The results of this project over the last year have shed new light on pathogen transmission by ACP, some of which is radically different from the outcomes of other previously published studies. We have demonstrated in both laboratory and field studies that psyllids acquire the Las pathogen more readily as nymphs than as adults when feeding on infected plants. Furthermore, just because a psyllid tests positive for the Las pathogen does not mean that it can successfully inoculate a healthy plant. In pathogen transmission experiments, a very low rate of successful pathogen inoculation occurred from feeding by single psyllids. Much higher numbers of Las carrying psyllids were required to successfully inoculate at an average rate of 70%. We have also demonstrated that low rates of transovarial passage (mother to egg) of Las does indeed occur. Thus, Las+ nymphs collected from plants not showing symptoms of HLB may not necessarily indicate that the plant is already infected, but could be the result of oviposition by a Las+ female. In studies where psyllids reared on Las+ plants were continually transferred to Las- plants over time, the percentage of psyllids retaining the Las pathogen decreased to as low as 20% after 24 days on Las- plants. This is evidence that Las may not replicate within the psyllid as previously suggested; psyllids may need continual access to Las+ plants for acquisition to be capable of vectoring the pathogen over extended periods of time. In experiments designed to determine the effects of Las presence on psyllid biology, there was a significantly higher rate of oviposition by Las+ female psyllids compared to female psyllids not carrying the pathogen. However, longevity of adult Las+ female psyllids was significantly reduced when compared with Las- females, perhaps as a consequence of increased energy expended for oviposition by Las+ females. We have completed two years worth of surveys determining the incidence of Las+ psyllids in commercial groves. The objective of the repeated sampling of ACP populations on a monthly basis was to determine if there was sufficient variation in the incidence of Las+ psyllids to adjust pesticide applications to only those times of the year when the potential for pathogen transmission was increased. While we observed variation in the incidence of Las+ psyllids throughout the year, the months with highest incidence of Las+ psyllids was not consistent year to year. While not accounted for in this study, we suspect that flushing patterns of HLB infected trees (which vary year to year) are the primary factor responsible for the fluctuations in Las+ psyllids. The sampling of citrus groves under different HLB/ACP management regimes also showed that there was a much higher incidence of Las+ psyllid in groves were little or no HLB management programs were in place thus providing some indirect evidence that HLB management programs are likely to be beneficial in terms of reducing rate of pathogen spread. In studies using an Electrical Penetration Graph (EPG) monitor to examine psyllid feeding behavior on insecticide treated versus untreated plants, we have found that the feeding behaviors responsible for pathogen transmission (inoculation) can be significantly disrupted or prevented for certain insecticides. Soil-applications of imidacloprid greatly reduced psyllid feeding behaviors including phloem salivation indicating that imidacloprid can reduce the likelihood of successful inoculation of uninfected plants with Las. However, soil-applications of aldicarb did not have any effect on psyllid feeding behaviors in the first 18 hours of feeding suggesting aldicarb will provide little protection from inoculation by Las+ psyllid migrating from surrounding areas. Thus growers should not rely solely on aldicarb for psyllid control but instead should incorporate foliar applications with the aldicarb which provides extended systemic control of developing nymphs. Foliar applications of fenpropathrin prevented phloem feeding (inoculation) behaviors due to the rapid mortality of adult psyllids exposed to residues.
USDA test 1. A new block of young, HLB’free citrus (Val on Carr) was planted on May 1, 2008 in a grove with substantial levels of HLB. Three ACP control treatments (programs) are being compared in this planting: 1) a monoculture of citrus receiving monthly insecticide applications (annual chemical cost of $198/acre); 2) citrus interplanted with jasmine with a relaxed insecticide program for the citrus (annual chemical cost of $156/acre), jasmine not treated with insecticides; and 3) citrus interplanted with jasmine with a relaxed insecticide program for the citrus and regular applications of imidacloprid to jasmine (total annual chemical cost of $213/acre). For plots with jasmine, a jasmine plant was planted between each citrus tree along some rows in each plot. Program 2 is being studied because ACP may be attracted to jasmine thus reducing numbers of ACP that go to citrus, and population levels of ACP natural enemies may be enhanced by having jasmine in the vicinity of citrus. Program 3 is being studied because ACP may be attracted to jasmine and killed, reducing numbers of ACP in citrus. HLB-infected trees are removed. None of the trees tested HLB positive just before planting in May 2008. One year later, a mean of 0.9, 0.6, and 0.6% of the trees were HLB-infected under programs 1, 2 and 3, respectively. During August 2009, a mean of 9.8, 3.9, and 4.2% of the trees were infected under programs 1, 2 and 3, respectively. The rapid increase in incidence of HLB in the plots between May and August 2009 was attributed to increases in ACP infestations during June and July, particularly in one program 1 plot that was adjacent to older citrus. In February 2010, a mean of 13%, 12% and 13% of the trees were HLB-infected under programs 1, 2 and 3. USDA test 2. Three ACP control programs are being compared for preventing HLB in a block of young, HLB-free citrus (Val on Carr): 1) citrus under a relaxed insecticide program (annual chemical cost of $173/acre); 2) citrus receiving monthly insecticide applications (annual chemical cost of $198/acre); and 3) citrus treated once every three weeks with spray oil [PureSpray Foliar (470, C27) and PureSpray Green (435, C23)] from February through November (plus a December application of Danitol) (annual chemical cost of $76/acre). There are two replications of each treatment. The experiment officially started in August 2009. In January 2010, 0%, 0.3% and 0.3% of the trees tested HLB positive under programs 1, 2 and 3, respectively. These trees were removed. UF test. The ability of systemic insecticides to protect a new planting of citrus from ACP (and consequently HLB infection) is being evaluated. 112 Hamlins were planted in two rows at 15 ft spacing on 03 Mar 2009. Half of each row was considered a replicate and divided into two main plots, treated and untreated. Treated plots were split into two subplots on 13 March, one receiving a liquid formulation of imidacloprid (Nuprid 2f @ 32 oz/acre ‘ 0.5 lbs a.i./acre) and the other receiving a solid formulation of the same (Suscon 13 @ 10 lbs/acre ‘ 0.5 lbs a.i./acre). On 11-Sep 2009, no significant treatment effects (Chi square = 3.61, P = 0.182) were found with respect to numbers of HLB trees. At that time, 10 of the 56 control (untreated) trees tested positive for HLB, 10 of the 28 trees treated with the granular slow release imidacloprid were positive, and 6 of the 28 trees treated with the liquid formulation of imidacloprid were positive. The same trees were resampled on 15 Jan, but all tested negative. PCR was run twice more on the DNA from both the Sep and Jan samples and the results were confirmed. Thus, the different result from Jan could only be attributed to effects of cold weather on titer and/or lack of ACP to reinoculate the trees.
