PhD in Plant Pathology working in a nonprofit research institution on research activities to better understand the spread of a insect-transmitted bacterium-associated vascular disease of citrus trees, in attempts to develop a warning system to improve disease control.
IntroductionThe severe Asian form of huanglongbing (HLB), a vascular disease associated with the ... more IntroductionThe severe Asian form of huanglongbing (HLB), a vascular disease associated with the phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’, is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri. Disease impacts are known for sweet oranges and acid limes but not lemons.MethodsIn a five-year study (2017–2021) we compared yield and fruit quality between naturally-infected and healthy 5-yr-old trees of Sicilian lemon ‘Femminello’, and shoot phenology on both lemon and ‘Valencia’ orange, both grafted onto ‘Swingle’ citrumelo, grown in southeastern São Paulo State, Brazil. HLB severity (percentage of tree canopy area with HLB symptoms) was assessed every 3–4 months, fruit yield and quality in May (2017 to 2019) or June/July (2020-2021), and vegetative and reproductive shoots fortnightly on 50-cm-long branches. The development of ACP on one-year-old seedlings of five lemon varieties, ‘Tahiti’ acid lime, ‘Valencia’ orange, and orange jasmine was evaluated.Resu...
Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus are two bacterial specie... more Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus are two bacterial species that cause huanglongbing (HLB) disease in citrus-growing regions of Brazil. A concentrated sampling plan of a grove in Matao, Brazil was initiated to evaluate the spatial and temporal spread of these bacteria. The exact location of each of 8697 trees was recorded, and each symptomatic tree was assessed by PCR for the presence of Ca. L. americanus and Ca. L. asiaticus during 17 different months from April 2006 to May 2008 (Fig. 1). In the first month, only five trees were confirmed to have Ca. L. asiaticus. The first trees with confirmed cases of Ca. L. americanus were not found until February 2007. By the end of the study, 43 trees were confirmed to have Ca. L. americanus, 1164 trees were confirmed to have Ca. L. asiaticus, and three trees were coinfected. For the new trees each month that were confirmed to have Ca. L. asiaticus, their distance (m) from previously infected trees was ca...
Huanglongbing (HLB) is the most destructive, yet incurable disease of citrus. Finding sources of ... more Huanglongbing (HLB) is the most destructive, yet incurable disease of citrus. Finding sources of genetic resistance to HLB-associated ‘CandidatusLiberibacter asiaticus’ (Las) becomes strategic to warrant crop sustainability, but no resistantCitrusgenotypes exist. SomeCitrusrelatives of the family Rutaceae, subfamily Aurantioideae, were described as full-resistant to Las, but they are phylogenetically far, thus incompatible withCitrus. Partial resistance was indicated for certain cross-compatible types. Moreover, other genotypes from subtribe Citrinae, sexually incompatible but graft-compatible withCitrus, may provide new rootstocks able to restrict bacterial titer in the canopy. Use of seedlings from monoembryonic species and inconsistencies in previous reports likely due to Las recalcitrance encouraged us to evaluate more accurately theseCitrusrelatives. We tested for Las resistance a diverse collection of graft-compatible Citrinae species using an aggressive and consistent challen...
In São Paulo State (SPS), the occurrence of several citrus diseases and pests has largely increas... more In São Paulo State (SPS), the occurrence of several citrus diseases and pests has largely increased fruit production costs. Huanglongbing (HLB), caused by two Candidatus Liberibacter species, is currently the most important citrus disease in the country. First reported in Brazil in 2004, HLB is already present in 268 municipalities affecting 24% of the more than 96 thousand citrus blocks. Control measures include elimination of symptomatic trees, insecticide application against the vector of the disease, Diaphorina citri, and planting healthy trees produced in screened nurseries. Tree elimination is mandatory but has not been adopted in all farms, where HLB incidence has increased considerably. This situation affected the effectiveness of disease control in their neighbors. Important research questions to improve disease management also need answers, despite the wide perception that only the increase in citrus resistance to liberibacter or D. citri will bring long-term solution to the HLB problem.
Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (c... more Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (citrus variegated chlorosis [CVC]) and coffee (coffee leaf scorch [CLS]) plants in Brazil. CVC and CLS occur sympatrically and share leafhopper vectors; thus, determining whether X. fastidiosa isolates can be dispersed from one crop to another and cause disease is of epidemiological importance. We sought to clarify the genetic and biological relationships between CVC- and CLS-causing X. fastidiosa isolates. We used cross-inoculation bioassays and microsatellite and multilocus sequence typing (MLST) approaches to determine the host range and genetic structure of 26 CVC and 20 CLS isolates collected from different regions in Brazil. Our results show that citrus and coffee X. fastidiosa isolates are biologically distinct. Cross-inoculation tests showed that isolates causing CVC and CLS in the field were able to colonize citrus and coffee plants, respectively, but not the other host, indicati...
Xylella fastidiosa comprises a diverse group of xylem-limited, insect-transmitted bacterial patho... more Xylella fastidiosa comprises a diverse group of xylem-limited, insect-transmitted bacterial pathogens. In Brazil, the citrus variegated chlorosis (CVC) and coffee stem atrophy (CSA) diseases are caused by X. fastidiosa subspecies pauca transmitted by common insect vectors. No simple protocol allowing strain discrimination exists, making epidemiological studies, which are important for devising control measures, difficult to undertake. Here, we show that both strains can easily be distinguished based on the pattern of leaf symptoms that they induce on pin prick-inoculated tobacco seedlings, namely small orange lesions and large necrotic lesions induced by the CVC and CSA strains, respectively. These differential responses allowed us to investigate whether mixed strain infections would occur in citrus or coffee trees in the field. Seedlings were individually inoculated with X. fastidiosa colonies recovered from citrus or coffee plants from various locations at three different times. N...
The phloem-limited ‘Candidatus Liberibacter asiaticus’ (Las) causes huanglongbing (HLB), a destru... more The phloem-limited ‘Candidatus Liberibacter asiaticus’ (Las) causes huanglongbing (HLB), a destructive citrus disease. Graft-inoculated potted plants were used to assess Las speed of movement in phloem in greenhouse (GH), and the impacts of temperature on plant colonization in growth chambers (GC) experiments. For assessment of Las speed, plants were inoculated at the main stem and assessed over time by qPCR or symptom at various distances from the inoculum. For colonization, the plants were inoculated in one of two opposite top branches, were maintained at 8 to 20, 18 to 30 or 24 to 38ºC daily range, and assessed by qPCR of samples taken from non-inoculated shoots. For all experiments, frequencies of Las-positive sites were submitted to ANOVA and binomial GLM and logistic regression analysis. Probabilities of detecting Las in GH plants were functions of time and distance from the inoculation site, which resulted in 2.9 and 3.8 cm day-1 average speed of movement. In GC, the temperat...
‘Candidatus Liberibacter asiaticus’ (Las) is an unculturable, phloem-limited, insect-transmitted ... more ‘Candidatus Liberibacter asiaticus’ (Las) is an unculturable, phloem-limited, insect-transmitted bacterium associated with the Asiatic form of huanglongbing (HLB), the most destructive citrus disease. In Asia and the Americas, it is transmitted by the Asian citrus psyllid (Diaphorina citri Kuwavama). Despite considerable research, little is known about the processes involved in plant infection and colonization by Las. This study was conducted to determine whether the basal portion (below girdling) of the plant is an important route for Las to move laterally from a point of inoculation on a branch to pathogen-free branches elsewhere in the canopy, and to quantify the influence of actively growing tissues on vertical upward (acropetally) or downward (basipetally) movement of Las. Nongirdled and fully or partially girdled stems of potted plants of ‘Pera’ sweet orange, graft-inoculated above or below girdling, were sampled in distinct regions and assessed by qPCR, 6 months postinoculation. Las invaded all regions of partially and nongirdled plants but remained restricted to the inoculated regions of fully girdled plants, evidence that in planta bacterium movement is limited to the phloem. In fully girdled plants, starch accumulated above the girdling site, probably because of changes in flow of phloem sap. To study the influence of actively growing tissues, inoculated ‘Valencia’ sweet orange plants were kept intact or were top- or root-pruned to force production of new tissues, and sampled at 15-day intervals. Las migrated rapidly and most predominantly toward newly developing root and leaf tissues. The rapid and predominant movement of Las to newly developed shoots and roots would explain failures of canopy heat treatments and pruning to cure HLB-affected trees, and reinforces the need to protect rapidly growing new shoots from feeding by D. citri in order to minimize transmission and spread of the pathogen by the vector within and between orchards.
