots in Tsirang.
Although the current study assessed only the incidence of the psyllids, the population
sizes and the foliar damage due to Cacopsylla heterogena appeared to decrease with
decreasing altitude.
The higher incidence of ‘CLas’ at the lower altitudes of 800 m to 1100 m ASL
compared to at 1200 m and above could be related to the presence of the vector,
Diaphorina citri, in these low areas.
The occurrence of ‘CLas’ at the higher
elevations was probably due to introduction of infected plants during orchard
establishment.
Therefore, in the absence of the vector and with appropriate
agronomic practices, it is possible to grow citrus in Bhutan above 1200 m, although
different varietiel selections would need to be grown to suit the conditions at the
higher elevation (Susanto et al. 2013; Rokaya et al. 2016).
Based on these results, factors other than ambient temperature, relative humidity and
rainfall limit the occurrence of Diaphorina citri and ‘CLas’ at ≥ 1200 m in Tsirang.
It is likely that UV radiation, could be a possible limiting factor.
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8.1.5 Cacopsylla heterogena and ‘CLas’
I observed Cacospylla heterogena, (a species originally described from China),
infesting mandarin in Tsirang in 2013 and, subsequently, this species was found also
on lime, lemons and orange trees in Tsirang, and on wild citrus in Wengkhar,
Mongar and in Basochhu, Wangdue Phodrang (Chapter 7). Around the same time, I
also observed two species of green psyllids on an unidentified Zanthoxylum sp. in
various places in Tsirang and in Baychhu, Wangdue Phodrang.
Morphological
identification of Cacopsylla heterogena was conducted by Susan Halbert
(Taxonomic Entomologist, Division of Plant Industry, Florida Department of
Agriculture and Consumer Services) and Luo Xinyu (Department of Entomology,
China Agricultural University).
One of the two species on Zanthoxylum was
identified as Cornopsylla rotundiconis (Luo et al. 2012) and the other is an
undescribed Cacopsylla sp. (Luo Xinyu, pers. comm.).
I investigated the phylogenetic relationships among the species of the green psyllids,
and investigated if these psyllids could harbour the HLB pathogen using qPCR. I
also obtained three specimens of Cacopsylla citrisuga from Yunnan, China through
Dr Cen Yijing, South China Agricultural University to compare with the other
psyllids in the molecular analyses. Cacospylla citrisuga is another green psyllid
described from China and is similar to Cacopsylla heterogena morphologically (Li
2011), and has been reported to harbour and transmit ‘CLas’ in China (Cen et al.
2012a; 2012b) but not formally published.
The molecular phylogenetic analyses based on two mitochondrial genes (COI &
16S), and one nuclear gene (ITS) revealed that three distinct species of green psyllids
occur in Bhutan: the green psyllid infesting citrus in Bhutan belonged to one species,
Cacopsylla heterogena; and two species on Zanthoxylum sp. (Cornopsylla
rotundiconis and an unidentified Cacopsylla sp). Both Cornopsylla rotundiconis and
the unidentified Cacopsylla sp. are morphologically and molecularly distinct from
Cacopsylla heterogena and Cacopsylla citrisuga.
However, the phylogenetic
relationship of Cacopsylla heterogena to Cacopsylla citrisuga could not be resolved
based on the current molecular analyses. Data based on the mitochondrial genes
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suggest that Cacopsylla heterogena is a separate species from Cacopsylla citrisuga
but analysis of the ITS region showed otherwise.
I tested DNA samples from adults and nymphs of Cacopsylla heterogena collected
from various locations for ‘CLas’ using qPCR. I also tested samples of the host
plants on which Cacopsylla heterogena was collected. In most locations, the host
plants of Cacopsylla heterogena tested positive for ‘CLas’.
However, my
investigation showed that none of the psyllid samples collected in 2015 were positive
for ‘CLas’, even though some insects were collected from an area where some of the
host plants were ‘CLas’-positive (e.g., some of the lemon trees in the RDC, Tsirang).
From the samples collected in 2016, only 2% of the adults sampled (total adults =
185) were positive for ‘CLas’. The host plants sampled from the same location as
the positive psyllids also tested positive for ‘CLas’. This indicates a low acquisition
rate by Cacopsylla heterogena. However, I did not obtain any positive results from
DNA extracts made from nymphs. Further, I could not obtain any evidence of
‘CLas’ in Cornospylla rotundiconis and the undescribed Cacopsylla sp. and their
host plant, Zanthoxylum sp., even though the plant sampled exhibited strong mottling
symptoms (Figure 8.1) and low FAM Ct values. Unlike Diaphorina communis,
Cacopsylla heterogena feeds and develops on citrus, which means that the nymphs
have the opportunity to acquire the pathogen. However, this does not seem to be the
case based on the current results—no detection or low detection in nymphs and
adults, respectively.
