®
Functional Plant Science and Biotechnology ©2012 Global Science Books
Sugarcane White Leaf Disease Characterization,
Diagnosis Development, and Control Strategies
Porntip Wongkaew
Plant Pathology Division, Department of Plant Science and Aricultural Resources, Faculty of Agriculture Khon Kaen University, Khonkaen 40002, Thailand
Corresponding author: * porwon@kku.ac.th
ABSTRACT
Sugarcane white leaf (SCWL) is the most destructive disease of sugarcane in Thailand where major cane and sugar production of the
world are situated. The severity degree and symptom variance are depended on soil fertility, temperature, cane sette quality, cultural
practice and the phloem-colonized phytoplasma amount. The leafhopper Matsumuratettix hiriglyphicus is the first known vector that
transmits SCWL phytoplasma. Recently at least other six leafhoppers have been found containing SCWL phytoplasma DNA. However,
only Yamatotettix flavovitatus has been closely investigated and found its transmissibility but with lesser extent. Outbreak of SCWL
disease is mainly activated by transportation and propagation of endemic cane setts. This SCWL phytoplasma is closely related to
Thailand sugarcane green grassy shoot (SCGGS) and India sugarcane grassy shoot (SCGS) with 96-98% similarity in their 16S-23S
rDNA sequences. Some diversity among SCWL phytoplasma isolates from different sources and growing locations has also been
indicated within the range of 89-98% similarity by the 16S-23S rDNA sequence analyses. In addition to existed conventional diagnosis
methods, the electrochemical DNA sensors have been proposed as a new tool for reliable routine practice. At present, control of the
SCWL disease is difficult due to their systemic nature and the lack of desirable resistant variety. Application of insecticide is ineffective
either. The disease control measures thus are mostly emphasized on the other strategies such as physical and chemical treatment of the
cane setts, disease free plant production via tissue cultures, sanitation and crop rotations, and regulatory quarantine. Cooperative
assistance among growers and involving association is strongly needed to accomplish effective control.
_____________________________________________________________________________________________________________
Keywords: control measures, disease outbreak, diversity, electrochemical DNA sensors, transmission
Abbreviations: AFM, atomic force microscope; CV, cyclic voltammetry; DAPI, 4-6-diamidino-2-phenylindole; DPV, differential pulse
voltammetry; ELISA, enzyme linked immunosorbent assay; PCR, polymerase chain reaction; TEM, transmission electron microscope
CONTENTS
INTRODUCTION........................................................................................................................................................................................ 73
SCWL DISEASE CONFIGURATION ........................................................................................................................................................ 74
Disease symptoms ................................................................................................................................................................................... 74
Disease transmission................................................................................................................................................................................ 74
Epidemic spread of the disease................................................................................................................................................................ 75
The causal SCWL phytoplasma and recent molecular characterization................................................................................................... 76
SCWL DISEASE DIAGNOSIS DEVELOPMENT..................................................................................................................................... 77
Conventional disease diagnosis ............................................................................................................................................................... 77
INNOVATIVE DNA BIOSENSORS IN SCWL PHYTOPLASMA DETECTION AND THEIR PERSPECTIVE..................................... 79
DISEASE CONTROL STRATEGIES ......................................................................................................................................................... 80
Hot water and tetracycline treatments...................................................................................................................................................... 81
Disease-free plant production and propagation........................................................................................................................................ 81
Sanitation and crop rotation..................................................................................................................................................................... 81
Insecticide application ............................................................................................................................................................................. 81
Regulatory quarantine.............................................................................................................................................................................. 81
Disease resistance .................................................................................................................................................................................... 81
CONCLUSIONS.......................................................................................................................................................................................... 82
ACKNOWLEDGEMENTS ......................................................................................................................................................................... 82
REFERENCES............................................................................................................................................................................................. 82
_____________________________________________________________________________________________________________
INTRODUCTION
In Thailand, approximately three million acres are belong to
sugarcane field which makes this amount to be the fifth
biggest planting area of the world and almost half of them
are located in the Northeast of the country. Although this
huge planting area can push Thailand to become the second
exporter of the world but the exact yield capability is still as
low as 20-25 tons per acre. One of the most serious factors
Received: 20 May, 2011. Accepted: 9 November, 2011.
of such low productivity is sugarcane white leaf disease.
This sugarcane white leaf (SCWL) disease has been considered as the most important disease of sugarcane in Thailand since the year 1962 (Kusalwong 1980). According to
the 2010 year report from Thailand Office of The Cane and
Sugar Board, over 30 million US dollars has been correspondent for the losses in Thai sugarcane industry each
year due to this disease. An economic threshold of SCWL
has also been reported in Taiwan, Bangladesh and Sri
Invited Review
Functional Plant Science and Biotechnology 6 (Special Issue 2), 73-84 ©2012 Global Science Books
SCWL DISEASE CONFIGURATION
Lanka (Leu 1983; Kumarrasingh and Jones 2001). The
common name sugarcane white leaf disease has been given
from dominant appearance of the complete white throughout the whole leaf of severely affected sugarcane. The evidence of SCWL disease was found on cv. ‘NCo 421’ grown
in North Thailand at Kao Ka district of Lampang Province
by the first sugarcane industry, Thai Lampang Sugar Industry Company and it was first reported epidemic with at least
50% yield losses in the year 1962-1964 to the Department
of Agriculture, Ministry of Agriculture and Cooperatives,
Thailand under a research program for Thailand Sugar
Industry (Chen 1974; Kusolwong 1980). Then the disease
was rapidly distributed to several provinces in the North,
the Central and especially in the East of Thailand by the
year 1974 to 1983 until all canes at the East were burnt out
from the fields (Kusalwong and Ouvanich 1993). The main
planting area has then shifted to the Northeast that is even
more suitable for SCWL disease development by its sandy
soil type. Serious destructive epidemic in this area was pronounced in 1989 with endemic cv. ‘F-154’. Intensive disease control program under a cooperation between Ministry
of Agriculture and Cooperatives and Ministry of Industry
established from 1989-1991 has demonstrated that the
losses could be effectively reduced by replacing the cv. ‘F154’ with a new cv. ‘Phil 58-260’ coupled with green
manure crop rotations (Kusalwong and Ouvanich 1993).
However, a few years later the disease reoccurred at much
higher incident and subsequently occupies all growing areas
throughout the country owing to the lack of disease free
cane setts by a rapid swift demand of sugarcane production
(Wongkaew et al. 1999; Wongkaew and Fletcher 2004).
The SCWL disease was also discovered in the south of
Taiwan by the year 1958 on cv. ‘NCo 310’ at Tainan, Kaochiung and Pingtung districts. The disease was further
spread to nearby districts and readily covering the island by
the use of infected cane setts for transplanting propagation
(Ling and Chaung-Yang 1962). But the crisis was substantially declined later by effective integrated control campaigns (Richi and Chen 1989). In Japan, this disease was
recognized in 1986 at Tanegashima island of Kagoshima
prefecture by Arai and Ujihara (1989), but all the infected
plants were eradicated which resulted in the disease disappearance ever since (Nakashima et al. 1999). Similar type
of the disease has been simultaneously observed in Bangladesh, India, Malaysia, Nepal, Pakistan and Sri Lanka. Indeed,
it has been first recorded by Barber in 1947 at Belapur
district of Bombay, Maharashtra in India (Vasudeva 1955).
The disease caused 10-40% economic losses and subsequently proceeded to Panjab, Uttar Pradesh, Haryana, Bihar,
West Bengal, Madhya Pradesh, Andhra Pradesh, Karnataka
and Tamilnadu (Agnihotri 1983). This disease is now a
great economic concern to both farmers and sugar industry
in India, Which the yield losses in ratoons reach their maximum in crops and up to 70% or higher incidence has been
arisen resulting in 100% yield losses (Rao et al. 2008). In
these countries, at least 6 different names were formerly
called such as albino, bunchy, grassy shoot, leafy tuft, new
chlorotic and yellowing disease. But the name “sugarcane
grassy shoot (SCGS)” was then accepted as a common
name according to a compilation study by Rane and Dakshindas (1962). The symptom appearances of this SCGS
disease are rather resembled the Taiwan and Thailand
SCWL than the sugarcane green grassy shoot (SCGGS)
disease investigated in Central Thailand that all the affected
leaves of bunchy cane in the latter case remain their normal
green (Lairungreung et al. 1995; Wongkaew et al. 1997).
Although it has been notified that these so-called SCWL,
SCGS, and SCGGS disease are likely caused by closely
related unculturable phytoplasmas from their 16S rDNA
and 16S-23S rDNA sequencing analysis (Wongkaew et al.
1997; Jung et al. 2003; Rao et al. 2008), still there are arguments on their true identity and their variability in different
sources, cultural practice condition and geographical attitudes.
