The Citrus Greening Bibliographical Database [ http://swfrec.ifas.ufl.edu/hlb/database/ ] is managed by the Entomology group at the University of Florida-IFAS in Immokalee, in collaboration with the Center for Library Automation at the University of Florida in Gainesville. The database was constructed from the ground up, and after less than a year contains 1,770 references, 880 of which are linked to original sources. With the help of our users, we are continually searching for new information, cross-referencing existing data for accuracy, and updating the database regularly. Ninety percent of the entries are in English, with the remaining 10% in Spanish, Portuguese, Afrikaans, Japanese, Chinese, French, German, Vietnamese, Dutch, Farsi, Arabic, Czech and Hebrew. The database includes references from refereed and non-refereed research and extension publications, presentations, websites, proceedings, grant reports, periodicals, dissertations, book chapters and complete books. Entries are the product research worldwide on the various aspects of Huanglongbing (HLB): the associated bacteria (Candidatus Liberibacter spp.), vectors [Diaphorina citri Kuwayama and Trioza erytrea (Del Guercio)], plant and vector effects of the disease, and management tactics. The database was designed as a tool for growers, researchers, and students to access HLB related information. It is also readily accessible to the general public through a user-friendly interface. This project has been promoted in presentations at several national and international meetings in the U.S and Mexico to invite researchers to use the database and to contribute to it by submitting their publications. We have received a positive response from many researchers attending these meetings, and several have sent us their presentations and publications to be included in the database. During 2009, the database received close to 5,000 visits. The majority of those connecting to the Internet Protocol (IP) addresses are from Florida and California. However, we have also received visits from IPs located in several other countries such as China, Mexico, Colombia, and Brazil. These results indicate that the database is becoming an integral source of information for a growing number of researchers, growers, and the citrus community. Pending continued funding, unfortunately not requested in the original proposal, our goal for the second year of the project is to continue providing this service by including the most current information as it becomes available, increasing the number of linked documents, and creating an interactive forum in which researchers and growers can exchange HLB related information. Presentations related to the citrus greening database database: – Arevalo, H. A., A. B. Fraulo, and P. A. Stansly. 2009. Update on the Citrus Greening Bibliographical Database. (Poster). HLB/ ZC meeting, McAllen Texas – Arevalo. H. A. and P. A. Stansly. 2009. Monitoreando el Psillydis Asiatico de los Citricos en el Campo y el Internet (Monitoring the Asian Citrus Psyllid in the field and the Internet). XIII Simposio Internacional de Citricultura. CD Victoria, Tamaulipas, Mexico. – Arevalo, H. A., A. B. Fraulo, and P. A. Stansly. 2009. Update on the Citrus Greening Bibliographical Database. (Poster). Florida Entomological Society Annual Meeting. Ft. Myers, FL July 2009 – Arevalo, H. A., G. Snyder, and P. A. Stansly. 2008. The citrus greening Bibliographical Database, a New Tool for Researchers, Students and Growers. (Poster). International Research Conference on Huanglongbing. Orlando, FL December 1-5, 2008
The Citrus Greening Bibliographical Database [ http://swfrec.ifas.ufl.edu/hlb/database/ ] is managed by the Entomology group at the University of Florida-IFAS in Immokalee, in collaboration with the Center for Library Automation at the University of Florida in Gainesville. The database was constructed from the ground up, and after less than a year contains 1,770 references, 880 of which are linked to original sources. With the help of our users, we are continually searching for new information, cross-referencing existing data for accuracy, and updating the database regularly. Ninety percent of the entries are in English, with the remaining 10% in Spanish, Portuguese, Afrikaans, Japanese, Chinese, French, German, Vietnamese, Dutch, Farsi, Arabic, Czech and Hebrew. The database includes references from refereed and non-refereed research and extension publications, presentations, websites, proceedings, grant reports, periodicals, dissertations, book chapters and complete books. Entries are the product research worldwide on the various aspects of Huanglongbing (HLB): the associated bacteria (Candidatus Liberibacter spp.), vectors [Diaphorina citri Kuwayama and Trioza erytrea (Del Guercio)], plant and vector effects of the disease, and management tactics. The database was designed as a tool for growers, researchers, and students to access HLB related information. It is also readily accessible to the general public through a user-friendly interface. This project has been promoted in presentations at several national and international meetings in the U.S and Mexico to invite researchers to use the database and to contribute to it by submitting their publications. We have received a positive response from many researchers attending these