Thus, the results of my studies indicate that Cacopsylla
heterogena poses little or no threat to citrus production in the higher elevations
where it usually occurs.
This complements the results of my studies on the
distribution of Diaphorina citri in Tsirang (Chapter 5) suggesting that it may be
feasible to grow citrus at higher elevations (1200 m and above) due to the absence of
Diaphorina citri. My results also indicate that the psyllids on Zanthoxylum sp. are
not a threat to citrus.
My work on the phylogenetics of Cacopsylla heterogena, Cornopsylla rotundiconis,
and the undescribed Cacopsylla sp. is the first of its kind. There is limited molecular
data on psyllids, and my results contribute to the information within molecular
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databases that can be used in studies of psyllid phylogenetics.
Molecular data
support morphological identification and support psyllid identification especially
when dealing with species complexes and cryptic species. During my work on the
detection of ‘CLas’ in the green psyllids, I also designed a set of primers, WGCaF
and WGCaR (based on the primers (WGf and WGr) of Li et al. (2008)) to amplify
the wg gene of the Cacopsylla species. This primer pair can be multiplexed with
TaqMan probes for wg (Li et al. 2008) and TaqMan probes for ‘CLas’ with the
primers for ‘CLas’ (Li et al. 2006) for the detection of ‘CLas’. This knowledge is of
use in diagnostic testing.
Figure 8.1. Leaflets of Zanthoxylum sp. with mottling symptoms.
8.1.6 Murraya species and ‘CLas’
My phylogenetic studies confirmed that the Murraya plants occurring in Baychhu,
Wangdue Phodrang and in Reldri along Rinchending-Pasakha at Phuentsholing were
Murraya elongata. Further, the analyses (using five cpDNA and the ITS region)
established that the accessions of Murraya elongata from Bhutan grouped with the
accessions from Yingde and Guangxi in China. The analyses also differentiated
Murraya elongata accessions from mainland Asia including the Bhutanese
accessions from accessions of Murraya sumatrana and Murraya heptaphylla that are
of Malesian or Australasian origin. Comparisons of leaf morphology differentiated
Murraya paniculata and Murraya heptaphylla from Murraya elongata and Murraya
sumatrana but indicated that leaves of the latter two species are morphologically
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similar. This similarity in leaf morphology emphasises the importance of sampling
flowers and fruits for morphological descriptions. The morphological differentiation
between Murraya sumatrana and Murraya elongata in my study was, therefore,
based on the shape of the fruit as plants of Murraya elongata from Bhutan and China
have globose fruit and seed whereas Murraya sumatrana produces long, ellipsoid
fruit with mammiform apex and spindle-shaped seeds as shown in Nguyen (2011).
The accession of Murraya that I collected from Zantopelri Park in Phuentsholing is
Murraya paniculata based on both molecular and morphological analyses; this plant,
commonly known as orange jasmine is grown as an ornamental around the world.
My study identified two species of Murraya (elongata and paniculata) occurring in
Bhutan, and provides the first description of fruits of Murraya elongata in addition to
molecular data available for future phylogenetic studies.
I observed adults of both Diaphorina citri and Diaphorina communis on Murraya
elongata during sample collection in Baychhu in 2014. I also found that the eggs
and young nymphs on Murraya elongata observed in April 2015 belonged to
Diaphorina citri based on the adults eclosed in the caged branches. This is the first
record of development of Diaphorina citri on Murraya elongata.
Previous reports have indicated that Murraya paniculata, the widely cultivated form,
is a reservoir of ‘CLas’ when evaluated under laboratory conditions (Deng et al.
2007; Zhou et al. 2007), in field experiments (Walter 2012a.) and in field-collected
samples (Lopes et al. 2010; Walter 2012b). Therefore, I tested the Murraya species
for ‘CLas’ using qPCR, followed by cPCR in an attempt to amplify and sequence
some of the samples deemed as positive by qPCR. However, the cPCR using
primers A2/J5 (Hocquellet et al. 1991) did not yield any amplicons from any of the
Murraya elongata samples; thus, no sequence data was obtained. Based on these
results, it can only be stated that Murraya elongata is a putative host of ‘CLas’ and
unlikely to be important in the spread of HLB. Populations of the psyllid I observed
on Murraya elongata were always low, suggesting that if it is the original host of
Diaphorina citri, populations of the psyllid in natural environments (in understories
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of forests) may also be low in contrast to populations that occur on Murraya exotica,
a plant introduced to the subcontinent.