Disease symptoms
The typical characteristics symptoms of the disease caused
by SCWL phytoplasma are bunchy white leaf proliferation
on the dwarfed shoots. The disease symptoms can be pronounced in every growth stages of sugarcane plants from
the earliest seed cane or cane sett germination until the
latest maturity. Variation among diseased plants is frequently exhibited in different plant stages and conditions including the disease development stages. There are subsistent levels of leaf chlorosis that vary degrees of the basic
white color which lead to the appearances such as pale
green, pale yellow, yellow plus white, pale white and yellow,
cream, and lastly pure white throughout all leaves of the
whole plants. Pathological effects of the disease on green
components of the leaf had been investigated by Wu et al.
(1969) and it has been concluded that the symptom expression of white leaf is considered to be directly related to
the development of chloroplast and chlorophyll biosynthesis. Quantity of the causal phytoplasma inside affected
plants was also shown to rule on symptoms extent by an
experiment on the relationship between chlorophyll losses
and SCWL phytoplasma amount (Nakashima et al. 1994).
The leaf color appearance is sometimes variegation and
becomes green again in certain environment. Profuse budding and shooting with bushy slim white leaves are arisen
in severely infected young plants. These affected plants are
soon dried as a result of heavy chlorosis and die before they
can produce desirable sugar juicy stems. Climate and temperature effects have been reported in Taiwan that the
symptoms were masked in winter and rapidly developed
severe white leaves in warmer season (Leu 1983). Observation on white leaf disease incidence in various locations and
preliminary screening for disease resistance indicated different scale of the disease symptoms that were depended on
the soil fertility and sugarcane cultivars (Ouvanich et al.
1990; Ouvanich and Kusalwong 1993). Thus the disease
severity is influenced by several factors such as the cane
sett quality, sugarcane vigor, cultivation practice, sugarcane
cultivar, soil type and fertilization, and the containing phytoplasma quantity.
Disease transmission
Transmission of SCWL phytoplasma to healthy plants has
been assumed to be possible in the tests with two leafhoppers, Matsumuratettix hiroglyphicus Matsumura (Matsumoto et al. 1969) and Yamatotettix flavovittatus Matsumura
(Hanboosong et al. 2006). Grafting experiment with sugarcane plantlets and periwinkle (Catharanthus roseus L.)
could also transmit the phytoplasma from infected sugarcane to each grafted plant (Wongkaew and Fletcher 2004).
While the attempts using mechanical injurie and dodder
(Cuscuta spp.) feeding were failed to transmit the SCWL
phytoplasma (Richi and Chen 1989; Sarindu and Clark
1993). However, a natural transmission in Taiwan and Thailand is likely happened via the most effective insect vector
M. hiroglyphicus according to its feeding preference, transmission efficiency from 55-100% and its population dynamics accompanying to monthly disease incidence (Matsumoto et al. 1969; Yang and Pan 1969; Pisitkul et al. 1989;
Hongspluk et al. 1993; Wongkaew 1999). Transovarian
transmission has been found in M. hiroglyphicus which
declare it to be a reservoir of SCWL phytoplasma (Hanboonsong et al. 2002). On the other hand, although the
phytoplasma could be detected by nested PCR and 16S-23S
rDNA gene sequencing in some other leafhoppers such as
Balcluta sp., Bhatia olivacea, Exitianus indicus, Hecalus
prasinus and Recilia sp. (Fig. 1), their transmissibility have
not yet been intensively investigated. While the leafhopper
Y. flavittatus has been ascertained it’s capable to transmit
the SCWL phytoplasma but with less efficiency. Their
population also fluctuates differently from the disease inci74
Sugarcane white leaf disease: Characterization, diagnosis and control. Porntip Wongkaew
Fig. 2 Life cycle of the sugarcane white leaf phytoplasma’s preference
insect vector, Matsumuratettix hiroglyphicus. Reprinted from Wongkaew P
(1999) Sugarcane White Leaf Disease Management, Thailand Research Fund,
Pimpatana Press, Khon Kaen, Thailand, 228 pp, with kind permis-sion from the
publisher.
Fig. 1 Sugarcane white leaf phytoplasma-containing leafhoppers detected by nested PCR and 16S-23S rDNA sequencing. (A) Balcluta sp.;
(B) Bhatia olivacia; (C) Exitianus indicus; (D) Hecalus prasinus; (E)
Recilia sp.; (F) Yamatotettix flavovittatus; (G) the first known insect vector, Matsumuratettix hiroglyphicus. Reprinted from Wongkaew P (1999)
Sugarcane White Leaf Disease Management, Thailand Research Fund, Pimpatana Press, Khon Kaen, Thailand, 228 pp, with kind permission from the
publisher.
dence recorded throughout the year (Hanboonsong et al.
2006; Wongkaew 1999).
The transmission by M. hiroglyphicus requires at least 3
h acquisition feeding on the diseased plant and 30 min inoculation on the target plant (Chen 1973; Pisitkul et al. 1989).
Efficiency in disease transmission by female adults is
65.6% while the transmission by males is 45.8% (Chen
1973). Incubation period of SCWL phytoplasma is 4-5
weeks in the insect body and 2-4 weeks in sugarcane plant
(Matsumoto et al. 1969). A life span of M. hiroglyphicus at
14°C is 92 days and at 35°C is 29.6 days. Hence its life
span observed in Taiwan is generally 42 days since egg
immergence including 25 days of the 5th instar development,
while the life span in Thailand is 30 days including 12-15
days of instar development as shown in Fig. 2 (Yang and
Pan 1969; Wongkaew 1999). The optimum condition for its
ovulation is an environment of sandy loam soil texture with
10% relative humidity at 30-35°C which as much as 200
eggs with at least 72% hatching capability by each female
can be produced. Eggs can be found in soil near the base of
sugarcane plant throughout the year but peaks of their population are usually seen in February to March, May to June,
and September to October (Yang 1972; Pisitkul et al. 1989).
While the adult population is abundant during June to October and reaches the highest peak in August along with the
peak of SCWL disease incidence (Yang and Pan 1969;
Pisitkul et al. 1989; Hongspluk et al. 1993; Wongkaew
1999).
Fig. 3 Epidemic spread of sugarcane white leaf disease following the
transportation and propagation of causal phytoplasma-embedded
cane setts. (A) Cane setts planting; (B) explicit disease symptoms since
the first immergence of sugarcane shoots; (C) severely affected sugarcane
fields.
Epidemic spread of the disease
As the disease is systemic due to a colonization of the fastidious phytoplasma in sugarcane phloem, thus it can easily
spread via vegetative propagation by cane setts. The transportation and the use of endemic cane setts for sugarcane
production are considered to be the most effective means
responsible for severe epidemic outbreak. An ignorance on
quality control of the cane setts prior to their growing in
large plantations is the main reason that execute the rapid
spread and extreme disease incidence. The typical white
leaf symptoms can be obviously seen since the first immergence of sugarcane shoots by this transplanting of the
phytoplama embedded cane setts. Secondary spread within
sugarcane field and nearby location is then accelerated by
the vector-leafhopper that rapidly distributes SCWL phytoplasma further on (Fig. 3). It has been confirmed that the
yearly population dynamic of the insect vector M. hiroglyphicus is correlated to the advance of the disease within the
75
Functional Plant Science and Biotechnology 6 (Special Issue 2), 73-84 ©2012 Global Science Books
grasses needs to be re-examined although the vector transmission feeding tests remain unsuccessful at present.
The causal SCWL phytoplasma and recent
molecular characterization
SCWL phytoplasma is localized within phloem sieve element of the diseased plant. An ultrastructural observation
under electron microscope has revealed its typical mollicute
cell structure that lacks cell and is surrounded by only a soft
cell membrane unit. Pleomorphic shapes of the phytoplasma
in sugarcane phloem are often seen with different sizes in
their diameter from 80-900 nm. Although it is not possible
to culture this phytoplasma in vitro but the progress of
molecular biology nowadays has allowed an opportunity to
analyze comparable genetic information involving its genotypic characterization. Roughly, the SCWL phytoplasma
has been classified into the Class Mollicutes of the Tenericutes Division under Prokaryotes Kingdom. Recent genomic analysis concerning the 16S rDNA sequence has categorized its position into the phylogenetic SCWL group that
the memberships of this group as shown in Table 1 include
each representative causal phytoplasma of SCWL in Thailand, SCGS in India, rice yellow dwarf (RYD) in Japan and
Thailand, nepier grass stunt in Kenya and Uganda, kalimantan coconut wilt in Indonesia, waligama coconut wilt in Sri
Lanka, coconut root wilt and areca palm yellow leaf (YLD)
in India, burmuda or cynodon grass white leaf (BGWL) in
Thailand, Australia, China, Italy and India, Delhi grass
white leaf (DicWL) in India, chlorotic streak and white leaf
of Oplismenus burmannii and Digitaria sanguinalis grasses
in India, coconut yellow decline (CYD) in Malaysia, brachiaria grass white leaf (BraWL) in China and Thailand,
sorghum grassy shoot (SGS) in Australia, BVK phytoplasma from Psammotettix cephalotes leafhopper, galactia
little leaf (GaLL) in Australia, and Circium arvense phyllody (CirP) in Germany. The 16S rDNA sequence similarity
among the phytoplasmas in Gramineae plants are 97% up
and the SCWL phytoplasma shows its similarity percentage
as high as 97.5-98.8 to SGS (Rao et al. 2008), 98 to Kenya
and Uganda Napier grass stunt (Jones et al. 2004; Nielsen et
al. 2007); 97.8-98.4 to BGWL (Jung et al. 2003; Marcone
Fig. 4 Matsumuratettix hiroglyphicus leafhopper population dynamic
and sugarcane white leaf disease incident percentage in growing areas
of Udornthani Province from monthly observations throughout 19961998. Reprinted from Wongkaew P (1999) Sugarcane White Leaf Disease
Management, Thailand Research Fund, Pimpatana Press, Khon Kaen, Thailand,
228 pp, with kind permission from the publisher.
fields as shown in Fig. 4 (Yang and Pan 1969; Wongkaew
1999).