meetings, and several have sent us their presentations and publications to be included in the database. During 2009, the database received close to 5,000 visits. The majority of those connecting to the Internet Protocol (IP) addresses are from Florida and California. However, we have also received visits from IPs located in several other countries such as China, Mexico, Colombia, and Brazil. These results indicate that the database is becoming an integral source of information for a growing number of researchers, growers, and the citrus community. Pending continued funding, unfortunately not requested in the original proposal, our goal for the second year of the project is to continue providing this service by including the most current information as it becomes available, increasing the number of linked documents, and creating an interactive forum in which researchers and growers can exchange HLB related information. Presentations related to the citrus greening database database: – Arevalo, H. A., A. B. Fraulo, and P. A. Stansly. 2009. Update on the Citrus Greening Bibliographical Database. (Poster). HLB/ ZC meeting, McAllen Texas – Arevalo. H. A. and P. A. Stansly. 2009. Monitoreando el Psillydis Asiatico de los Citricos en el Campo y el Internet (Monitoring the Asian Citrus Psyllid in the field and the Internet). XIII Simposio Internacional de Citricultura. CD Victoria, Tamaulipas, Mexico. – Arevalo, H. A., A. B. Fraulo, and P. A. Stansly. 2009. Update on the Citrus Greening Bibliographical Database. (Poster). Florida Entomological Society Annual Meeting. Ft. Myers, FL July 2009 – Arevalo, H. A., G. Snyder, and P. A. Stansly. 2008. The citrus greening Bibliographical Database, a New Tool for Researchers, Students and Growers. (Poster). International Research Conference on Huanglongbing. Orlando, FL December 1-5, 2008
Obj. 1. Carry out DNA bar coding to establish the identity and diversity (if it exists) in south Florida (Stansly, Brown). PCR using mtCOI primers amplify a 850 bp fragment from colony psyllid adults; DNA sequencing confirmed. FL field collections from citrus and other spp. obtained from cooperator collections in previous years; 2010 spring and summer FL (and elsewhere) field collections are planned when psyllid dispersal is underway. Obj. 2. Employ qPCR to detect Ca. Liberibacter presence (or absence) in the psyllid colony cohorts over different AAPs, for immatures and adults (Roberts, SWFREC, HLB diagnostics lab). The FL psyllid rearing system is fully functional and Ca. Liberibacter-infected and bacterium-free colonies are being maintained. Climatic factors affect growth of colony host plants (Citrus, Murraya) and seem to influence bacterial titer in plants and in the psyllid vector instars. Using qPCR (DNA isolation) we are monitoring over time the detectable bacterial titer in psyllid colonies and the host plant, toward quantitative detection in fresh flush plant tissue and in whole insect bodies. Obj. 3a. Carried out exploratory analysis of psyllid organs (guts) for HLB detection using qPCR (consulted methods of: Li et al. 2006 for plant material; Manjaunth et al. 2008 for psyllids). Obj. 3. Using the colonies (analyzed above by qPCR), localize Ca. Liberibacter in whole psyllids and dissected organs to develop a gross anatomical road map of Ca. Liberibacter accumulation in organs, tissues, and cells (adult and 5th instar psyllids) (Cicero, Brown). [In our hands, D. citri does not render a signal when processed with the standard protocol proven for whiteflies (Gottlieb et al 2006)]. We have observed positive fluorescence by in-situ hybridization (FISH) with a Cy5-HLB 16S rDNA probe in variously prepared samples. We have observed a robust signal in infected adult psyllids cut in half to improve infiltration. These results represent extensive experimentation to develop and optimize fixation, dehydration, decolorization and hybridization procedures for this species. After replicated experimentation and further validation of this and several other probes, probes can then be applied to thick sections, which in turn can be compared to ultrathin TEM sections (gold probes) (Obj. 4) for precisely localizing interactions between HLB and the organs involved in transmission. Obj. 4. Using the resultant thick section road map, elucidate at the TEM level, specific organs, tissues, and cells where Ca. Liberibacter accumulates (Cicero, Brown). Using TEM we observed bacterial cells on the outside of the esophagus and of the PSGs. By SEM, bacteria were observed on the outside and inside of the midgut epithelial cells. This suggests that Ca. Liberibacter bacterial colonies exist in multiple locations, perhaps adhering to the upper alimentary canal and midgut, and elsewhere. We also have observed by TEM groups of phage-like particles, intercellularly in Liberibacter-infected adult psyllids (unknown significance). We have compiled an anatomical, serial, ultrathin-section library of infected, 5th instar D. citri larvae. It is apparent that our focus must include both adult and late immature instars based on recent results of transmission studies. Therefore we are developing procedures for adult and immature instars to extirpate and SEM-stage digestive organs, followed by TEM. We also have carried out initial SEM exploration of extirpated guts from adults and 5th immature instars.