1. Localization of Liberibacter asiaticus (Las) in the hemolymph and other tissues and organs of the Asian citrus psyllid (ACP). [A]. Fluorescence In situ hybridization (FISH) studies are continuing using three oligonucleotide primers (including a newly designed one) based on the following sequences of Las: Primer 1 (HLBr): TCG AGC GCG TAT GCA ATA CG, Primer 2 (LL-REV): TCC CTA TAA AGT ACC CAA CAT CTA GGT AAA; Primer 3 (Cr-RDR-1): TAT ATT GTT CAC ATG AGG GAG CAT TTA ACC. Several FISH protocols have been tested on hemolymph smears and dissected organs of ACP and on leaf sections and extracts from HLB-diseased plants. Green fluorescence, indicating Las, was detected in the hemolymph, filter chamber and midgut of field-collected ACP, but not in healthy controls. It was also detected in leaf sections from HLB-diseased citrus and periwinkle plants but not in those from healthy plants. We are continuing to refine our FISH protocols to reduce the background fluorescence and to increase the permeabilization of insect tissues. [B]. Quantitative RT-PCR of dissected insect organs: We tested two different RT-PCR procedures for detection of Las in dissected salivary glands, alimentary canals and other parts of individual ACP adults. In five successive experiments, Las was detected in an average of 13-24% of the alimentary canals, 12-16% of the salivary glands, and in 16-25% of the rest of the body from psyllids collected from HLB-infected trees in Fort Pierce, FL. To our knowledge, this is the first direct demonstration of Las (using PCR) in the alimentary canal and salivary glands of ACP. Additionally, percentage of infection did not differ significantly between the three insect parts in males but was significantly lower in the salivary glands than in the alimentary canals of females suggesting that in the latter the salivary glands may act as transmission barriers. [C]. Transmission electron microscopy (TEM): We are continuing to use a combination of TEM and RT-PCR, comparing the ultrastructure of psyllid adults that have never been exposed to infected plants with those collected from HLB-infected trees and tested positive for Las with RT-PCR. This is providing a very useful library on the ultrastructure of the alimentary canal, salivary glands and other organs of ACP as well as its bacterial symbiotes, and will form the basis for future immunogold-labeling TEM studies on Las pathogen-vector interactions in ACP at the cellular, tissue and organ levels. 2. Clarification of various acquisition and transmission parameters between ACP and Las. [A] Among ACP adults field collected from HLB-infected citrus trees, averages of 37% females and 38% males have tested positive by PCR (not significantly different). Significantly higher percentages of field-collected adult ACP tested positive for Las during November 2009 (38.9%) than during late March 2010 (22.1%), and CT values associated with adults collected during November (mean 24.8) were significantly lower than for adults collected during late March 2010 (mean 30.5). [B] An average of 16% individual HLB-infected adult ACP (field-collected, mean CT of 26.4) transmitted HLB to young potted citrus trees. Taken together, the data in 2A and 2B, combined with those in1B above, support our hypothesis that transmission barriers to Las in ACP (including the alimentary canal and salivary glands) may play an important role in HLB transmission, since much higher percentages of ACP adults proved PCR-positive compared to those that were actually able to transmit the disease to citrus plants. Our studies are continuing to elucidate the roles of these and other psyllid organs in HLB transmission by its vector.
March, 2010 – 15 McPhail traps were established in locations where Mustang had been sprayed for Asian Citrus Psyllid control, 15 McPhail traps were set where Malathion had been sprayed for Asian Citrus Psyllid control and 15 McPhail traps were set in the control area. GPS locations were recorded and a helper was trained. The servicing schedule was set up and the results of each service date were recorded.