A wild species of cane Saccharum spontaneum has been
suggested to be an alternate host for SCWL phytoplasma
from the transmissibility test with M. hiroglyphicus. Several
other grass species have also been investigated but most of
them could not attract this insect feeding. Survival after
rearing from egg to the 5th instar of this insect were 13.2%
on burmuda grass (Cynodon dactylon L.), 3.7% on Euphorbia hirta L. and 2.7% on Cyperus roduntus L. but no adult
could be developed (Yang 1972). Among 90 species of
sugarcane weeds that were checked for an existence of
SCWL phytoplasma, only burmuda grass, brachiaria grass
(Brachiaria distachya L.), and crowfoot grass (Dactylocteneum aegyptium L.) showed their containing phytoplasmas
which closely related to SCWL phytoplasma in the 16S
rDNA restriction fragment polymorphism and the 16S-23S
rDNA sequence (Wongkaew et al. 1997; Wongkaew 1999;
Jung et al. 2003). Still, the alternate host capacity of these
Table 1 The 16S rDNA sequence similarity percentages between SCWL phytoplasma from Udornthani, Thailand (SCWL-Ud) with other phytoplasmas
within closely related group.
Phytoplasma
Associated plant disease
Origin
% Similarity
Reference/Investigator
to SCWL-Ud
SCWL-T
Sugarcane white leaf
Central Thailand
99.8
Jung et al. 2003
SCGS
Sugarcane grassy shoot
India
97.5-98.8
Rao et al. 2008
Candidatus Phytoplasma Rice yellow dwarf (RYD)
Japan, Thailand
97.6
Jung et al. 2003
oryzae
Nepier grass stunt
Kenya, Uganda
98
Jones et al. 2004; Nielsen et al. 2007
Kalimantan wilt disese of coconut Indonesia
98
Warokka 2005
Nejat et al. 2009; Nejat and Vadamalai 2010
Waligama wilt disese of coconut
Sri Lanka
98
Root wilt disese of coconut
India
99
Manimekalai et al. 2010b
Yellow leaf disease (YLD) of areca India
99
Manimekalai et al. 2010a
palms
Jung et al. 2003; Marcone et al. 2004; Salehi
Candidatus Phytoplasma Burmuda grass white leaf (BGWL) Khon Kaen and Central 97.8-98.4
et al. 2009
cynodontis
Thailand, Australia,
China, Italy, Iran
India
Rao et al. 2007b; Snehi et al. 2008
Delhi grass white leaf (DicWL)
India
97.9
Nasare et al. 2007; Rao et al. 2009
India
97
Rao et al. 2010
Chlorotic streaks and white leaf
disease of Oplismenus burmannii
and Digitaria sanguinalis grasses
Nejat et al. 2009; Nejat and Vadamalai 2010
Malaysia
98
Coconut yellow decline (CYD)
diseases in cv. ‘Malayan Tall’ and
‘Malayan Red’
BraWL
Brachiaria grass white leaf
Thailand, China
97.9-98.4
Jung et al. 2003; Marcone et al. 2004
SGS
Sorghum grassy shoot
Australia
97.1
Blanche et al. 2003
BVK
Phytoplasma from Psammotettix
Germany
96.1
Seemüller et al. 1998; Rao et al. 2008, 2009
cephalotes
GaLL
Galactia little leaf
Australia
95.5
Jung et al. 2003
CirP
Circium phyllody
Germany
94.6
Jung et al. 2003; Rao et al. 2008, 2009
76
Sugarcane white leaf disease: Characterization, diagnosis and control. Porntip Wongkaew
Table 2 The 16S-23S rDNA gene spacer sequence similarity percentages between sugarcane white leaf phytoplasma from Udornthani (SCWL-Ud) with
other SCWL from different sources and with some related grass species.
Phytoplasma isolates
Associated disease
Origin
% Similarity
No. bases
Reference/
to SCWL-Ud sequenced
Investigator
SCWL-Kk
Sugarcane white leaf
Khon Kaen, Thailand
98-100
210, 810
Wongkaew 1999
SCWL infected M.
SCWL-Insect vector
Udornthani, Thailand
97
810
Wongkaew 1999
hiroglyphicus leafhopper
SCWL-TC
Sugarcane white leaf
Tissue cultured plantlets,
93
810
Wongkaew 1999
Khon Kaen, Thailand
SCWL-Nr
Sugarcane white leaf
Nakornrachasima, Thailand 89
810
Wongkaew 1999
SCWL-Sk
Sugarcane white leaf
Sra Kaew, Thailand
89
810
Wongkaew 1999
SCGGS
Sugarcane green grassy shoot Suphanburi, Thailand
96-98
210, 810
Wongkaew et al. 1997;
Wongkaew 1999
SCGGS
Sugarcane green grassy shoot Southern Thailand
87
452
Sadudee 2004
SCGS
Sugarcane grassy shoot
Sri Lanka
98
452
Ariyarathna et al. 2007
SCGS
Sugarcane grassy shoot
India
86.9
240
Rao et al. 2007a
97.9
210
Rao et al. 2008
SGS
Sorghum grassy shoot
Australia
86.9
452
Ariyarathna et al. 2007
Candidatus Phytoplasma
Burmuda grass white leaf
Australia, India, Italy,
83-87
210, 250, 810 Wongkaew 1999;
cynodontis
(BGWL)
Thailand
Marcone et al. 2004;
Rao et al. 2007b, 2008
Firouzabud burmuda grass
210
Salehi et al. 2009
Iran
90.8
white leaf (FBGWL)
Juyom burmuda grass white
91.2
leaf (JBGWL)
BraWL
Brachiaria grass white leaf
China, Thailand
83-87
210, 810
Wongkaew 1999;
Rao et al. 2008
Candidatus Phytoplasma oryzae Rice yellow dwarf (RYD)
Japan, Thailand
79.8
210
Rao et al. 2008
et al. 2004; Rao et al. 2007; Snehi et al. 2008; Salehi et al.
2009), 97.9-98.4 to BraWL (Jung et al. 2003; Marcone et al.
2004), 97.9 to DicWL (Nasare et al. 2007; Rao et al. 2009),
97.1 to SGS (Blanche et al. 2003) and 97 to O. burmannii
and D. sanguinalis chlorotic streak and white leaf (Rao et al.
2010). Moreover, the analyses of 16S rDNA sequences of
phytoplasmas affected coconut and other palm species in
some Asian countries have revealed their close relationship
to SCWL phytoplasma with as high as 98-99% similarities
such as the phytoplasma in kalimantan coconut wilt
(Warokka 2005), waligama coconut wilt (Nejat et al. 2009;
Nejat and Vadamalai 2010), coconut root wilt and areca
palm YLD (Manimekalai et al. 2010) and coconut CYD
(Nejat et al. 2009; Nejat and Vadamalai 2010).
Determination of 16S-23S rDNA spacer region sequence of SCWL phytoplasma and its closely related strains
provide similar trend of their relationship. The similarity
percentages in reference to the SCWL-Ud from Udornthani
Province, Thailand are summarized in Table 2 on the basis
of the sequenced 210 bases (bp) level (Wongkaew et al.
1997; Wongkaew 1999; Rao et al. 2008, 2009; Salehi et al.
2009), 240 bp level (Rao et al. 2007a), 250 bp level (Rao et
al. 2007b), 452 bp level (Ariyarathna et al. 2007) and 810
bp level (Wongkaew 1999, 2000). The SCWL phytoplasmas
from Udornthani and adjacent Khon Kaen Province of Thailand exhibit an identical or almost identical sequence with
98-100% similarity in 210-810 bp level sequencing. A very
close relationship has also obtained with the India and Sri
Lanka SCGS at 97.9-98% in 210-452 bp level and with the
SCGGS from central Thailand at 96-98% in 210-810 bp
levels. The relationships of SCWL-Ud with Southern Thailand-SCGGS, SGS, RYD, BGWL and BraWL have been
shown up to be 87, 86.7, 79.8, and 83-87% of similarity,
respectively. It is interesting that there is also diversity
among the SCWL phytoplasma 16S-23S rDNA sequenced
from its insect vector and different sources or locations of
the SCWL-diseased sugarcane collected. As the sequence
similarity between SCWL-Ud and SCWL from other source
such as SCWL-Khon Kaen, SCWL in M. hiroglyphicus,
SCWL in infected tissue culture plantlets, SCWL-Nakornrachsima Province, and SCWL-Srakaew Province have
been found to be 98, 97, 93, 89 and 89%, respectively. Thus,
it seems that this diversity may possibly caused by cultural
environment, host species or cultivars and geographical dif-
ferences. However, further details such as their cross interaction, the whole genome sequencing, environmental effects
and other biological behaviors are still necessary to justify
the true classification and effective disease control.