This work will determine if certain alternative plant species are better hosts for the suspected HLB bacterial pathogens (Ca. Liberibacter asiaticus (Ca. Las), Ca. Liberibacter americanus (Ca. Lam) and Ca. Liberibacter africanus (Ca. Laf)) and can serve as a reservoir hosts for infection to citrus. During this quarter work was again was performed at all four investigator locations. At the University of Florida, CREC Lake Alfred, quantitative real time PCR (qPCR) was done on Severinia buxifolia (orange boxwood) and showed that it was a good host for the Las bacterium. HLB healthy Asian citrus psyllids were allowed to feed on HLB infected S. buxifolia and 20-30% of them were found PCR positive. Initial PCR tests of sweet orange plants that psyllid transmissions were previously done from the infected Severinia produced some positive plants. Additional PCR testing will be done after further incubation. This research was done by Hao Hu a graduate student studying with Dr. Brlansky. Real time PCR results on rough lemon (C. jambhiri) showed infection of both symptomatic as well as asymptomatic shoots and leaves with similar PCR values for all shoots. Rough lemon continues to grow even when infected with Ca. Las. Rootstocks from field experiments are being tested to determine infection. Various sweet orange cultivars were inoculated with Florida Ca. Las and one cultivar tested PCR negative in multiple tests. At the Texas A&M Citrus Center, Weslaco psyllid feeding tests continued on the rutaceous plants that are established there. More replications of psyllid feeding experiments were done with Esenbeckia berlandieri (jopoy), Amyris madrensis (torchwood), Choisya ternata and C. arizonica. All are feeding hosts for the psyllid. As previously reported egg laying was found on torchwood but the psyllids did not complete development. Egg laying and nymphal development to adults were found on C. ternata. This work is part graduate student Jose Sandoval’s research with Dr. da Graca. At the USDA, ARS, Beltsville quarantine greenhouse. Graft inoculations were done to Murraya paniculata with exotic Ca. Ca. Lam and Ca. Laf isolates and will be tested soon. No symptoms are apparent. Work with dodder as an alternative host was completed studying the plant infection process and for its use to transmit all Liberibacters to plants that are not graft compatible with citrus. A manuscript was submitted for publication in Phytopathology by Drs. Hartung and Brlansky. In this publication dodder became infected and phloem necrosis occurred similar to that in citrus. In addition we found that the Liberibacter exists in two morphological forms in the dodder similar to that seen in citrus and periwinkle. In addition we found the intermediate between the two forms which proves that both forms are the same bacterium. At the USDA, ARS, FDWSRU, Ft. Detrick, MD a manuscript entitled ‘The relevance of Murraya paniculata and related species as potential hosts and inoculum reservoirs of ‘Candidatus Liberibacter asiaticus’, causal agent of Huanglongbing (HLB)’ was completed and accepted for publication. Authors from the grant include Drs. Damsteegt, Brlansky and Schneider. The details of this work were reported in earlier reports. Psyllid transmissions from HLB (Ca. Laf and Ca. Las (Thailand strain)) infected sweet orange to Severinia buxifolia are underway.