The first year of a 2-year research proposal (FDACS Contract Number 58-1920-9-925 #40) has been completed. Our research objectives were: (1) Devise and perform alternative methods (microinjection and membrane uptake) to complete Koch’s postulates using a pure culture of bacteria isolated and cultured in our laboratory and healthy psyllids as a transmission tool; and, (2) following successful inoculation or loading of the psyllids, we will complete Koch’s postulates. The cultures of two strains of ‘Candidatus Liberibacter asiaticus’ (Las) from China (China1) and Taiwan (B239) were produced in our and a collaborator’s laboratory and determined to be Las by PCR and sequencing. We prepared stretched parafilm membrane sachets containing sucrose solutions in 1XTE buffer in which the cultured bacteria were suspended. Sucrose solutions from 5-20% were tested, however, most experiments were done with 10% sucrose. The titer of the bacteria in sucrose was roughly assessed using real time PCR (Ct values ranged from 20 to 38). Membrane sachets were suspended over 15 ml or 50 ml conical tubes within which 15-20 healthy adult psyllids and 5th instar nymphs were allowed to probe the membranes. After 48 – 72 hr the feeding psyllids were transferred onto sweet orange seedlings and the remaining culture fluid in the sachets was assayed by real time PCR. Psyllids and nymphs were allowed to feed on the sweet orange seedlings for 14 days after which they were removed, and assayed for Las by real time PCR. From more than 30 membranes attempted, nine test membranes, each containing 250 ‘ls of culture in 0.25 ‘ 0.5 ml sucrose solution, were successful in uptake of bacterial culture by the psyllids. The orange seedlings were observed for symptom development and assayed three months post-inoculation by real time PCR. Although no symptoms were observed at 3 months, Madam Vinous sweet orange seedlings inoculated with infectious culture had Ct values ranging from 19.5 to 43.8. Typical HLB leaf symptoms were observed by six months. Test plants are being monitored for additional symptoms and plans are to re-isolate the bacteria from positive seedlings, assay for Las titer, sequence the bacteria to validate the strain originally inoculated, and grow the re-isolated bacteria in culture media. This would complete Koch’s Postulates for Las as the causal agent of Huanglongbing. A second approach used direct microinjection of the culture fluid into the hemolymph of adult and 5th instar nymphs of Diaphorina citri. Psyllids were immobilized with a low velocity stream of C02 and approximately 0.01’l of bacterial culture (Ct value of the bacterial culture suspensions ranged from 32.9 to 38.3) was injected into the abdomen of each adult psyllid. Microinjection of 5th instar nymphs was attempted but mortality was very high. Psyllids were allowed to recover and then placed on young sweet orange seedlings for recovery feeding for 14 days. Surviving psyllids were captured, assayed by real time PCR for the presence of Las, and plants were observed for 3 months for symptom development. While this does not serve as additional confirmation of Koch’s postulates for Las, it does provide important insights into the interaction of Las with the vector. Continued refinement of the microinjection technique is underway. We have developed a usable membrane uptake mechanism of transferring cultured bacteria of strains of Las. Our primary objective for year 1 was to complete Koch’s postulates on a pure culture of Las and we are on the threshold of completing that objective. This work is not only demonstrating the causal relationship of Las to HLB, it is providing important insights about the nature of the pathogen-vector interaction.
the project goals were to establish issue culture cell lines derived from the citrus greening vectoring psyllid, D. citri and we have over a dozen actively growing insect cell lines. the two major cell types in the lines are heterogenous adherant cells and more homogenous suspension cells. Most lines display an asymmetric growth pattern in which cell populations appear to grow slowly, then rapidly, then slowly again. We have also developed antibiotic free cell lines for use in culturing the Liberibacter organism. Project goals that have been met include determining the optimal conditions for passage of the cell lines, measuring the stability of the cell lines and storage/preservation of the cell lines (in progress). A new project for funded for the 2010 fiscal year (Application of Asian Citrus psyllid, Diaphorina citri, tissue culture cell lines) will seek to disseminate the cell line for various purposes. current groups that have requested and recieved cell lines/materials include Dr. Davis (Lake Alfred Citrus Research Center) and Dr. J. Brown (University of Arizona, Dept. of Plant Sciences). It is anticipated that the major project goals will be complete within the 2010 funding period.
Objective 1. Develop baseline susceptibility information for commonly used insecticides for ACP control. The baseline susceptibility data (LD50) of laboratory colony of Asian citrus psyllid (ACP) have been developed for 14 insecticides that are labeled for use in Florida citrus. Also, the baseline sensitivity data of acetylcholinesterase (AChE) to inhibition by selected OP and carbamate insecticides have been generated using biochemical assays. All goals for Objective 1 have been completed. Objective 2. Compare the insecticide susceptibility levels of field ACP populations in different parts of the state to monitor resistance development. The susceptibility of field collected ACP from 5 FL counties has been determined for 14 insecticides from Objective 1 using topical bioassays. all five ACP populations collected from five Counties showed decreased susceptibility to all the tested compounds (especially imidacloprid) when compared with laboratory strain. Also, the baseline sensitivity data of AChE to inhibition by OP and carbamate insecticides was determined for these insecticides.AChE of all five ACP populations collected from five Counties showed decreased sensitivity to inhibition to all the tested insecticides compared with laboratory strain with few exceptions. These data corroborate with insecticide susceptibility data obtained from bioassays and will help monitor changes in resistance levels in field populations both at physiological level for all insecticides and at the target site level for OP and carbamate insecticides. Objective 3. Select ACP for imidacloprid (other insecticide) resistance and determine the mechanisms of resistance. For this objective, two ACP field colonies have been established in a greenhouse and are being subjected to imidacloprid or chlorpyrifos selection pressure in every generation for developing resistant colonies. Thus far, we have selected for up to 50 fold resistance to imidacloprid and 25 fold resistance to chlorpyrifos in the laboratory. Further selection of future generations and determining the mechanisms of resistance by toxicological and biochemical studies are in progress. These colonies will be used for further studies on determining the rate of resistance and cross-resistance development potential in ACP and mechanisms of resistance. Objective 4. Determine the cross-resistance potential in imidacloprid (other insecticide)-selected ACP strain. This objective will be started in the 2nd year once the highly resistant ACP colonies for imidacloprid and chlorpyrifos insecticides are selected in the greenhouse, which is currently in progress as described in objective 3. In the 2nd year of this project, we will continue screening more field ACP populations collected from 4-5 different locations in Florida for determining the changes in insecticide resistance levels both at physiological and target site which will be helpful in monitoring insecticide resistance levels. We will also continue selection of two field colonies for imidacloprid and chlorpyrifos resistance in the greenhouse. The information from the objectives 2, 3 and 4 should help monitor the onset and levels of resistance in ACP to different insecticides. Also, it is helpful in determining the rate of resistance development and cross resistance potential to various insecticides. This will enable us to design, develop and implement an effective insecticide resistance management program for ACP.