8.1.7 Identity of wild citrus and testing of accessions for ‘CLas’
During the course of my research, a wild citrus taxon, having leaves with large,
winged petioles and infested with Cacopsylla heterogena was observed at near
Wengkhar in eastern Bhutan and at Basochhu in Wangdue Phodrang.
Four
accessions of this wild citrus (NCBT.1, 5, 7 & 9) together with a further wild citrus
accession that had ovate leaves and petioles that are not winged (NCBT. 3, sourced
from Rimchhu) were molecularly and morphologically characterised. However, I
could not obtain sufficient evidence to resolve the identity of these wild citrus
accessions. The molecular data showed that NCBT.1, 5, 7, & 9 share the same
maternal parent with NCBT.3 but have a different paternal parent. The results of my
study also suggested that these accessions may not be true Citrus species but rather
hybrids: NCBT. 1, 5, 7 & 9 are possibly hybrids of Citrus latipes and Citrus
cavaleriei, and NCBT.3, may be a hybrid of Citrus latipes on the maternal side and
Citrus hystrix or Citrus medica on the paternal side. I have no evidence of the
presence of ‘CLas’ in any of the wild citrus accessions that I tested. However, this
could be because the wild citrus plants were located in wild areas where ‘CLas’ may
not be present, and does not rule out the potential of these plants to harbour the
pathogen.
8.2
Future research
8.2.1 Diaphorina communis and ‘CLas’
Many factors influence vector-pathogen relationships and vector competence such as
variation in environmental factors, genetic variation associated with the pathogen or
the vector, pathogen dose, and age of the vector (Tabachnick 2015). For Diaphorina
citri, higher acquisition and transmission rates are associated with acquisition by the
psyllid in its nymphal stages (Inuoe et al. 2009; Pelz-Stelinski et al. 2010; Ammar et
al. 2016) and with longer acquisition periods experienced by adults (Pelz-Stelinski et
al. 2010; Ammar et al. 2016).
These observations seem to explain the low
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acquisition of ‘CLas’ by Diaphorina communis in the current study; however, it
would be interesting to explore the:
gut flora of Diaphorina communis in comparison with that of Diaphorina
citri to determine whether it could have a role in ‘CLas’ suppression; and,
the mechanism of resistance to ‘CLas’, if any, in Diaphorina communis that
can be exploited in Diaphorina citri.
8.2.2 ‘Candidatus Liberibacter asiaticus’ and Ct values
The most widely used diagnostic method for detection of ‘CLas’ uses real-time PCR
(qPCR) and the primers, probes and conditions developed by Li et al. (2006); this
method was used in the current studies. In my studies, I interpreted all results based
on Ct values set out in the guideline by USDA-APHIS (2012) as being ‘CLas’
positive for plant assay if the FAM Ct values (flurophore for ‘CLas’ DNA) were in
the range of 0.00 < FAM Ct ≤ 36 or 0.00 < FAM Ct < 32 for psyllid assays. For
plant assays, Ct = 36 is said to achieve greater sensitivity (the probability of detection
in a truly infected plant material) with minimal loss of specificity, the probability of
non-detection in a healthy plant (Turechek et al. 2012). Values between 36–40 or
32–40 for plants and psyllids, respectively, are said to be inconclusive which requires
further evaluation. In the current studies, regardless of the Ct value threshold cut-off,
I further evaluated samples that produced FAM Ct values > 0.00 through sequencing
with primers A2/J5 (Hocquellet et al. 1999). I obtained sequencing data in few
instances where the FAM Ct values for plant samples were above the Ct threshold
(e.g., 37.46 and 37.75) but failed to obtain amplification for samples with FAM Ct
values of ~34 to 35 (Chapter 5). This reaffirms the interpretation that samples
producing Ct values between 36 and 40 are inconclusive but casts doubt over those
that produce Ct values < 36, yet fail to yield amplicons when subjected to cPCR; are
these false positives or do these values mean that the bacterial titres are below
detection level by cPCR? If so, then what about the samples that produce Ct values
greater than 36 and, yet, give amplicons. The instance where the samples of the
mottled Zanthoxylum sp. produced FAM Ct values of 20.98 and 23.18 (Chapter 6), is
another case where the samples did not yield any amplicons when subjected to cPCR
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and, therefore, were classified as false positives.