We have now developed a series of flexible stochastic models to predict the temporal increase and spatial spread of diseases. The models were initially characterized for citrus canker spreading in plantation and urban (backyard) environments. They have subsequently been extended to HLB in this project. The models can be used in a number of ways: to predict spread and to analyze the effectiveness of control strategies. Most attention has been given to spread within plantations, including allowance for proliferation of infection along boundaries in response to vector behavior. The models can readily be extended to consider spread at larger scales including spread through heterogeneous environments up to State-wide scales. We have also considered the effects of uncertainties in the distribution of host crops for example the effects of small areas of crop that may not be recorded but which can act as ‘bridges’ in transmitting disease. The effects of uncertainty in parameter estimates for dispersal parameters and transmission rates have also been included. Additional computer-friendly formulations of the models have also been developed to aid in education of stake-holders to illustrate the effects of uncertainty in predicting future disease spread and the effectiveness of alternative methods of control. Estimation of parameters for dispersal of HLB poses considerable statistical challenges, especially where trees may become infectious before they are symptomatic/detected. Here we use an SEIDR model (Susceptible, Exposed (latently infected but not yet infectious), Infectious but not yet symptomatic/detected, Detected and infectious and Removed trees). Using MCMC methods, and extensive data from Southern Gardens for successive snapshots of the occurrence of symptomatic/detected trees in known populations of susceptible trees, we are able to estimate model parameters for the transmission rates and dispersal kernel for the disease. Current work is focused on the differential effects of host age on epidemiological parameters as well as variability across the plantation. From these it is possible to allow for uncertainty in the parameters as well as variability over time and through space. The uncertainty is then incorporated in models to predict spread and to allow for uncertainty in the efficiency and comparison of control methods.
Methods and models for the control of HLB disease of citrus. Citrus huanglongbing (HLB) is the most serious disease of citrus worldwide and presently for the very existence of citrus industry of Florida. The approach is two-fold: First was the examination of the effect of various control strategies on HLB, in control plots, established in 2007. I this phase of the study, five treatments were examined: Minimal control, Insecticide vector control, Roguing, Roguing via PCR+, and Comprehensive. Results indicated that although treatments were significantly different, there was no benefit of any control treatment over another. Small differences were due to plot location, not treatment effects. The tests are being repeated, however, the results point to a need for regional control strategy and that small plantings that can’t control neighbors cannot control the disease. Our estimates indicated that for each tree with visual symptoms, there were an average of 13 (range 2-52) that were infected but asymptomatic, i.e., infections that have occurred over the duration of the epidemic but that have not yet expressed symptoms. New plots have been established at the USDA, ARS , in Fort Pierce, Florida and the data collection is under way. Psyllids are also being trapped (in each plot) to estimate populations and correlate with disease progress in each plot. To date 3 HLB+ trees have been identified within the plots, but no statistical difference among plots is yet apparent. Differences should become noticeable in 2010. The second approach is to develop epidemiological models of HLB disease dynamics which improve the understanding of vector-driven disease transmission and analyze disease control policies aimed at disrupting vector population dynamics. In previous and current work we have developed a model for citrus canker. This is the basis upon which we have built a preliminary model for HLB. The HLB simulation model is stochastic based on biological, epidemiological, and meteorological parameters using Markov Chain Monte-Carlo simulation methods and SIR modeling protocols. The model is fit to the HLB data collected in various observed epidemics from Florida and SE Asia by thousands of simulations. Linked-differential equations are used that describe the temporal increase in HLB infected trees and explicitly characterize the population dynamics of the vector. The effectiveness of different disease control measures such as intercropping with guava, roguing and insecticide use will eventually be analyzed via this model and a suite of mathematical tools to identify the most effective strategies. HLB data sets will be correlated with various disease mitigation strategies/events from our epidemiology trials in Florida. The stochastic models allow testing of multiple disease management strategies in thousands of simulated epidemics to determine which will have the optimum effect and in what combination these methods can best be deployed for maximal disease control. The HLB model will continue to be augmented and improved over the next 1.5 years but are well near completion. Data continue to be generated for multiple test plots to parametrize the models. Models are being validated against actual data to ensure correct estimation of disease dynamics. New data sets have been acquired from south Florida and will be used for validation and further model improvement during the first part of 2010. We are also setting up an extensive field experiment at Fort Piers in 2010 to examine the latency period between transmission and infection. This has never been documented with precision in field trees and is a key component in he model.