During the past year, 4 main objectives are being pursued with the goal of better understanding the psyllid/host plant interaction to improve psyllid management using alternative control tactics to broad-spectrum insecticides. 1) Psyllid fitness on various host plants – investigations were initiated to examine the fitness of the Asian citrus psyllid when reared on 8 different citrus varieties. Experiments conducted in both the greenhouse and growth chambers examined psyllid longevity, number of eggs produced, immature survival and development rate for each of these eight varieties examined. During the first set of evaluations, it was found that psyllids were not able to complete development on one of the root stock varieties evaluated, Cleopatra mandarin. Because of the potential significance of this finding, this objective was temporarily set aside to focus more in depth and confirm that Cleopatra mandarin was indeed not a suitable host for the psyllid (see objective 2 below). Work has now resumed on this first objective. Results to date suggest that not all citrus varieties are equally suitable for psyllid development. An electrical penetration graph (EPG) monitor is being used to record psyllid feeding behavior on these 8 host plants to determine if there is a difference in psyllid feeding behavior that can be correlated with difference in psyllid fitness on certain host plants. Preference experiments using an olfactometer are being used to determine if plant volatiles may also be responsible in part for psyllid preference of certain varieties under field conditions. 2) Survival of psyllid on Cleopatra mandarin – based on the preliminary results from objective 1, we conducted a series of replicated experiments to confirm our findings that Cleopatra mandarin is an unsuitable host plant for ACP. Survival of ACP on Cleopatra mandarin was less than 5% compared to greater than 60% survival on sour orange. Female ACP also laid significantly fewer eggs on Cleopatra compared to other host plants. The factors affecting the “unsuitability” of Cleopatra mandarin as a host for psyllid are being further investigated. Working with the CREC plant improvement program (Fred Gmitter), we are conducting evaluations of ACP survival on numerous Cleopatra hybrids in the CREC field collection to determine if any differences in suitability for ACP exiists between these hybrids. Any differences in suitability for ACP will be further examined to determine the responsible factors. Such factors could be exploited in breeding programs for psyllid resistant plants. 3) Effects of host plant nutrition on ACP fitness – the effects of fertilization practices on psyllid population dynamics was examined in greenhouse studies. High levels of nitrogen fertilization resulted in more rapid psyllid development, whereas high levels of potassium significantly slowed down psyllid development. There was also a similar effect for psyllid body mass. Ongoing studies are examining psyllid fecundity and longevity under the varying fertilization regimes. Field trials are planned for 2010 to confirm these findings under typical commercial growing conditions. The goal of this work is to determine if adjusting nutrient applications (within acceptable limits for commercial groves) can be used to help minimize psyllid populations. 4) Effects of boron application on psyllid populations – Based on initial findings from Objective 3 (above), investigations were undertaken to determine the effects of Boron application on ACP populations, particularly adult ACP mortality. Evaluations of differing rates of Boron in both greenhouse and growth chamber experiments demonstrated that Boron alone caused significant mortality of ACP. Filed experiments are underway currently to investigate the use of Boron as an alternative to traditional pesticides for ACP control.