SCWL DISEASE DIAGNOSIS DEVELOPMENT
Conventional disease diagnosis
1. Diagnosis based on symptoms
The disease can be easily distinguished by its predominant
symptoms of thorough white leaf expression and bunchy
stunt, although some variations from white to green color
are presented within the scope of classical symptoms. Physical finding in this type of diagnosis are based on close
speculations following the progress of disease development
in growing cane and suspected mask symptom accounts. In
this scheme the cane setts prepared from suspected stems
are transplanted separately into each sandy soil filling pot in
greenhouse condition and the growing plants are investigated daily to notify the development of the disease. Typical
symptoms of SCWL disease usually arise since the sprout
of their first leaf and are obviously displayed within 2-4
weeks from the infected cane setts. On the other hand, diagnosis can also be done by observing the remission of SCWL
disease symptoms of cane plants after infiltration of the
tetracycline antibiotic into main stems (Wongkaew and
Fletcher 2004). This method depends on factors such as the
extent of accompanying phytoplasma, indigenous cane sett
health, planting season, and other environmental circumstances. Thus, diagnosis by the symptom basis is considered
less efficient; however it still can be used to ensure the
visual characterization of the disease.
2. Microscopy techniques
Microscopic methods that have been used to detect phytoplasma in the infected sugarcane plants include observations using light microscope, fluorescence microscope and
transmission electron microscope (TEM). In light microscopy, the phloem cells of sugarcane infected with phytoplasma exhibit a distinguish deep blue coloration (Fig. 5A)
that is absent in healthy sieve tissues (Fig. 5B) after staining
77
Functional Plant Science and Biotechnology 6 (Special Issue 2), 73-84 ©2012 Global Science Books
Fig. 6 Detection of sugarcane phytoplasma containing in the top, the
middle and the base of sugarcane stem by dot blot hybridization using
16S-23S rDNA probe. Simplified detection using direct plant sap (on the
right) produces similar dot blot reaction as the use of DNA extract (on the
left), the H columns represent healthy plant and the D columns represent
diseased plant. Reprinted from Wongkaew P (1999) Sugarcane White Leaf
Disease Management, Thailand Research Fund, Pimpatana Press, Khon Kaen,
Thailand, 228 pp, with kind permission from the publisher.
ease and green grassy shoot disease of sugarcane and the
white leaf disease of grasses from other phytoplasma in diseased dicots such as cowpea phyllody and sesame phyllody.
But the antisera also showed high cross reaction signal and
low sensitivity which limited their use for desirable diagnosis. Recently, monoclonal antibodies have been introduced to improve detection efficiency and the SCWL phytoplasma detection kit has then been developed commercially for field diagnosis by a collaborative program of
National Center for Genetic Engineering and Biotechnology,
Thailand (Patent No. 9582). However, it has not yet widely
accepted because of a considerable high cost due to its
complicate preparation and high investment.
Fig. 5 Observation for the existence of sugarcane white leaf phytoplasma within plant tissues by microscopic techniques. (A) Dienes’
staining-light microscopy of infected tissue; (B) Dienes’ staining-light
microscopy of healthy tissue; (C) DAPI staining-fluorescence microscopy
of infected tissue; (D) DAPI staining-fluorescence microscopy of healthy
tissue; (E) ultrastructural micrograph of the phytoplasma cells localized in
sugarcane phloem sieve cells revealed by transmission electron microscopy. Arrows indicate the presence of phytoplasma.
4. Dot blot-DNA hybridization techniques
Detection of the hybridization between complementary
DNA by dot blot technique has been employed for SCWL
disease diagnosis in the last decade. The DNA probes used
in the assays include randomly cloned fragments of chromosomal and extrachromosomal DNA of SCWL phytoplasma and the complementary ribosomal DNA (Klinkong
and Seemüller 1993; Nakashima et al. 1994; Wongkaew et
al. 1995; Wongkaew 1999). The resulted signal color intensity is highest with the cloned extrachromosomal DNA in
both peroxidase-labeled gene detection system and digoxigenin colorimetric gene assay, while the faintest signal is
from 16S-23S rDNA probe. All probes are capable to rank
the phytoplasma amount in various parts of sugarcane stem
approximately by their reaction color intensity. The cloned
DNA probe detects phytoplasma DNA in SCWL diseased
plants and the insect vector M. hiroglyphicus as well as
phytoplasma DNA from other white leaf diseased Gramineae plants such as brachiaria grass, burmuda grass, crowfoot grass and yellow dwarf diseased rice plants. Dot blotDNA hybridization techniques have been successfully simplified for field survey detection with sugarcane sap stream
from freshly cutting as displayed in Fig. 6 (Wongkaew et al.
1998). Diagnosis of SCWL disease has been done by this
dot blot-DNA hybridization for several years, but recently it
is rarely used because of its complexity and time consuming
process in comparison to a subsequently developed polymerase chain reaction technique.
the tissue sections with Dienes’ stain proposed by Deeley et
al. (1979). Florescence microscopy in combination with
DNA-specific fluorochrome, 4-6-diamidino-2-phenylindole
(DAPI) staining of tissue sections is a sensitive and reliable
technique for rapid and precise localization of phytoplasma
in phloem sieve of the infected sugarcane. By this technique,
the infected phloem cells show a diffuse fluorescence brighter than the one typical of the nuclei of parenchyma cells
and these bright spots are not visible in healthy tissues (Fig.
5C, 5D). In case of transmission electron microscopy
(TEM), it reveals the presence of phytoplasma cells in sieve
and neighboring cells of the infected plants (Fig. 5E). The
ultrastructural characteristics of phytoplasma and host cell
modifications induced by phytoplasma can be demonstrated
by this technique (Nakashima et al. 1999; Wongkaew and
Fletcher 2004). It has been shown by this TEM illustration
that the SCWL phytoplasma shapes are pleomorphic due to
the lack of cell wall and are variable in size ranging from
80-900 nM in diameters. Although these traditional microscopy techniques have permitted only morphological characters that are indistinguishable among phytoplasma strains,
but their performances remain valuable for preliminary
diagnosis and cytological investigation on host-parasite
interactions.
3. Serology-based techniques
A serological method using conventional polyclonal antibodies has been tested for the detection of SCWL phytoplasma in the last decade (Sarindu and Clark 1993; Wongkaew 1999). The antisera rose up to 1:100 titers that were
adequate for an indirect enzyme linked immunosorbent
assay (ELISA). This method can differentiate the phytoplasma in diseased gramineae plants such as white leaf dis-
5. Polymerase chain reaction based techniques
Polymerase chain reaction (PCR) has distinct advantages
over dot blot-DNA hybridization by its higher sensitivity
and specificity. The technique requires less processing time
and complicates operation. A single copy of a target DNA
78
Sugarcane white leaf disease: Characterization, diagnosis and control. Porntip Wongkaew
Fig. 7 Electrophoresis gel expression in the detection of sugarcane white leaf phytoplasma by traditional polymerase chain reaction and nested
polymerase chain reaction techniques. (A) indication of the phytoplasma DNA in samples by the appearance of 16S rDNA at 1,400 bp; (B) indication
of the phytoplasma DNA in samples by the appearance of 16S-23S rDNA at 810 bp; (C) nested PCR indication of the phytoplasma DNA in samples by
the appearance of 16S-23S rDNA at 210 bp. Reprinted from Wongkaew P (1999) Sugarcane White Leaf Disease Management, Thailand Research Fund, Pimpatana Press, Khon Kaen, Thailand, 228 pp, with kind permission from the publisher.
sequence can be amplified one million fold within an hour
prior to an electrophoresis illustration. Thus, PCR has been
widely used for SCWL phytoplasma detection in the concerning laboratories (Nakashima et al. 1996; Wongkaew et
al. 1997; Sdoodee et al. 1999; Kumarasinghe and Jones
2001). A variety of primer pairs have been designed according to the ribosomal gene alignment including the universal phytoplasma primers from 16S rDNA and the narrowed group specific primers from 16S-23S rDNA sequences. The universal primers produce a DNA band size of
about 1.4 kb that indicates the presence of non-specific
phytoplasma 16S rDNA in sample. While the group specific
primers are capable to demonstrate the phytoplasma in
sugarcane and gramineae white leaf diseased samples which
the DNA band at either 210 or 810 bp can be seen depending on the selected primers (Fig. 7). Detection of SCWL
phytoplasma usually employs a common one-round PCR
cycle using any pair of those universal or more specific primers for the target plant samples and a nested PCR which
re-amplifies the first round PCR with an internal primers
specific to the phytoplasma for the target insect vector and
suspected-very low amount of phytoplasma containing samples. In spite of a powerful capability, these techniques need
special skills to carry out an operation and are too much
expensive for routine detection in sugarcane industry. The
techniques thus are being used only for certain confirmation
in laboratories and research activities.