These findings make the
interpretation of results problematic whether it is for diagnostic or research purposes.
Recent studies on ‘CLas’ transmission and detections are based on qPCR Ct value
thresholds ranging from 32 to ≥ 40 as being positive for ‘CLas’ (Li et al. 2007; PelzStelinski et al. 2010; Walter et al. 2012a; Ramadugu et al. 2015; Ammar et al. 2016)
and, additionally, establishing a standard curve with a known quantity of ‘CLas’
DNA as a reference DNA sample (Li et a. 2006; Li et al. 2008; Manjunath et al.
2008; Lopes et al 2013). Studies by Kunta et al. (2014) conducted comparative
assays using different primer sets for both conventional and real-time PCR for
detection of ‘CLas’ in leaf and root samples of ‘Valencia’ sweet orange grafted on
sour orange. Their studies showed the root samples that yielded ‘CLas’ positive Ct
values (22.90 to 31.78) with the 16S primers-probe set of Li et al. (2006) were false
positives, and the 16S primers-probe produced non-specific amplification due to
shared similarity with the -glucosidase gene of several Proteobacteria. Kunta et al.
(2014) noted that the primer sets Las606/LSS and A2/J5 for cPCR, and LJ900fpr and
CQULA for qPCR are more reliable for root samples. Coy et al. (2014) indicated
that the ‘CLas’ 16S primer-probe set is not consistent in its performance compared to
the wg gene in psyllid DNA. For instance, the Ct values for 16S were about 24.5 for
all four components of a ten-fold dilution series of a psyllid sample, whereas the
serial dilution for the wg gene from the same sample was normal, and overall found
no correlation in the efficiencies for most psyllid samples between the 16S and wg
genes. Coy et al. (2014) hypothesised that some contaminants that are co-extracted
with the target DNA along with other factors caused this problem.
While the
contribution of the current diagnostic methods developed by many studies, especially
that of Li et al. (2006), is indisputable, it is vital to examine and evaluate the results
obtained in this study more closely. It is possible that there are some compounds in
the samples that interfere with the amplification of the target gene, and it is also
possible that there are differences in the isolates of the pathogen in Bhutan that is not
detected by the A2/J5 primers. Therefore, it is pertinent to determine whether the
current observations are due to the techniques used or variation in the pathogen by
analysing the current samples using other primer-probe sets for qPCR, and other
primers for cPCR.
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8.2.3 Incidence of ‘CLas’ and Diaphorina citri at higher elevations
Different studies have attributed different factors to the distribution of insects along
altitude gradients such as low temperature affecting psyllid development, disruption
of synchrony between insect and its host plant due to changes in temperature,
increased parasitism, and changes in plant biochemistry due to UV-B (Hill &
Hodkinson 1992; Hodkinson 2005; Jenkins et al. 2015). I found that incidences of
‘CLas’ was low at 1200 m and above while Diaphorina citri rarely occurred at 1200
m and above in Tsirang. What is interesting is comparing the results obtained at
1100 m, where both ‘CLas and high population of Diaphorina citri exist, with those
from 1200 m where the psyllid rarely occurs. Different elevation delimitation for
Diaphorina citri had been observed in different places. As discussed in Chapter 5,
Diaphorina citri has been reported as high as 1700 m in Taif in Saudi Arabia (Bové
2014), though elevation in Google Earth suggest that the observations in Taif were at
elevations possibly ~1450 m. In Puerto Rico, Jenkins et al. (2015) did not observe
the psyllid above 600 m, but observed few psyllids at Adjuntas which is about 580 m
ASL where Diaphorina citri was first detected in 2001 by Philip Stansly and Robert
Rouse (Halbert & Núñez 2004). Flores et al. (2009) also observed the psyllid at
Adjuntas in contrast to Pluke et al (2008) who did not detect the psyllid during
surveys conducted in 2004–2005. Based on the results of my studies in Tsirang, and
the early records of Diaphorina citri, ambient temperatures, RH, and rainfall, it
would be useful to evaluate the differences in UV index at these different elevations
and to test samples of plants from different elevations for differences in phenolic
compounds (e.g., flavonoids, tannins and coumarins). The phenolic compounds are
said to play important roles in UV protection and have anti-microbial and antiherbivore effects (Kuhlmann & Müller 2011; Katerova et al. 2012). Identification of
genes responsible for such changes, and potential manipulation of those genes in
either plants or psyllids, would be another avenue to explore for management of
HLB.