The first year of a 2-year research proposal (FDACS Contract Number 58-1920-9-925 40) has been completed. Our research objectives were: (1) Devise and perform alternative methods (microinjection and membrane uptake) to complete Koch’s postulates using a pure culture of bacteria isolated and cultured in our laboratory and healthy psyllids as a transmission tool; and, (2) following successful inoculation or loading of the psyllids, we will complete Koch’s postulates. The cultures of two strains of ‘Candidatus Liberibacter asiaticus’ (Las) from China (China1) and Taiwan (B239) were produced in our and a collaborator’s laboratory and determined to be Las by PCR and sequencing. We prepared stretched parafilm membrane sachets containing sucrose solutions in 1XTE buffer in which the cultured bacteria were suspended. Sucrose solutions from 5-20% were tested, however, most experiments were done with 10% sucrose. The titer of the bacteria in sucrose was roughly assessed using real time PCR (Ct values ranged from 20 to 38). Membrane sachets were suspended over 15 ml or 50 ml conical tubes within which 15-20 healthy adult psyllids and 5th instar nymphs were allowed to probe the membranes. After 48 – 72 hr the feeding psyllids were transferred onto sweet orange seedlings and the remaining culture fluid in the sachets was assayed by real time PCR. Psyllids and nymphs were allowed to feed on the sweet orange seedlings for 14 days after which they were removed, and assayed for Las by real time PCR. From more than 30 membranes attempted, nine test membranes, each containing 250 ‘ls of culture in 0.25 ‘ 0.5 ml sucrose solution, were successful in uptake of bacterial culture by the psyllids. The orange seedlings were observed for symptom development and assayed three months post-inoculation by real time PCR. Although no symptoms were observed at 3 months, Madam Vinous sweet orange seedlings inoculated with infectious culture had Ct values ranging from 19.5 to 43.8. Typical HLB leaf symptoms were observed by six months. Test plants are being monitored for additional symptoms and plans are to re-isolate the bacteria from positive seedlings, assay for Las titer, sequence the bacteria to validate the strain originally inoculated, and grow the re-isolated bacteria in culture media. This would complete Koch’s Postulates for Las as the causal agent of Huanglongbing. A second approach used direct microinjection of the culture fluid into the hemolymph of adult and 5th instar nymphs of Diaphorina citri. Psyllids were immobilized with a low velocity stream of C02 and approximately 0.01’l of bacterial culture (Ct value of the bacterial culture suspensions ranged from 32.9 to 38.3) was injected into the abdomen of each adult psyllid. Microinjection of 5th instar nymphs was attempted but mortality was very high. Psyllids were allowed to recover and then placed on young sweet orange seedlings for recovery feeding for 14 days. Surviving psyllids were captured, assayed by real time PCR for the presence of Las, and plants were observed for 3 months for symptom development. While this does not serve as additional confirmation of Koch’s postulates for Las, it does provide important insights into the interaction of Las with the vector. Continued refinement of the microinjection technique is underway. We have developed a usable membrane uptake mechanism of transferring cultured bacteria of strains of Las. Our primary objective for year 1 was to complete Koch’s postulates on a pure culture of Las and we are on the threshold of completing that objective. This work is not only demonstrating the causal relationship of Las to HLB, it is providing important insights about the nature of the pathogen-vector interaction.
Knowledge about factors that determine transmission efficiency of Candidatus Liberibacter asiaticus (CLas) by the Asian citrus psyllid (ACP) is important for understanding epidemiology and improving current HLB management strategies. The goal of this project is to investigate how transmission of CLas is influenced by: 1) vector developmental stage, 2) pathogen titer in source plants, 3) plant phenology, 4) inoculation access periods and 5) insecticide treatment. In study 1, we are comparing acquisition efficiency of CLas by different ACP nymphal stadia (1st, 2nd, 3rd, 4th and 5th instars) and adults (1 wk old). After an acquisition access period (AAP) of 48 h on young shoots of infected (source) plants, the insects of each age treatment were first transferred to healthy citrus seedlings for a latent period of 3 wks and then submitted to real-time PCR for detection and quantification of CLas in their bodies. Results of three replications of this experiment using independent CLas-infected plants as sources indicated that nymphs of all development stadia can efficiently acquire the pathogen; adults can also acquire it, but with a lower efficiency. We are still analyzing the data to determine if bacterial multiplication in the vector and transmission efficiency were also influenced by vector age during acquisition. In study 2, we investigated the effect of leaf age (young and asymptomatic x symptomatic mature leaves) in source plants of CLas on acquisition efficiency and vector probing behavior. ACP adults acquired CLas with higher efficiency on fully-expanded young (asymptomatic) leaves (20.4% infective individuals) than on mature leaves (0%). We repeated this experiment with inclusion of a third leaf age treatment (not fully-expanded young leaves), and observed that acquisition efficiency on mature leaves was lower (6.0% infective individuals) than on fully-expanded (25.1%) or not fully-expanded (9.1%) young leaves. By comparing the probing behavior of ACP females on mature x young leaves of infected citrus by the Electrical Penetration Graph (EPG) technique, we noted that phloem ingestion was longer and more frequent on young leaves. On mature leaves, the insects spent most of the time with the stylets in the parenchyma (pathway phase) or non-probing. The higher frequency and longer duration of phloem ingestion appears to explain at least in part the higher acquisition efficiency of CLas when ACP is confined on young asymptomatic leaves. In study 3, we evaluated acquisition efficiency of CLas by ACP adults on infected citrus plants with variable pathogen population. Fifty citrus plants were graft-inoculated with Clas at the same time, and groups of five independent plants were used as source for acquisition assays at 60, 130, 145, 165, 180, 200, 235, and 280 days after inoculations (DAI). Clas was acquired by ACP from both symptomatic and asymptomatic citrus, and a positive linear relationship was observed between pathogen population within the source plant and the percentage of infective psyllid samples. Similarly, the bacteria population within the plant increased over time and became stable at 235 and 280 DAI when HLB symptoms became more evident. In 2010 we will be analyzing the transmission data of this experiment in relation to CLas population in the source plants. The results obtained so far indicate that vector control should be targeted to young shoots of citrus trees, where developing ACP nymphs can efficiently acquire the pathogen and subsequently spread the pathogen as emerging adults. Because ACP can acquire CLas on symptomless plants, vector control should be an important component in HLB management, in addition to eradication of symptomatic trees. Experiments related to studies 4 and 5 will be set up in 2010 and will help to clarify if insecticide treatment can prevent transmission.