Fig. 8 Schematic diagram of DNA biosensor for sugarcane white leaf
phytoplasma detection using voltammetry method and visualization
of the hybridization event by atomic force microscopy.
that transform the recognizing reaction into recordable signal for electronic processor to interpret and display the output result. Biosensors have been widely applied in various
types of research and commercial industry for monitoring
the target or bioterrorists in an environmental atmosphere
such as food, medicine, metabolite, and pathogenic infection. Among several types of biosensors, the DNA biosensor in combination of an electrochemical method has provided the utmost desirable tool for several biological analyses including disease diagnosis because of its simplicity,
rapidity, low cost and ability to work with turbid samples.
Techniques mainly used in electrochemical transduction
include amperometric, conductometric, impedimetric, and
potentiometric that based on corresponding signal measurement as current, conductance, impedance and potential, respectively. The specificity and unlimited sensitivity of this
biosensor type can be easily enhanced in coupled with a
modification of biosensing platform and other biosensing
techniques (Teles and Fonseca 2008). The electrochemical
DNA biosensors have been successfully developed following the rule of DNA hybridization for rapid detection of
several microorganisms such as hepatitis B virus (Erdem et
al. 1999, 2000; Ye and Ju 2003; Guo et al. 2007), Micro-
INNOVATIVE DNA BIOSENSORS IN SCWL
PHYTOPLASMA DETECTION AND THEIR
PERSPECTIVE
The term “biosensor” implies for an analytical system that
integrates a biological component with a physicochemical
detector to yield a measurable electric signal. It generally
consists of three components: a biological sensing element,
a transducer or detector element and a signal processor. This
system takes advantage of the ability of a biomolecule to
specifically recognize the target substance. The biological
sensing element can be created from biological materials
such as enzymes, antibodies, nucleic acid, cell organelles,
microorganisms, biomimics, and etc. that specifically recognize the target in the analyzing condition and produce a
signal related to the concentration. The transducer or detector element can be each of or combination of physicochemical techniques such as optical, thermometric, piezoelectric,
magnetic, micromechanical and electrochemical techniques
79
Functional Plant Science and Biotechnology 6 (Special Issue 2), 73-84 ©2012 Global Science Books
10
11
Fig. 9 Detection of sugarcane white leaf phytoplasma DNA from
sugarcane plants using SCWL-ssDNA probe by differential pulse (A)
and cyclic (B) voltammograms. (a) Hybridization with ssDNA from
healthy plant, (b) SCWL-ssDNA probe only, (c) hybridization with
ssDNA from diseased plant. The signal current of an intercalator methyllene blue was determined in 20 mM Tris buffered saline (pH 7.0) at 100
mVs-1 scan rate.
cystis spp. (Erdem et al. 2002), native yeast (Ju et al. 2004),
human immunodeficiency virus (Zhang et al. 2010), Yersinia enterocolitica (Sun et al. 2010) and several food-borne
pathogenic bacteria (Velusamy et al. 2010).
The electrochemical DNA biosensor has been applied in
the detection of SCWL phytoplasma (Fig. 8). It has been
tried first with a capacitance measurement of the hybridization reaction using a pair of 21 bp each known sequence
from phytoplasma 16S rDNA designed by Namba et al.
(1993) as complementary DNA probes and oligochitosanmodified glassy carbon electrode as conductive platform.
The charging current signal and total capacitance obtained
from hybridization reaction between phytoplasma 16S
rDNA probes with ssDNA of SCWL diseases plant target
have shown to be obviously higher than the reaction with its
noncomplementary ssDNA of healthy sugarcane plant
(Wongkaew and Poosittisak 2008). Subsequently, a labelfree DNA biosensor using the whole chromosomal ssDNA
of SCWL phytoplasma that immobilized on chitosan-modified glassy carbon electrode (GCE) has been developed in
couple with methylene blue intercalator for specific indication of the SCWL-DNA hybridization. Progressive detection capability can be achieved by cyclic and differential
pulse voltammetry through the three electrode system
potentiostat controlled with general purpose electrochemical system software (Fig. 9). Quantitative determination of
SCWL phytoplasma DNA in the target sample could be
operated in these experiments with a detection limit of 0.1
nM. The corresponding DNA hybridization event has also
been visible through atomic force microscopic (AFM) observation as shown in Fig. 10 (Wongkaew and Poosittisak
2010). These performances thus allow a feasibility of the
DNA biosensor for practical use in SCWL phytoplasma
detection and encourage further studied on the efficiency
enhancement and optimization for real time field monitoring by a reliable, cost effective and convenient portable
device.
Fig. 10 Atomic force microscopic observation of the hybridization event
directed by sugarcane white leaf DNA biosensor. (A) Hybridization with
complementary phytoplasma DNA from diseased plant; (B) reaction with
non-complementary DNA from healthy plant. Arrows indicate observed
DNA strands. Fig. 11 Production of disease-free sugarcane plants and cane
setts from apical meristem tissue cultures. (A) Cultured apical meristem
section; (B) shooting derivation; (C) developing plantlet cultures; (D)
acclimatization of plantlets transplanting; (E) field-grown plants intensively prepared for disease cane setts production. Reprinted from Wong-
DISEASE CONTROL STRATEGIES
kaew P (1999) Sugarcane White Leaf Disease Management, Thailand Research
Fund, Pimpatana Press, Khon Kaen, Thailand, 228 pp, with kind permission
from the publisher.
Several control strategies have been introduced including
80
Sugarcane white leaf disease: Characterization, diagnosis and control. Porntip Wongkaew
use of disease free cane setts strategies have made Taiwan
completely control the disease to a harmless condition ever
since (Leu and Kusalwong 2000).
In tropical country such as Thailand which there is not
much different in year round temperature, it is insignificant
for planting time differences. The most appropriate strategies are likely the sanitation, the disease free cane setts utilization and the crop rotation with economic green manure
plants. As there has been an evidence that the SCWL disease could be successfully controlled in a defined research
area at Udornthani Province of Northeast Thailand during
1989-1991 using pigeon pea (Cajanus canja L.) and sword
bean (Canavalia gladiata Jacq.) as green manures for crop
rotation and healthy new cane variety for a replacement of
the weak old variety (Kusalwong and Ouvanich 1993). Five
years later, the intensive control program has to be repeatedly performed during 1996-1999 against a higher epidemic
incidence of SCWL disease reoccurrence due to an ignorance of such proper cultural practices (Wongkaew 1999).
Although the losses were effectively reduced through this
latter program until the end of the project period, however,
the SCWL disease has resettled soon after that by similar
former reasons.
eradication of the phytoplasma in sugarcane setts by hot
water and tetracycline treatment, disease free plant production and propagation, sanitation, crop rotation, insecticide
application, regulatory quarantine, and disease resistance.
Hot water and tetracycline treatments
It has been reported that treatment of sugarcane setts with
hot water only could not eliminate SCWL phytoplasma
(Ling and Chaung-Yang 1963a; Liu et al. 1963). Unsuccessful therapy has been indicated from the heat treatment
studies by dipping infected cane setts into hot water at 50°C
for 2-4 h and at 53°C for 1 h. Somewhat curing effect could
be seen when the cane setts were treated with hot water at
52°C for 8 h and 54°C for 40 min, but most of the cane setts
could not withstand nor produce regular shoot buds following such high temperature in that given times. Better results have been achieved in the treatment with tetracycline
antibiotic. Shigata et al. (1969) reported that treatment of
cutting infected sugarcane with tetracycline for 72 h could
reduce SCWL phytoplasma multiplication and the growing
shoots remained free of white leaf disease symptoms for
three months. Similarly, temporary reduction of the disease
symptoms by tetracycline has been confirmed in an investigation performed by Mongkolsuk and Sutrabutra (1976) in
Thailand. In this case curing effect was shown up within 2
weeks after dipping the cutting in tetracycline solution at
200-500 ppm for 24 h. The leaves became completely green
in the 4th week and stayed green for 8 weeks. Remission of
SCWL symptoms has also been demonstrated in tissue culture system that about 70-100% of plantlets sustained the
green appearance through 5-8 subcultures or at least 8
months of sequential serial transfers from their mother
plantlets cultures that were grown in 200-500 ppm of oxytetracycline (Wongkaew and Fletcher 2004).
Insecticide application
Application of insecticides to kill the insect vector in the
field infested with SCWL disease has proved ineffective.