Murraya paniculata and Swinglea glutinosa are aurantioid hosts of the Asian citrus psyllid (ACP) ... more Murraya paniculata and Swinglea glutinosa are aurantioid hosts of the Asian citrus psyllid (ACP) Diaphorina citri, the principal vector of ‘Candidatus Liberibacter asiaticus’ (Las). Las is the pathogen associated with huanglongbing (HLB), the Asian form of which is the most devastating disease of Citrus species and cultivars (Rutaceae: Aurantioideae). M. paniculata is a common ornamental and S. glutinosa is grown as an ornamental, a citrus rootstock, and a hedgerow fence plant. Because of the uncertain status of these plants as reservoirs of Las, a series of cross-inoculation bioassays were carried out in different environments, using infected Valencia sweet orange (Citrus × aurantium) infected shoot tops as a source of inoculum and D. citri nymphs and adults reared on M. paniculata and S. glutinosa to inoculate pathogen-free Valencia orange plantlets. In contrast to sweet orange, Las was more unevenly distributed and reached much lower titers in M. paniculata and S. glutinosa. Infe...
Citrus variegated chlorosis (CVC) disease, caused by the xylem-limited and insect-transmitted bac... more Citrus variegated chlorosis (CVC) disease, caused by the xylem-limited and insect-transmitted bacterium Xylella fastidiosa, has caused severe losses in orange production in Brazil. Disease control requires insecticide applications, tree removal, and pruning of symptomatic branches. Pruning success has been erratic, especially in areas of high disease incidence. In this work, in planta X. fastidiosa distribution and the effectiveness of severe pruning procedures for curing diseased adult trees were investigated. Most sampled upper parts of the trees contained X. fastidiosa, but at higher frequencies in symptomatic branches. Removal of all main branches (decapitation) was not effective and revealed a 20 to 30% incidence of latent infections. Trunk decapitation resulted in a higher number of healthy scions but killed 10 to 30% of the remaining trunks. Removal of all scion and grafting the newly sprouted shoots of ‘Rangpur’ lime (Citrus limonia Osbeck) or ‘Cleopatra’ (Citrus reshni Hort...
The biology and behaviour of the psyllid Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Li... more The biology and behaviour of the psyllid Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Liviidae), the major insect vector of bacteria associated with huanglongbing, have been extensively studied with respect to host preferences, thermal requirements, and responses to visual and chemical volatile stimuli. However, development of the psyllid in relation to the ontogeny of immature citrus flush growth has not been clearly defined or illustrated. Such information is important for determining the timing and frequency of measures used to minimize populations of the psyllid in orchards and spread of HLB. Our objective was to study how flush ontogeny influences the biotic potential of the psyllid. We divided citrus flush growth into six stages within four developmental phases: emergence (V1), development (V2 and V3), maturation (V4 and V5), and dormancy (V6). Diaphorina citri oviposition and nymph development were assessed on all flush stages in a temperature controlled room, and in...
Huanglongbing (HLB) is a difficult-to-control and highly destructive citrus disease that, in Braz... more Huanglongbing (HLB) is a difficult-to-control and highly destructive citrus disease that, in Brazil, is associated mainly with the bacterium ‘Candidatus Liberibacter asiaticus’ transmitted by the psyllid Diaphorina citri. The aim of this study was to improve our understanding of the ‘Ca. L. asiaticus’ infection process by exposing excised, fully expanded, immature citrus leaves in 50-ml Falcon tubes to one, four, or eight adults from a ‘Ca. L. asiaticus’-exposed colony for 1-, 3-, 7-, or 15-day periods for access to inoculation (IAP). The leaves were incubated at 26°C for 1, 3, 7, 15, and 21 days (incubation period [IP]). Infection frequencies and ‘Ca. L. asiaticus’ titers were assessed by quantitative polymerase chain reaction (qPCR). ‘Ca. L. asiaticus’ infection was a function of leaf age, number of insects, IAP, and IP. In general, higher infection rates were observed on younger leaves inoculated with higher numbers of insects and after longer IAP and IP. The immature excised lea...