Knowledge about factors that determine transmission efficiency of Candidatus Liberibacter asiaticus (CLas) by the Asian citrus psyllid (ACP) is important for understanding epidemiology and improving current HLB management strategies. The goal of this project is to investigate how transmission of CLas is influenced by: 1) vector developmental stage, 2) pathogen titer in source plants, 3) plant phenology, 4) inoculation access periods and 5) insecticide treatment. In study 1, we are comparing acquisition efficiency of CLas by different ACP nymphal stadia (1st, 2nd, 3rd, 4th and 5th instars) and adults (1 wk old). After an acquisition access period (AAP) of 48 h on young shoots of infected (source) plants, the insects of each age treatment were first transferred to healthy citrus seedlings for a latent period of 3 wks and then submitted to real-time PCR for detection and quantification of CLas in their bodies. Results of three replications of this experiment using independent CLas-infected plants as sources indicated that nymphs of all development stadia can efficiently acquire the pathogen; adults can also acquire it, but with a lower efficiency. We are still analyzing the data to determine if bacterial multiplication in the vector and transmission efficiency were also influenced by vector age during acquisition. In study 2, we investigated the effect of leaf age (young and asymptomatic x symptomatic mature leaves) in source plants of CLas on acquisition efficiency and vector probing behavior. ACP adults acquired CLas with higher efficiency on fully-expanded young (asymptomatic) leaves (20.4% infective individuals) than on mature leaves (0%). We repeated this experiment with inclusion of a third leaf age treatment (not fully-expanded young leaves), and observed that acquisition efficiency on mature leaves was lower (6.0% infective individuals) than on fully-expanded (25.1%) or not fully-expanded (9.1%) young leaves. By comparing the probing behavior of ACP females on mature x young leaves of infected citrus by the Electrical Penetration Graph (EPG) technique, we noted that phloem ingestion was longer and more frequent on young leaves. On mature leaves, the insects spent most of the time with the stylets in the parenchyma (pathway phase) or non-probing. The higher frequency and longer duration of phloem ingestion appears to explain at least in part the higher acquisition efficiency of CLas when ACP is confined on young asymptomatic leaves. In study 3, we evaluated acquisition efficiency of CLas by ACP adults on infected citrus plants with variable pathogen population. Fifty citrus plants were graft-inoculated with Clas at the same time, and groups of five independent plants were used as source for acquisition assays at 60, 130, 145, 165, 180, 200, 235, and 280 days after inoculations (DAI). Clas was acquired by ACP from both symptomatic and asymptomatic citrus, and a positive linear relationship was observed between pathogen population within the source plant and the percentage of infective psyllid samples. Similarly, the bacteria population within the plant increased over time and became stable at 235 and 280 DAI when HLB symptoms became more evident. In 2010 we will be analyzing the transmission data of this experiment in relation to CLas population in the source plants. The results obtained so far indicate that vector control should be targeted to young shoots of citrus trees, where developing ACP nymphs can efficiently acquire the pathogen and subsequently spread the pathogen as emerging adults. Because ACP can acquire CLas on symptomless plants, vector control should be an important component in HLB management, in addition to eradication of symptomatic trees. Experiments related to studies 4 and 5 will be set up in 2010 and will help to clarify if insecticide treatment can prevent transmission.
This research project is directed towards controlling psyllids using biologically-based control strategies that employ the use of RNAi technology against key biological control pathways, peptide hormones and protein inhibitors that, if expressed in transgenic citrus, would enhance plant resistance to psyllids feeding. DIET: Both protein-based and RNAi strategies were tested by feeding psyllids artificial diets. Both protein-based and RNAi strategies were tested using artificial diets on which pysllids were fed. Psyllids in nature, feed on phloem content of citrus and its relatives. Thus, psyllids do not tolerate many alterations to diet composition that is drastically different than the phloem content. Addition of high concentrations of proteins or single stranded and double stranded RNA (ssRNA and dsRNA) reduces psyllids survival. Therefore, we determined the acceptable concentrations of each molecule and cofactor that was added including a suitable buffer to allow continuous feeding and maintenance of a physiological pH that was not detrimental to psyllids. We also identified an antimicrobial agent that was added to the diet and prevented fungal growth but did not harm the psyllids or their associated and obligate symbiotic microflora. Prior to the identification of the antifungal agent, fungal contamination of the diet caused unacceptable high level of psyllids mortality because the fungus is carried by the psyllids and can enter the diet through the psyllids feeding process. Control experiments showed that addition of dsRNA molecules, that did not target psyllids transcripts, at up to ~16 ng/uL improved psyllids performance, but above this concentration, the non-specific dsRNA would reduce psyllids survival. Therefore, comparisons of efficacy of specific psyllids gene targeting dsRNA were done with dsRNA that did not target psyllids genes. PROTEIN: In separate experiments, mosquito peptide hormone, TMOF, and Diaprepes abbreviates (citrus root weevil) cysteine protease inhibitor (CPI) were added to an artificial diet that was fed to psyllids. TMOF and CPI were tested at concentrations of 10 ‘g/’L and 3 ‘g/’L, respectively. After 10 days of feeding, all the psyllids that were fed diets containing either TMOF or CPI died, whereas only 40% mortality was observed in psyllids that fed on the control diets. TMOF caused 15% mortality after 4 days of feeding as compared with less than 5% mortality in the control group. Psyllids that were fed CPI did not show significantly higher mortality than the controls until after 7 days of feeding, because CPI was tested at less than . the concentration that was used for the TMOF because of limited availability. During the second year of the grant’s period more CPI will be synthesized and purified to study dose effect and optimal concentration, as well as, potential for synergistic effects when both proteins are present within the same diet. RNAi: Ten psyllids genes representing three gene families of cathepsins (five genes), vacuolar ATPases (four genes), and tubulin (one gene) were targeted and their dsRNA (16 ng/’L) fed to psyllids using artificial diets. The earliest effects were observed at ~4 days after feeding and feeding continued until day 10. At day 5 after feeding, two cysteine protease dsRNAs targeting cathepsin L and cathepsin B genes had the highest mortality 2-fold higher than controls. At day 10, three vacuolar ATPases and three cathepsins (B, L and F) showed significantly higher mortality than the controls. The current results reflect the use of reduced concentration of dsRNA because some transcripts that are active at higher concentrations in the diet do not show significant activity when reduced to 16 ng/’L. Dose curves and combinatorial experiments are in progress to determine if combinations of these active dsRNA molecules can provide synergistic effects. QRT-PCR experiments are in progress to determine the influence of dsRNA on cognate psyllid transcript levels. Our results show that specific dsRNA and proteins cause psyllids mortlity. Using these observations it is possible now to develop transgenic citrus trees that are resistant to psyllids and express these moieties in their phloem.