The attempt on spraying an insecticide malathion since the
young stage of sugarcane plant has failed to control the
vector and the disease incidence (Leu, 1974). In the sugarcane white leaf management project, over 3.2 hectares of
each sugarcane plantation were thoroughly treated with carbosulfan insecticide every two weeks from April to June
before the insect vector population could reach its regular
peak in July and August. But neither the insect vector nor
the disease has been successfully controlled by this insecticide application (Wongkaew 1999).
Disease-free plant production and propagation
The strategy of control through disease-free plants is considered to be one of the most effective methods for controlling a pathogen that has systemically colonized the plant
such as the case of SCWL phytoplasma. Intensive production programs by conventional selection and tissue culture
technique carried out in Taiwan have proved to be highly
success in breaking off the disease epidemic (Leu 1978; Liu
1981). In Thailand, the disease free plants could be extensively produced from tissue culture of meristem tip as well
(Wongkaew and Fletcher 2004). An extension in the field
use of disease free tissue culture plants and their derived
cane setts has been established during 1996-1999 by sugarane white leaf management research project as concluded in
Fig. 11 (Wonkaew 1999). Selves’ preparation of disease
free healthy cane setts by growers have also intensively persuaded although this propaganda has not yet fully accepted
for their routine practicing.
Regulatory quarantine
While sugarcane is a prohibit material by plant quarantine
regulation, a strict inspection is still needed for the local
SCWL disease to limit further distribution of the disease.
This cooperative practice has contributed successful control
of SCWL disease in Taiwan (Leu 1983). However, regular
inspection of cane setts and growing plants in the fields including mother plants for propagation has not yet widely
applied in Thailand, thus sustains yearly serious epidemic
spread and economic losses by this disease.
Disease resistance
Although the use of disease resistant varieties is the most
desirable mean for growers, however it is unavailable in
sugarcane to the white leaf disease. The screening test for
SCWL disease resistance from at least 400 varieties including a wild cane Saccharum spontaneum L. by insectary
method has displayed inopportunity in obtaining the required resistant character (Leu 1974). Another test with 158
sugarcane varieties and 24 hybrids in Thailand in 19881991 has also shown susceptible response in all populations
(Ouvanich and Kusalwong 1993; Ouvanich et al. 1988).
Recent observations on sugarcane varieties including their
hybrids and wild canes in Thailand during 2004-2007 have
confirmed the unavailability of resistance or tolerance to
this SCWL disease (Sa-Nguanrangsirikul et al. 2007). Subsequent investigations by natural selection screening and
breeding programs have still proceeded by various associations involve such as the sugarcane research experiment stations of the Ministry of Industry and the Ministry of Agriculture and Cooperatives including private sugarcane associations and universities, but so far there is no report on the
evidence of SCWL disease resistance up to the present.
Sanitation and crop rotation
The main activity for sanitation to eliminate the re-infective
risk of SCWL phytoplasma is concentrated on removal and
disposal of infected plants and debris from the fields. Crop
rotation with other plant species seemed to be unnecessary
in an adequate water supply land of Taiwan which other
effective plans have been occupied. According to the lower
disease occurrence record during spring season, the sugarcane growers have been suggested to change their planting
time from ordinary autumn to the planting in spring time.
As the population of M. hiroglyphicus insect vector and the
multiplication of SCWL phytoplasma are limited by low
temperature during winter season. This situation, thus deteriorate the infection efficacy in the following spring (Ling
and Chaung-Yang 1963b; Pan and Yang 1970). However, in
unavailable water supply areas, a substitution of sugarcane
planting with green manure plants from season to season is
strongly recommended. Combining these actions with the
81
Functional Plant Science and Biotechnology 6 (Special Issue 2), 73-84 ©2012 Global Science Books
CONCLUSIONS
demic spread throughout the country. Cooperative assistances among involving associations and intensive researches
for innovative tools should be emphasized in coupled with a
proper subsidy to accomplish the successful disease control.
SCWL disease is named after the complete white color
developed appearance of the whole leaf in severely affected
plants in Taiwan and Thailand. The disease has also been
reported for its sporadically occurrence in Bangladesh,
Japan, Pakistan and Sri Lanka. Variation within the white
leaf symptoms and their severity degree are often shown up
due to several factors such as soil fertility, temperature,
cane sett quality, cultural practice and the causal phytoplasma amount. Transmission of this phloem sieve-colonized phytoplasma by insect vector has been confirmed
firstly with M. hiroglyphicus leafhopper (Matsumoto et al.
1969; Yang and Pan 1969; Pisitkul et al. 1991; Wongkaew
and Fletcher 2004). The SCWL phytoplasma DNA has also
been detected in at least six other leafhoppers but at present
only Y. flavovittatus has been experimentally found to
posses SCWL phytoplasma transmissibility (Wongkaew
1999; Hanboonsong et al. 2006). Epidemic outbreak of
SCWL disease is primarily activated by transportation and
the use of endemic cane setts for sugarcane production and
secondary spread within growing fields and nearby areas is
then accelerated by insect vectors. The causal phytoplasma
has been placed into phylogenetic group of SCWL concerning the 16S rDNA sequence. The reports on 16S-23S
rDNA sequence analyses have indicated that SCWL and
SCGGS of Thailand and SCGS of India are closely related
with 96-98% similarity. While some diversity according to
the 16S-23S rDNA sequences has also been found among
SCWL phytoplasma isolates from different sources and
locations within 89-98% similarity range (Wongkaew 1999,
2000). The evidence of this diverseness thus remains to be
clarified.
The SCWL disease has formerly diagnosed by its symptoms expression. Microscopic techniques include light
microscopy with Dienes’ staining, fluorescence microscopy
with DAPI staining and transmission electron microscopy
have then and still been used for morphological identification and investigation on cytological interaction during
pathogenesis. Diagnosis of the disease can be done by other
conventional methods such as serology-based techniques,
dot blot DNA hybridization and PCR but they need high
investment and special skill with laborious production and
detection process. Recently, preliminary experiments on
electrochemical analyses of SCWL-DNA biosensors for
specific DNA hybridization have proved their advantages as
new desirable diagnostic tool. As significance differences in
charging current signals and total capacitances have been
displayed in the reaction after hybridization of the SCWL16S rDNA probe with healthy and diseased plant DNA
(Wongkaew and Poosittisak 2008). Discrimination between
DNA extracts from healthy and SCWL diseased plants has
also been pronounced by cyclic and differential pulse voltammetry using the whole chromosomal DNA probe for
specific DNA biosensor. This difference in hybridization reaction event has similarly revealed by visible images under
atomic force microscopy (Wongkaew and Poosittisak 2010).
These results hence provide the feasibility to construct the
new rapid, specific, quantifiable, simple and cost effective
portable tool for routine use in early warning and control of
the SCWL disease.
The strategies for SCWL disease control are mostly
relied on cultural methods because disease resistant cultivars are unavailable and physico-chemical therapy including insecticide application are ineffective. Excellent control of SCWL disease has been achieved in Taiwan by
means of field sanitation and disease-free cane sett transplanting in spring time instead of an ordinary planting season in autumn (Pan and Yang 1970). Integration of disease
disinfestations, green manure crop rotation and re-transplanting with disease free cane setts have also proved effectively control SCWL disease in Thailand during the
intensive disease management programs (Kusalwong and
Ouvanich 1993; Wongkaew 1999). But these strategies have
been neglected and hence result in persisting serious epi-
ACKNOWLEDGEMENTS
The Thailand Research Fund and the supporting fund from Khon
Kaen University to the SCWL Project and the Biosensing Research
Group are acknowledged. Special thanks are also consigned to
Coax Group Corporation Ltd., for their AFM assistances and R.
Viswanathan for his kind advices.