IntroductionThe severe Asian form of huanglongbing (HLB), a vascular disease associated with the ... more IntroductionThe severe Asian form of huanglongbing (HLB), a vascular disease associated with the phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’, is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri. Disease impacts are known for sweet oranges and acid limes but not lemons.MethodsIn a five-year study (2017–2021) we compared yield and fruit quality between naturally-infected and healthy 5-yr-old trees of Sicilian lemon ‘Femminello’, and shoot phenology on both lemon and ‘Valencia’ orange, both grafted onto ‘Swingle’ citrumelo, grown in southeastern São Paulo State, Brazil. HLB severity (percentage of tree canopy area with HLB symptoms) was assessed every 3–4 months, fruit yield and quality in May (2017 to 2019) or June/July (2020-2021), and vegetative and reproductive shoots fortnightly on 50-cm-long branches. The development of ACP on one-year-old seedlings of five lemon varieties, ‘Tahiti’ acid lime, ‘Valencia’ orange, and orange jasmine was evaluated.Resu...
Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus are two bacterial specie... more Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus are two bacterial species that cause huanglongbing (HLB) disease in citrus-growing regions of Brazil. A concentrated sampling plan of a grove in Matao, Brazil was initiated to evaluate the spatial and temporal spread of these bacteria. The exact location of each of 8697 trees was recorded, and each symptomatic tree was assessed by PCR for the presence of Ca. L. americanus and Ca. L. asiaticus during 17 different months from April 2006 to May 2008 (Fig. 1). In the first month, only five trees were confirmed to have Ca. L. asiaticus. The first trees with confirmed cases of Ca. L. americanus were not found until February 2007. By the end of the study, 43 trees were confirmed to have Ca. L. americanus, 1164 trees were confirmed to have Ca. L. asiaticus, and three trees were coinfected. For the new trees each month that were confirmed to have Ca. L. asiaticus, their distance (m) from previously infected trees was ca...
Huanglongbing (HLB) is the most destructive, yet incurable disease of citrus. Finding sources of ... more Huanglongbing (HLB) is the most destructive, yet incurable disease of citrus. Finding sources of genetic resistance to HLB-associated ‘CandidatusLiberibacter asiaticus’ (Las) becomes strategic to warrant crop sustainability, but no resistantCitrusgenotypes exist. SomeCitrusrelatives of the family Rutaceae, subfamily Aurantioideae, were described as full-resistant to Las, but they are phylogenetically far, thus incompatible withCitrus. Partial resistance was indicated for certain cross-compatible types. Moreover, other genotypes from subtribe Citrinae, sexually incompatible but graft-compatible withCitrus, may provide new rootstocks able to restrict bacterial titer in the canopy. Use of seedlings from monoembryonic species and inconsistencies in previous reports likely due to Las recalcitrance encouraged us to evaluate more accurately theseCitrusrelatives. We tested for Las resistance a diverse collection of graft-compatible Citrinae species using an aggressive and consistent challen...
In São Paulo State (SPS), the occurrence of several citrus diseases and pests has largely increas... more In São Paulo State (SPS), the occurrence of several citrus diseases and pests has largely increased fruit production costs. Huanglongbing (HLB), caused by two Candidatus Liberibacter species, is currently the most important citrus disease in the country. First reported in Brazil in 2004, HLB is already present in 268 municipalities affecting 24% of the more than 96 thousand citrus blocks. Control measures include elimination of symptomatic trees, insecticide application against the vector of the disease, Diaphorina citri, and planting healthy trees produced in screened nurseries. Tree elimination is mandatory but has not been adopted in all farms, where HLB incidence has increased considerably. This situation affected the effectiveness of disease control in their neighbors. Important research questions to improve disease management also need answers, despite the wide perception that only the increase in citrus resistance to liberibacter or D. citri will bring long-term solution to the HLB problem.
Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (c... more Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (citrus variegated chlorosis [CVC]) and coffee (coffee leaf scorch [CLS]) plants in Brazil. CVC and CLS occur sympatrically and share leafhopper vectors; thus, determining whether X. fastidiosa isolates can be dispersed from one crop to another and cause disease is of epidemiological importance. We sought to clarify the genetic and biological relationships between CVC- and CLS-causing X. fastidiosa isolates. We used cross-inoculation bioassays and microsatellite and multilocus sequence typing (MLST) approaches to determine the host range and genetic structure of 26 CVC and 20 CLS isolates collected from different regions in Brazil. Our results show that citrus and coffee X. fastidiosa isolates are biologically distinct. Cross-inoculation tests showed that isolates causing CVC and CLS in the field were able to colonize citrus and coffee plants, respectively, but not the other host, indicati...
Xylella fastidiosa comprises a diverse group of xylem-limited, insect-transmitted bacterial patho... more Xylella fastidiosa comprises a diverse group of xylem-limited, insect-transmitted bacterial pathogens. In Brazil, the citrus variegated chlorosis (CVC) and coffee stem atrophy (CSA) diseases are caused by X. fastidiosa subspecies pauca transmitted by common insect vectors. No simple protocol allowing strain discrimination exists, making epidemiological studies, which are important for devising control measures, difficult to undertake. Here, we show that both strains can easily be distinguished based on the pattern of leaf symptoms that they induce on pin prick-inoculated tobacco seedlings, namely small orange lesions and large necrotic lesions induced by the CVC and CSA strains, respectively. These differential responses allowed us to investigate whether mixed strain infections would occur in citrus or coffee trees in the field. Seedlings were individually inoculated with X. fastidiosa colonies recovered from citrus or coffee plants from various locations at three different times. N...
The phloem-limited ‘Candidatus Liberibacter asiaticus’ (Las) causes huanglongbing (HLB), a destru... more The phloem-limited ‘Candidatus Liberibacter asiaticus’ (Las) causes huanglongbing (HLB), a destructive citrus disease. Graft-inoculated potted plants were used to assess Las speed of movement in phloem in greenhouse (GH), and the impacts of temperature on plant colonization in growth chambers (GC) experiments. For assessment of Las speed, plants were inoculated at the main stem and assessed over time by qPCR or symptom at various distances from the inoculum. For colonization, the plants were inoculated in one of two opposite top branches, were maintained at 8 to 20, 18 to 30 or 24 to 38ºC daily range, and assessed by qPCR of samples taken from non-inoculated shoots. For all experiments, frequencies of Las-positive sites were submitted to ANOVA and binomial GLM and logistic regression analysis. Probabilities of detecting Las in GH plants were functions of time and distance from the inoculation site, which resulted in 2.9 and 3.8 cm day-1 average speed of movement. In GC, the temperat...
‘Candidatus Liberibacter asiaticus’ (Las) is an unculturable, phloem-limited, insect-transmitted ... more ‘Candidatus Liberibacter asiaticus’ (Las) is an unculturable, phloem-limited, insect-transmitted bacterium associated with the Asiatic form of huanglongbing (HLB), the most destructive citrus disease. In Asia and the Americas, it is transmitted by the Asian citrus psyllid (Diaphorina citri Kuwavama). Despite considerable research, little is known about the processes involved in plant infection and colonization by Las. This study was conducted to determine whether the basal portion (below girdling) of the plant is an important route for Las to move laterally from a point of inoculation on a branch to pathogen-free branches elsewhere in the canopy, and to quantify the influence of actively growing tissues on vertical upward (acropetally) or downward (basipetally) movement of Las. Nongirdled and fully or partially girdled stems of potted plants of ‘Pera’ sweet orange, graft-inoculated above or below girdling, were sampled in distinct regions and assessed by qPCR, 6 months postinoculation. Las invaded all regions of partially and nongirdled plants but remained restricted to the inoculated regions of fully girdled plants, evidence that in planta bacterium movement is limited to the phloem. In fully girdled plants, starch accumulated above the girdling site, probably because of changes in flow of phloem sap. To study the influence of actively growing tissues, inoculated ‘Valencia’ sweet orange plants were kept intact or were top- or root-pruned to force production of new tissues, and sampled at 15-day intervals. Las migrated rapidly and most predominantly toward newly developing root and leaf tissues. The rapid and predominant movement of Las to newly developed shoots and roots would explain failures of canopy heat treatments and pruning to cure HLB-affected trees, and reinforces the need to protect rapidly growing new shoots from feeding by D. citri in order to minimize transmission and spread of the pathogen by the vector within and between orchards.