Our longterm goal is to identify and then use specific psyllid RNA sequences to induce RNA interference (RNAi) activity resulting in a negative phenotype (even death) in recipient psyllids. We envision that successful application of RNAi towards the Asian citrus psyllid (ACP, D. citri) will negatively impact their ability to colonize and/or reproduce on selected plants and thereby interfere with their ability to transmit C. Liberibacter spp., the causal agents of HLB. This strategy could then be an important component of HLB and/or ACP control. To make rapid progress we are using the tomato psyllid (Bactericerca cockerelli), which colonizes herbaceous plants (tomatoes, tobaccos) and transmits another Liberibacter spp. (C. L. psyllaurous; C. L. solanacearum) to several plant species. This psyllid:Liberibacter systems is very similar to the ACP:L. asiaticus complex associated with HLB. During this year we have established high quality colonies of B. cockerelli and identified three solanaceous plant species (tomato, Nicotiana benthamiana, N. tabacum) for use in our experiments. These plants are good hosts for the virus we will use to deliver interfering RNAs to plants, Tobacco mosaic virus (TMV), and for B. cockerelli and C. L. solanacearum. Furthermore, C. solanacearum induces specific and characteristic symptoms in these plants. We are using all three plant species for our ongoing efforts and by using recombinant TMV we have shown that we can induce production of specific siRNAs corresponding to the recombinant sequence in these plant species. We can also detect the presence of the recombinant RNAs in psyllids after feeding on these plants. We have utilized two complementary approaches to generate psyllid interfering RNAs for our work. The first has been by mining existing GenBank data for potentially useful D. citri sequences, and the second has been by constructing a new, normalized cDNA library for B. cockerelli mRNAs directly in the TMV-based virus delivery system. Both approaches have yielded a number of potential RNAi targets. We analyzed the D. citri EST database and identified 179 RNAs which are predicted to be expressed in D. citri midguts. Based on these analyses we cloned 80 midgut sequences (partial sequences varying in size between 500-1000bp) from D. citri. By BLAST comparative sequence analyses we next cloned 36 D. citri homologs from B. cockerelli. Sequence comparisons showed these to be highly homologous (80 ‘ 90%) to corresponding D.citri sequences. RT-PCR analysis was used to assess RNA presence in psyllids and confirmed our predictions showing that 25 of these were abundant in B. cockerelli gut RNA extracts. We believe that gut RNAs are potentially useful targets, and their knockdown effects can be specifically monitored in gut tissues. Sequence-based analyses identified some, including ion transporter and ATPase sequences, which are ideal candidates for RNA interference. We have developed three approaches for delivering candidate interfering RNAs to psyllids including TMV-based expression, micro-injection of dsRNAs into the psyllid hemocoel, and artificial membrane feeding in vitro. We are presently using all three to determine their abilities to induce RNAi effects in B. cockerelli. RNAi effects are monitored by RNA knockdown (northern hybridization and qRT-PCR) and by phenotype in recipient psyllids. We also generated and are presently analyzing a normalized B. cockerelli cDNA library. The library sequences were cloned directly into our TMV-based plasmid for expression in plants. Nucleotide sequence analysis has allowed us to confirm the quality of the library. Homologs corresponding to NADH dehyrogenase subunit, elongation factor Tu, glutathione peroxidase, peritrophin, alpha-keto reductase etc. have been identified within the library and these are being tested.