REFERENCES
Agnihotri VP (1983) Disease of Sugarcane, Oxford and IBH Publishing Co,
New Delhi, 383 pp
Arai K, Ujihara K (1989) Studies on sugarcane white leaf disease occurred in
Tanegashima-island. Bulletin of the Faculty of Agriculture, Kagoshima University 39, 9-16
Ariyarathana HACK, Everard JMDT, Karunanayake EH (2007) Diseased
sugarcane in Sri lanka is infected with sugarcane grassy shoot and/or sugarcane white leaf phytoplasma. Australasian Plant Disease Notes 2, 123-125
Chen CT (1973) Insect transmission of sugarcane white leaf by single leafhopper, Matsumuratettix hiroglyphicus (Matsumura). Republic of Taiwan Sugar
Research Institute Report 60, 25-33
Chen CT (1974) Sugarcame white leaf disease in Thailand and Taiwan. Sugarcane Pathology Newsletter 11/12, 23
Deeley J, Stevens WA, Fox RTV (1979) Use of Dienes’ stain to detect plant
diseases induced by mycoplasmalike organisms. Phytopathology 69, 11691171
Erdem A, Kerman K, Meric B, Akarca US, Ozsoz M (1999) Electrochemical
biosensor for the detection of short DNA sequences related to the hepatitis B
virus. Electroanalysis 11, 586-587
Erdem A, Kerman K, Meric B, Akarca US, Ozsoz M (2000) Novel hybridization indicator methylene blue for the electrochemical detection of short
DNA sequences related to the hepatitis B virus. Analitica et Chimica Acta
422, 139-149
Erdem A, Kerman K, Meric B, Ozkan D, Kara P, Ozsoz M (2002) DNA biosensor for Microcystis spp. sequence detection by using methylene blue and
ruthenium complex as electrochemical hybridization label. Turkish Journal of
Chemistry 26, 851-862
Guo M-D, Li Y-Q, Guo H-X, Wu X-Q, Fan L-F (2007) Electrochemical
detection of short sequences related to the hepatitis B virus using MB on
chitosan-modified CPE. Bioelectrochemistry 70, 245-249
Hanboonsong Y, Choosai C, Panyim S, Damak S (2002) Transovarial transmission of sugarcane white leaf phytoplasma in the insect vector Matsumuratettix hiroglyphicus (Matsumura). Insect Molecular Biology 11, 97-103
Hanboonsong Y, Ritthison W, Choosai C, Sirithorn P (2006) Transmission of
sugarcane white leaf phytoplasma by Yamatotettix flavovittatus, a new leafhopper vector. Journal of Economic Entomology 99, 1531-1537
Hongspluk W, Kusalwong A, Ouvanich W (1993) Population dynamic of
sugarcane white leaf disease vector. In: Proceedings of the 1st National Conference on Sugarcane and Sugar, 14-16 September 1993, Bangkok, Thailand
Society of Sugarcane and Technologies, Thailand, pp 621-633
Jones P, Devonshire BJ, Holman TJ, Ajanga S (2004) Napier grass stunt: A
new disease associated with a 16SrXI group phytoplasma in Kenya. Plant
Pathology 53, 519
Ju H, Ye B, Gu J (2004) Supermolecular interaction of ferrocenium with yeast
DNA and application in electrochemical sensing for hybridization recognition of yeast DNA. Sensors 4, 71-83
Jung HY, Sawayanagi T, Wongkaew P, Kakizawa S, Nishigawa H, Wei W,
Oshima K, Miyata SI, Ugaki M, Hibi T, Namba S (2003) Candidatus Phytoplasma oryzae, a novel phytoplasma taxon associated with rice yellow
dwarf disease. International Journal of Systematic and Evolutionary Microbiology 53, 1925-1929
Klinkong S, Seemüller E (1993) Detection and differentiation of the mycoplasma-like organism associated with sugarcane white leaf disease using
cloned extrachromosomal DNA probe. Kasetsart Journal 27, 98-103
Kumarrasinghe NC, Jones P (2001) Identification of white leaf disease of
sugarcane in Sri Lanka. Sugar Tech 3, 55-58
Kusalwong A (1980) Sugarcane disease. In Suriyapan P (Ed) Sugarcane,
Monograph Number 1, Department of Agriculture, Ministry of Agriculture
and Cooperatives, Bangkok, Thanapradith Press, Thailand, pp 131-144
Kusalwong A, Ouvanich W (1993) Example model for sugarcane white leaf
disease control. Proceeding of the 31st Kasetsart University Conference, 3-6
February 1993, Bangkok, Kasetsart University, Thailand, pp 348-352
Lairungreung C, Srising S, Pa-oplek S, Pliansinchai U (1995) Studies on the
evidence of sugarcane green grassy shoot disease in cane varieties collection
fields. In: Annual Report of Supanburi Agricultural Crop Research Center,
Special Issue for Sugarcane, Supanburi, Thailand, pp 220-232
Leu LS (1974) An insectary method for testing sugarcane varieties for resis-
82
Sugarcane white leaf disease: Characterization, diagnosis and control. Porntip Wongkaew
Disease Notes 4, 56-58
Rao GP, Mall S, Marcone C (2010) Candidatus Phytoplasma cynodontis’
(16SrXIV group) affecting Oplismenus burmannii (Retz.) P. Beauv. and Digitaria sanguinalis (L.) Scop. in India. Australasian Plant Disease Notes 5, 9395
Rao GP, Raj SK, Snehi SK, Mall S, Singh M, Marcone C (2007a) Molecular
evidence for the presence of 'Candidatus Phytoplasma cynodontis', the Bermuda grass white leaf agent, in India. Bulletin of Insectology 60, 145-6
Rao GP, Sangeeta S, Singh M, Marcone C (2007b) Phylogenetic relationships
of sugarcane grassy shoot phytoplasma with closely related agents. Bulletin
of Insectology 60, 347-348
Rao GP, Srivastava S, Gupta PS, Sharma SR, Singh A, Singh S, Singh M,
Marcone C (2008) Detection of sugarcane grassy shoot phytoplasma infecting sugarcane in India and its phylogenetic relationships to closely related
phytoplasmas. Sugar Tech 10, 74-80
Richi N, Chen CT (1989) Grassy shoot and white leaf diseases. In: Ricaud C,
Ecan BT, Gillaspie Jr. AG, Hughes CG (Eds) Disease of Sugarcane, Major
Diseases, Elsevier Publisher, Amsterdam, pp 289-300
Sadoodee R, Schneider B, Padovan AC, Gibb KS (1999) Detection and genetic relatedness of phytoplasmas associated with plant diseases in Thailand.
Journal of Biochemistry, Molecular Biology and Biophysics 3, 133-140
Salehi M, Izadpanah K, Siampour M, Taghizadeh M (2009) Molecular characterization of burmuda grass white leaf phytoplasma in Iran. Journal of
Plant Pathology 91, 655-661
Sa-Ngaunrangsirikul S, Sansayawichai T, Polpakdee W, Srising S, Vorastit
N, Vanich M (2007) A survey of sugarcane white leaf disease and the disease
tolerance in wild cane and sugarcane hybrids. Annaul Report of Khon Kaen
Agricultural Crop Research Center, Khon Kaen, Thailand, pp 132-138
Sarindu N, Clark MF (1993) Antibody production and identify of MLOs associated with sugarcane white leaf disease and Bermuda grass white leaf disease from Thailand. Plant Pathology 42, 396-402
Snehi SK, Khan MS, Raj SK, Mall S, Singh M, Rao GP (2007) Molecular
identification of 'Candidatus Phytoplasma cynodontis' associated with Bermuda grass white leaf disease in India. New Disease Reports 16, 3
Seemüller E, Marcone C, Lauer U, Ragozzino A, Göschl M (1998) Current
status of molecular classification of the phytoplasmas. Journal of Plant
Pathology 80, 3-26
Shigata E, Teng WS, Matsumoto T (1969) Mycoplasma or PLT-like microorganism detected in leaf of sugarcane plant infected with white leaf disease
and suppression of the disease symptoms by the antibiotics of tetracycline
group. Journal of the Faculty of Agriculture, Hokkaido University 56, 70-90
Sun W, Qin P, Gao H, Li G, Jiao K (2010) Electrochemical DNA biosensor
based on chitosan/nano-V2O5/MWCNTs composite film modified carbon
ionic liquid electrode and its application to the LAMP product of Yersinia
enterocolitica gene sequence. Biosensors and Bioelectronics 25, 1264-1270
Teles FRR, Fonseca LP (2008) Trends in DNA biosensors. Talanta 77, 606623
Vasudeva RS (1955) Stunting of sugarcane. Commonwealth Phytopathological
Society News 1, 11
Velusamy V, Arshak K, Korostynska O, Oliwa K, Adley C (2010) An overview of foodborne pathogen detection: In the perspective of biosensors. Biotechnology Advances 28, 232-254
Warokka J (2005) Studies on the etiology and epidemiology of kalimantan wilt
disease of coconut in Indonesia. PhD thesis, University of Nottingham,
Nottingham, UK, 179 pp
Wongkaew P (1999) Sugarcane White Leaf Disease Management, Thailand
Research Fund, Pimpatana Press, Khon Kaen, Thailand, 228 pp
Wongkaew P, Fletcher J (2004) Sugarcane white leaf phytoplasma in tissue
culture: Long-term maintenance, transmission, and oxytetracycline remission.
Plant Cell Reports 23, 426-434
Wongkaew P, Hanboonsong Y, Sirithorn P, Boonkrong S, Choosai C (1998)
Simplified techniques for field diagnosis of sugarcane white leaf phytoplasma by DNA probe. Proceedings of the 3rd National Conference on Sugarcane and Sugar, 6-8 May 1998, Khon Kaen, Thailand Society of Sugarcane
and Technologies, Thailand, pp 50-61
Wongkaew P, Hanboonsong Y, Sirithorn P, Choosai C, Boonkrong S, Tinnangwattana T, Kitchareonpanya R, Damak S (1997) Differentiation of
phytoplasmas associated with sugarcane and gramineous weed white leaf disease and sugarcane grassy shoot disease by RFLP and sequencing. Theoretical and Applied Genetics 95, 660-663
Wongkaew P, Poosittisak S (2008) Direct electrochemical DNA sensor for
sugarcane white leaf disease diagnosis using label free DNA probes and
oligochitosan self assembled monolayer-modified glassy carbon electrodes.