299
8.2.4 Phylogeny of Cacopsylla heterogena
Although, the green psyllid on citrus was identified as Cacopsylla heterogena based
on morphological descriptions, I could not obtain sufficient evidence to separate
Cacopsylla heterogena from Cacopsylla citrisuga. Therefore, future work should
include more samples of Cacospylla citrisuga and of Cacopsylla heterogena from
Yunnan, and other locations. Additional molecular markers, especially those derived
from nuclear genes, should be used to compare and resolve the taxonomic status of
Cacopsylla heterogena and Cacopsylla citrisuga.
8.2.5 Cacopsylla heterogena and ‘CLas’
Although the results of my study indicate that Cacopsylla heterogena poses
minimum threat as a vector of ‘CLas’, there are certain questions that I did not have
the opportunity to explore. Therefore, it would be useful to examine and evaluate
these further:
Firstly, a larger number of psyllid samples and the corresponding host plant
samples must be collected and tested for the presence of ‘CLas’.
Psyllid
samples should consist of 4th–5th instar nymphs and adults. This will be helpful
to determine whether the nymphs can acquire ‘CLas’. In my studies, I collected
both nymphs and adults from areas where some of the host plants tested positive
for ‘CLas’, but I was not able to sample nymphs from plants known to be
infected.
Secondly, it should be determined whether Cacopsylla heterogena can transmit
‘CLas’. This may be achieved by releasing adults on ‘CLas’-infected plants and
monitoring for ‘CLas’ infection in both nymphs and eclosing adults. In the field,
Cacopsylla heterogena is found on mandarin, lime, lemon and oranges so
development on citrus is not a limitation as was found for Diaphorina communis
(Chapter 3). Subsequently, if found to be able to acquire ‘CLas’, the resulting
populations (preferably second generation adults that have fed on ‘CLas’infected plants as nymphs) should be placed in cages with healthy plants to
determine whether Cacopsylla heterogena can transmit the pathogen.
Determination of whether Cacopsylla heterogena can transmit ‘CLas’ is
300
important, because ‘CLas’ is present at higher elevations where Cacopsylla
heterogena is found. I suspect these ‘CLas’-infection may have resulted from
the planting of infected material when the orchards were established. This has
implications if the focus of citrus production moves to higher altitudes in
Bhutan, and is relevant to other countries with similar conditions.
8.2.6 Murraya species
Although Murraya elongata and Murraya sumatrana can be separated based on their
molecular biology, the failure to differentiate between Murraya elongata and
Murraya sumatrana based on leaflet morphology emphasises the need to include the
flowers and fruits in morphological descriptions. My studies found the fruits of
Murraya elongata to be globose based on the pictures of fruits of Murraya elongata
from Bhutan and China, and compared with the picture of fruits of Murraya
sumatrana in Nguyen (2011).
Therefore, more plant samples collected from
different parts in Asia need to be assessed to confirm the formation of globose fruit
by Murraya elongata.
Although I found that the eggs and young nymphs of Diaphorina citri on the plants
of Murraya elongata that I observed in April 2015, there was no opportunity to
determine the presence of ‘CLas’ in the adults that laid these eggs, as the adults were
not present. The few nymphs that were present were young instars and were unlikely
to harbour the pathogen. Therefore, further work must also determine whether M.
elongata can be a host of the pathogen and whether Diaphorina citri can acquire the
pathogen. This is important in the context of management of HLB and psyllids.
8.2.7 Wild citrus
My studies on the wild citrus species found in Bhutan included four cpDNA and one
nuclear gene; however, to resolve the status of wild citrus, a larger number of genes
from a wide range of possible parents should be assessed. Additionally, both flowers
and fruits of the true citrus species that are possible parents of these accessions
should be examined and described, as the current descriptions of the flowers and
301
fruits of both Citrus cavaleriei (syn. Citrus ichangensis) and Citrus latipes made by
different authors (Swingle & Reece 1967; Grierson 1991; Zhang et al. 2008) vary
(Tables 8.1 and 8.2). From these descriptions, it appears that the flowers of Citrus
cavaleriei can be single or in racemes and the fruit globose or pyriform, and the
flowers of Citrus latipes may be solitary or in racemes. These descriptions suggest
that each may be referring to more than one taxon.