Murraya paniculata and Swinglea glutinosa are aurantioid hosts of the Asian citrus psyllid (ACP) ... more Murraya paniculata and Swinglea glutinosa are aurantioid hosts of the Asian citrus psyllid (ACP) Diaphorina citri, the principal vector of ‘Candidatus Liberibacter asiaticus’ (Las). Las is the pathogen associated with huanglongbing (HLB), the Asian form of which is the most devastating disease of Citrus species and cultivars (Rutaceae: Aurantioideae). M. paniculata is a common ornamental and S. glutinosa is grown as an ornamental, a citrus rootstock, and a hedgerow fence plant. Because of the uncertain status of these plants as reservoirs of Las, a series of cross-inoculation bioassays were carried out in different environments, using infected Valencia sweet orange (Citrus × aurantium) infected shoot tops as a source of inoculum and D. citri nymphs and adults reared on M. paniculata and S. glutinosa to inoculate pathogen-free Valencia orange plantlets. In contrast to sweet orange, Las was more unevenly distributed and reached much lower titers in M. paniculata and S. glutinosa. Infe...
Citrus variegated chlorosis (CVC) disease, caused by the xylem-limited and insect-transmitted bac... more Citrus variegated chlorosis (CVC) disease, caused by the xylem-limited and insect-transmitted bacterium Xylella fastidiosa, has caused severe losses in orange production in Brazil. Disease control requires insecticide applications, tree removal, and pruning of symptomatic branches. Pruning success has been erratic, especially in areas of high disease incidence. In this work, in planta X. fastidiosa distribution and the effectiveness of severe pruning procedures for curing diseased adult trees were investigated. Most sampled upper parts of the trees contained X. fastidiosa, but at higher frequencies in symptomatic branches. Removal of all main branches (decapitation) was not effective and revealed a 20 to 30% incidence of latent infections. Trunk decapitation resulted in a higher number of healthy scions but killed 10 to 30% of the remaining trunks. Removal of all scion and grafting the newly sprouted shoots of ‘Rangpur’ lime (Citrus limonia Osbeck) or ‘Cleopatra’ (Citrus reshni Hort...
The biology and behaviour of the psyllid Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Li... more The biology and behaviour of the psyllid Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Liviidae), the major insect vector of bacteria associated with huanglongbing, have been extensively studied with respect to host preferences, thermal requirements, and responses to visual and chemical volatile stimuli. However, development of the psyllid in relation to the ontogeny of immature citrus flush growth has not been clearly defined or illustrated. Such information is important for determining the timing and frequency of measures used to minimize populations of the psyllid in orchards and spread of HLB. Our objective was to study how flush ontogeny influences the biotic potential of the psyllid. We divided citrus flush growth into six stages within four developmental phases: emergence (V1), development (V2 and V3), maturation (V4 and V5), and dormancy (V6). Diaphorina citri oviposition and nymph development were assessed on all flush stages in a temperature controlled room, and in...
Huanglongbing (HLB) is a difficult-to-control and highly destructive citrus disease that, in Braz... more Huanglongbing (HLB) is a difficult-to-control and highly destructive citrus disease that, in Brazil, is associated mainly with the bacterium ‘Candidatus Liberibacter asiaticus’ transmitted by the psyllid Diaphorina citri. The aim of this study was to improve our understanding of the ‘Ca. L. asiaticus’ infection process by exposing excised, fully expanded, immature citrus leaves in 50-ml Falcon tubes to one, four, or eight adults from a ‘Ca. L. asiaticus’-exposed colony for 1-, 3-, 7-, or 15-day periods for access to inoculation (IAP). The leaves were incubated at 26°C for 1, 3, 7, 15, and 21 days (incubation period [IP]). Infection frequencies and ‘Ca. L. asiaticus’ titers were assessed by quantitative polymerase chain reaction (qPCR). ‘Ca. L. asiaticus’ infection was a function of leaf age, number of insects, IAP, and IP. In general, higher infection rates were observed on younger leaves inoculated with higher numbers of insects and after longer IAP and IP. The immature excised lea...
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