In: Proceedings of the 2nd Technology and Innovation for Sustainable Development Conference, 28-29 January 2008, Khon Kaen, Khon Kaen University,
Thailand, pp 504-507
Wongkaew P, Poosittisak S (2010) A label-free electrochemical biosensor for
sugarcane white leaf disease diagnosis based on the causal phytoplasma DNA
and chitosan matrix using methylene blue as hybridization indicator. The 20th
Anniversary World Congress on Biosensors, 26-28 May 2010, Elsevier Inc.,
Glasgow, United Kingdom, P3.1.018
Wongkaew P, Sirithorn P, Chaleeprom W, Nakashima K, Hayashi T, Koizumi M (1995) Detection of sugarcane white leaf mycoplasma-like organism
tance to white leaf disease. In: Proceedings of the XV International Society of
Sugarcane Technologists Congress, 13-29 June 1974, Durban, The International Society of Sugarcane Technologists, South Africa, pp 266-274
Leu LS (1978) Apical meristem culture and redifferentiation of callus masses to
free some sugarcane disease. Taiwan Plant Protection Bulletin 20, 77-82
Leu LS (1983) White leaf disease. In: Agnihotri VP (Ed) Disease of Sugarcane,
Oxford and IBH Publishing Co, Delhi, pp 250-267
Leu LS, Kusalwong A (2000) Control of sugarcane white leaf disease in Taiwan and Thailand. In: Proceeding of the 6th International Society of Sugarcane Technologists Pathology Workshop, 16-23 July 2000, Petchaburi, The
International Society of Sugarcane Technologists, Thailand, Ph-1
Ling KC, Chaun-Yang C (1962) A preliminary study on the white leaf disease
of sugarcane. Report of the Republic of Taiwan Sugar Experiment Station 28,
139-172
Ling KC, Chaun-Yang C (1963a) Studies on the white leaf disease of sugarcane, II. Effect of hot-water treatment in the disease. Report of the Republic
of Taiwan Sugar Experiment Station 30, 75-89
Ling KC, Chaun-Yang C (1963b) Studies on the white leaf disease of sugarcane, III. Evidence of spreading of the disease under natural condition. Report of the Republic of Taiwan Sugar Experiment Station 30, 91-94
Liu HP, Lee SM, Teng WS (1963) Studies on the effect of cane yield and the
heat treatment of white leaf disease of sugarcane. Report of the Republic of
Taiwan Sugar Experiment Station 32, 103-110
Liu MC (1981) In vitro methods applied to sugarcane improvement. In: Thorpe
TA (Ed) Plant Tissue Culture Methods and Applications in Agriculture, Academic Press, New York, pp 299-323
Manimekalai R, Sathish Kumar R, Soumya VP, Thomas GV (2010a) Molecular detection of phytoplasma associated with yellow leaf disease in areca
palms (Areca catechu) in India. Plant Disease 94, 1376
Manimekalai R, Soumya VP, Sathish Kumar R (2010b) Molecular detection
of 16SrXI group phytoplasma associated with root (wilt) disease of coconut
(Cocos nucifera) in India. Plant Disease 94, 636
Marcone C, Schneider B, Seemüller E (2004) Candidatus Phytoplasma
Cyanodontis, the phytoplasma associated with Bermuda grass white leaf.
International Journal of Systematic and Evolutionary Microbiology 54, 10771082
Matsumoto T, Lee CS, Teng WS (1969) Studies on sugarcane white leaf disease of Taiwan, with special reference to the transmission by a leafhopper,
Epitettix hiroglyphicus Mats. Annals of the Phytopathological Society of
Japan 35, 251-259
Mongkolsuk Y, Sutabutra T (1976) Mycoplasma-like organism in white leaf
disease of sugarcane. Journal of Science Society of Thailand 2, 139-141
Nakashima K, Chaleeprom W, Wongkaew P, Sirithorn P (1994) Detection of
mycoplasmalike organism associated with white leaf disease of sugarcane in
Thailand using DNA probes. JIRCAS Journal 1, 57-67
Nakashima K, Wongkaew P, Chaleeprom W, Sirithorn P, Hayashi T (1999)
Molecular detection and characterization of phytoplasmas that cause sugarcane white leaf disease. JIRCAS Journal 7, 1-17
Namba S, Oyaisu H, Kato S, Iwanami S, Tsuchizaki T (1993) Phylogenetic
diversity of phytopathogenic mycoplasma like organisms. International Journal of Systematic Bacteriology 43, 461-467
Nasare K, Yadev A, Singh AK, Shivasharanappa KB, Nerkar YS, Reddy VS
(2007) Molecular and symptom analysis reveal the presence of new phytoplasmas associated with sugarcane grassy shoot disease in India. Plant Disease 91, 1413-1418
Nejat N, Sijam K, Abdullah SNA, Vadamalai G, Dickinson M (2009) Molecular characterization of a phytoplasma associated with coconut yellow decline (CYD) in Malaysia. American Journal of Applied Sciences 6, 1331-1340
Nejat N, Vadamalai G (2010) Phytoplasma detection in coconut palm and
other tropical crops. Plant Pathology 9, 112-121
Nielsen SL, Ebong C, Kabirizi J, Nicolaisen M (2007) First report of a
16SrXI group phytoplasma (Candidatus Phytoplasma oryzae) associated
with Napier grass stunt disease in Uganda. Plant Pathology 56, 1039-1039
Ouvanich W, Kusalwong A (1993) Red rot-Fusarium stem rot and white leaf
disease incidence on some sugarcane hybrids. In: Proceedings of the 31st
Kasetsart University Conference, Bangkok, 3-6 February 1993, Bangkok,
Kasetsart University, Thailand, pp 561-569
Ouvanich W, Kusalwong A, Hongspluk W, Serapan S, Titatan S (1990) Studies on the epidemiology of sugarcane white leaf disease. In: Proceedings of
the 28th Kasetsart University Conference, 29-31 January 1990, Bangkok,
Kasetsart University, Thailand, pp 267-278
Pan YS, Yang SL (1970) Studies on the bionomics of Matsumuratettix hiroglyphicus (Matsumura), an insect vector of sugarcane white leaf disease. I. Seasonal abundance and dispersal of the adult. Report of the Republic of Taiwan
Sugar Experiment Station 50, 65-71
Pisitkul S, Kanta C, Wongkaew S, Neera P, Chaioy P, Chetarash C (1989)
Studies on the insect vector of sugarcane white leaf disease and their control.
Khon Kaen Agriculture 17, 164-172
Rane MS, Dakshindas DG (1962) The sugarcane disease "albino" or "grassy
shoot"? Indian Sugar 12, 179-180
Rao GP, Mall S, Singh M, Marcone C (2009) First report of a “Candidatus
Phytoplasma cyanodontis”-related strain (group 16SXIV) associated with
white leaf disease of Dichanthium annulatum in India. Australasian Plant
83
Functional Plant Science and Biotechnology 6 (Special Issue 2), 73-84 ©2012 Global Science Books
Yang SL (1972) Bionomics of Matsumuratettix hiroglyphicus Matsumura, an
insect vector of sugarcane white leaf disease. III. A study on the relationship
between environment factors and oviposition of Matsumuratettix hiroglyphicus Matsumura. Republic of Taiwan Sugar Experiment Station Report 57, 6574
Yang SL, Pan YS (1969) Ecology and morphology of Epitettix hiroglyphicus
Matsumura. Republic of Taiwan Sugar Experiment Station Report 48, 25-35
Ye Y, Ju H (2003) DNA electrochemical behaviors, recognition and sensing by
combining with PCR technique. Sensors 3, 128-145
Zhang D, Peng Y, Qi HQ, Zhang C (2010) Label-free electrochemical DNA
biosensor array for simultaneous detection of the HIV-1 and HIV-2 oligonucleotides incorporating different hairpin-DNA probes and redox indicator.
Biosensors and Bioelectronics 25, 1088-1094
in field plants and tissue cultures by DNA probes. In: Matangkasombat P,
Yoshida T (Eds) Microbial Utilization of Renewable Resources, ICCR Biotechnology 9, pp 406-419
Wongkaew P, Sirithorn P, Hanboonsong Y, Tinnangwattana T, Chareonkitpanya R (1999) Preliminary survey on the white leaf disease predicament in
the Northeast Thai. Thai Journal of Cane and Sugar 6, 36-52
Wongkaew P, Sirithorn P, Kusalwon A (2000) Phylogenetic relationship of
sugarcane white leaf phytoplasma with other phytoplasmas in sugarcane
fields. In: Proceedings of the 6th International Society of Sugarcane Technologists Pathology Workshop, 16-23 July 2000, Petchaburi, The International Society of Sugarcane Technologists, Thailand, Ph-4
Wu L, Wu R-Y, Li HW, Chu P-N (1969) Influence of white leaf disease of
sugarcane on the chloroplast development and chlorophyll biosynthesis.
Botanical Bulletin of Academia Sinica 10, 23-26
84