302
Table 8.1. A comparison of the descriptions of the flowers of Citrus cavaleriei and Citrus latipes.
Authority
Citrus cavaleriei
Citrus latipes
Swingle & Reece (1967)
Flowers borne singly in the leaf
axils; 2.5-3 cm diameter, white,
5-numerous
Flowers borne in small axillary
racemes with 5–7 flowers, 4numerous
Zhang et al. (2008)
Flowers solitary or to 9 in
fascicles, 3–3.5 cm in diameter;
Petals 4 or 5,
–
Grierson (1991)
–
Flowers solitary or in short
racemes, 4-numerous,
Table 8.2. A comparison of the descriptions of the fruit of Citrus cavaleriei and Citrus latipes.
Authority
Citrus cavaleriei
Citrus latipes
Swingle & Reece (1967)
Fruits small (3–4 diameter in
dried state, 3.5-5cm when
fresh), with 7–9 locules,; pulp–
vesicles very few; seeds few,
large, very thick,
Fruit almost globular with
compressed ends, oil cells
moderately small, fruit peel
much thicker (5–6 mm) than
that of Citrus cavaleriei (2–4
mm); seeds are smaller and
more numerous than Citrus
cavaleriei and arranged in 5–7
in each segment.
Zhang et al. (2008)
Fruit oblate, globose, or
pyriform, usually 3–5 × 4–6 cm
but when pyriform to 9–10 × 7–
8(–12) cm, with narrow
longitudinal grooves, oil dots
large
and
conspicuously
prominent, base rounded, apex
rounded, dimpled, and with or
without a papilla; pericarp to 2
cm thick but usually much less;
sarcocarp in 7–13 segments;
seeds 30 or more, subglobose to
irregularly pyramidal,
–
Grierson (1991)
–
Fruit subglobose, c 5cm
diameter, peel somewhat thick,
segment c 9, seed numerous
303
8.3
Implications for citriculture in Bhutan
Based on the findings of my studies and observations made during the study period,
the following recommendations are made:
It is essential that pathogen-free planting materials are produced under
conditions protected from Diaphorina citri.
Moreover, production facilities
should not be located in areas where the psyllid occurs e.g., Bhur in Gelephu,
Sarpang, or Bajo in Wangdue Phodrang.
The current situation of HLB in
Bhutan resulted from establishment of orchards with plants infected with HLB
and infested with Diaphorina citri. Records indicate the presence of Diaphorina
citri and HLB in orchards at Phuentsholing as early as the 1970s although the
presence of ‘CLas’ was not confirmed until 2002 after orchards in Punakha
Dzongkhag (~1200 m ASL) started to exhibit severe HLB-like symptoms in the
mid-1990s. The disease though more prevalent at lower altitude still prevails in
places where Diaphorina citri does not occur reaffirming the use of infected
materials.
Citrus production based on suitable varieties should be viable from 1200 m to
1700 m in the absence of Diaphorina citri and HLB, or where Diaphorina citri
rarely occurs, and with effective management of powdery mildew. Therefore,
importance should be given to plant breeders and plant protection personnel
collaborating to improve citrus varieties suitable for higher elevations.
Production below 1200 m is also feasible if all citrus below 1200 m in Bhutan is
progressively removed (most trees are already dead or dying) and replanted at
high densities with certified pathogen-free trees under strict quarantine.
Likewise all Bergera koenigii and Murraya paniculata plants within and in close
proximity to production regions below 1200 m must be removed before
pathogen free trees are planted.
Insecticides can be used to slow the spread of HLB but will not prevent spread.
Area-wide management is essential in places where many orchards are present
close to each other. However, spraying in most orchards in Bhutan is difficult
due to tall trees, rugged terrains, low availability of water and difficulties with
accessing suitable spray equipment.
304
Nevertheless, mineral oils should be
evaluated for suppression of Cacopsylla heterogena populations in higher
elevations as high populations of this psyllid cause severe foliage damage.
Control of Diaphorina citri will also be required on immature trees planted
below 1200 m and application of mineral oils and other chemicals during flush
growth cycles should be evaluated for this purpose.
Impacts of Cacopsylla heterogena damage on the growth of immature trees
should be assessed and studies undertaken to identify its natural enemies (no
parasitoids were observed in my study, possibly because the galls are so tightly
closed that oviposition by parasitoids is severely impeded) and impacts of these
natural enemies on populations of the psyllid.
305
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