The purpose of this project has been to develop an effective repellent for the Asian citrus psyllid (ACP). We recently identified DMDS as a metabolite produced in large quantities by wounded guava leaves, which is a repellent of ACP. Volatiles from guava leaves significantly inhibited attraction of ACP to normally attractive host-plant (citrus) volatiles. A similar level of inhibition was recorded when synthetic DMDS was co-released with volatiles from citrus leaves. In addition, the volatile mixture emanating from a combination of intact citrus and intact guava leaves induced a knock-down effect on adult ACP. Compounds similar to DMDS including dipropyl disulfide, ethyl-1-propyl disulfide, and diethyl disulfide did not affect the behavioral respomse of ACP to attractive citrus host plant volatiles in laboratory olfactometer assays. Our field experiments confirmed the results of our laboratory olfactometer assays. Deployment of synthetic DMDS from polyethylene vials (Alpha Scents) and SPLAT wax dispensers (ISCA technologies) reduced populations of ACP in an unsprayed citrus orchard for up to 3 weeks following deployment. Given that population densities were equivalent among plots prior to the deployment of DMDS treatments, we hypothesize that DMDS repelled adult ACP from treated plots. However, we cannot exclude the possibility that a proportion of the ACP populations in DMDS-treated plots may have been reduced due to direct intoxication. By the fourth week, there was no remaining DMDS in the dispensers, which likely explains why populations were once again equivalent in treated and control plots. Given the volatility of DMDS, one of the main obstacles to the development of a practical DMDS formulation for ACP management will be development of a slow-release device that maintains the chemical above a behaviorally active threshold for long periods. The polyethylene vials and SPLAT dispensers evaluated in these initial proof-of-concept investigations will likely not be economically practical for releasing DMDS for control of ACP in their current form. Both the number of dispensers required per acre (~200) as well as the amount of active ingredient required per three weeks (~3 kg) would likely be economically prohibitive for a hand applied dispenser. Furthermore, the dispensers evaluated in this study resulted in a ~2/3 decrease in field populations of ACP, which would be insufficient for effective control of this pest as a stand alone treatment. Another immediate logistical hurdle for developing DMDS into a practical psyllid management tool is the chemical’s strong and unpleasant odor. This may render field application difficult and potentially limit the use of DMDS depending on fruit harvesting schedules or proximity to urban areas. Ideally, a slow-release dispenser needs be developed that could achieve 150-200 d of behaviorally efficacious release. ACP populations are much more prevalent on crop borders and thus targeted applications of DMDS to those areas may be immediately useful with a dispenser that is not yet optimized. Our current efforts are focussing on further optimizing these dispensers to increase the duration of efficacy. ISCA has recently developed four new formulations of SPLAT dispensers that we will be evaluating in 2010. Most recently, we have identified new compounds that are ACP repellents. Trisulfides (dimethyl trisulfide) inhibited the response of ACP to citrus volatiles more than disulfides (dimethyl disulfide, allyl methyl disulfide, allyl disulfide). Monosulfides did not affect the behavior of ACP adults. A blend of dimethyl trisulfide (DMTS) and dimethyl disulfide (DMDS) in 1:1 ratio showed an additive effect on inhibition of the response of ACP to citrus volatiles and was more effective than DMDS alone in the lab. We met the objectives proposed for year 1 of this project. We hope to continue development of a psyllid repellent in year two by investigating the new formulations developed for sulfur compounds as well as blend of the newly identified chemicals. ISCA technologies (Riverside, CA) is working with CRDF/FCPRAC to register a DMDS-based product for ACP control in Florida.
The main objective of this series of investigations has been to develop an effective attractant for Tamarixia radiata, the main parasitic wasp attacking Asian citrus psyllid (ACP) in Florida. Development of an effective attractant for this insect will allow for accurate monitoring of this beneficial insect and it will allow us to recruit and establish high populations of this beneficial insect to improve biological control of ACP. This first objective of this research was to describe the antennal capabilities of this insect. This was promptly accomplished and was published in a peer-reviewed scientific journal (Onagbola, E.O., D.R. Boina, S.L. Herman, and L.L. Stelinski. 2009. Annals of the Entomological Society of America. 102: 523-531). Next, we moved onto conducting an in depth analysis of the chemicals produced by both sexes of this parasitoid. We discovered that both male and female ACP parasitoids release several volatile compounds. In laboratory behavioral tests, we found that male parasitoids were attracted to .-Butyrolactone to the same degree as to female parasitoids, indicating that this is likely the sex-attractant pheromone females produce and release to attract males. Subsequently, we developed a polyethylene-tube dispenser for releasing this chemical in collaboration with our industry partner, Alpha Scents. Field trapping tests indicated that in the early summer traps baited with .-Butyrolactone caught more Tamarixia radiata than unbaited traps, but this trend did not hold up in the late summer months. In addition to investigating the chemicals that Tamarixia radiata produces, we conducted an initial investigation with another chemical that is known to attract natural enemies of insect pests and reduce their populations. Methyl salicylate (MeSA) is a known herbivore-induced plant volatile which has been shown to attract natural enemies (e.g. parasitoids, coccinellid beetles, predatory mites) and repel plant pests (e.g. mites, herbivorous beetles, aphids). The treatments compared were plots treated with MeSA versus untreated control plots; all treatments were replicated five times. Two dispensers were deployed per tree in April of 2009 and populations of psyllids and their natural enemies were monitored through September. Our preliminary data indicate that treatment of citrus plots with MeSA increased populations of natural enemies such as lady beetle and fly predators of ACP and well as the ACP parasitoid, Tamarixia radiata. In addition, populations of ACP were lower in MeSA-treated plots compared with untreated controls. Finally, we have described the host searching and mating behavior of this ACP natural enemy. We examined the behavior of T. radiata to in response to volatiles from citrus and ACP or ACP feeding on citrus. We also examined the behavioral response of male and female T. radiata to conspecifics of the opposite sex to determine whether olfactory signals mediate mate location. T. radiata adults exhibited a sexually dimorphic response to volatiles emanating from ACP and citrus. Female T. radiata were attracted to the odors emanating from ACP nymphs. However, female wasps did not respond to odors emanating from ACP adults, ACP honey dew secretions or intact citrus. Odors emanating from ACP-damaged citrus were not attractive to T. radiata but stimulated attraction of wasps to ACP feeding on damaged plants. T. radiata females were not attracted to ACP immatures when they were presented as visual cues. Odors emanating from ACP immatures or adults, their honey dew secretions or citrus plants were not attractive to male T. radiata. Male T. radiata were attracted to the odor of female conspecifics compared with blank controls in the absence of associated citrus host plant volatiles. Collectively, our results provide behavioral evidence that : 1) female T. radiata uses volatiles from ACP nymphs to locate its host and : 2) female T. radiata release a volatile pheromone that attracts males. In summary, we not completed all of the objectives of this research and actually exceeded our goals. Based on this proposal, we will be continuing the development of an MeSA dispenser to recruit T. radiata and improve biological control of ACP in 2010.
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