Disease control of citrus huanglongbing (HLB) by interplanting with guava. HLB is the most devastating disease of citrus worldwide and presently threatens the existence of the citrus industry in Florida. In Vietnam guava has been shown to be an effective deterrent to HLB. For all plots and experiments, Guava trees, (Vietnamese white cultivar) were propagated and grown to appropriate size requiring about one year. Guava vs no guava nurseries: Two nursery sites, a guava protected citrus nursery versus and unprotected nursery, have been established. Disease free, PCR-negative citrus trees (2 sweet orange and 1 grapefruit cultivars) were located in the protected and unprotected plots in June 2009. The guava were established over a year ago and grown to appropriate size as indicated in Vietnam. Trees are assayed for HLB every 60 days, and are in their second assay. Psyllid populations are also being monitored continuously every two weeks within plots to document any repulsion of the vector due to guava. To date no HLB+ plants have been identified in the nursery plantings after multiple assays. Citrus/guava interplantings: 2 commercial plantings with multiple replications each have been established. This has taken considerable time. Guava trees were propagated and grown to transplant size. These were then out planted and grown for a year per Vietnam protocols. One trial was established in a commercial orchard with collaborators in Southern Gardens Citrus. A second trial planting was established at the USHRL Picos Farm in Fort Pierce. To date no HLB+ plants have been identified in the USHRL plantings after multiple assays. A severe frost last winter affected both the USHRL and the Southern Gardens plots causing a delay in the experiment. Damage was extensive in both plots. The damage to the guava was overcome by pruning and replanting of damaged guava trees. Renovation of the USHRL plot was less, and the guava have now been interplanted with the citrus as of August 2009 in the USHRL Picos Farm plot. To date no HLB+ plants have been identified in the USHRL plantings after multiple assays. In the Southern garden plots, damage was more severe and the guava have now been renovated sufficiently that the Souther Gardens plot will be interplanted with citrus. The guava have now been interplanted with the citrus as of November 2009 in the USHRL Picos Farm plot. To date no HLB+ plants have been identified in the SG plantings after multiple assays. Both nursery and field citrus trees are assayed for HLB every 60 days, and are in their second assay. Psyllid populations are also being monitored continuously every two weeks within interplanted plots to document any repulsion of the vector due to guava. Data collection continues and is currently ongoing.
Cultural Practices to Prolong productive Life of HLB Infected Trees. Through the summer of 2009 trees receiving the nutritional/SAR trees were vigorous and green with only normal HLB symptoms typical of HLB infected trees. This fall of 2009 at our 100% infected trial site we have observed numerous visual HLB symptoms in multiple sectors of the trees. Although our other two sites with lower infection rates of 50% and 15-20% had increased visual symptoms of HLB this fall and winter typical of greening trees, the symptomatic leaves were not as abundant as in the more infected trees. Fruit color break from green to yellow in Valencia oranges began early (late September) at the site with 100% infected trees and was not observed at the two other sites where fruit remained green as typical in uninfected trees. Fruit drop has been observed this fall only in the 100% infected trial. Data collected show treatments with the complete Maury Boyd cocktail, with or without hydrogen peroxide, and the foliar feed macronutrients in a typical range of 2 to 5% fruit drop. The other treatments ranged from 8 to 17% fruit drop. No abnormal coloring of fruit or fruit drop has been recorded in sites with 40-50% or 15-20% HLB infection. Evaluation of Systemic Acquired Resistance Inducers Combined with Psyllid Control to Manage Greening infected Groves We are monitoring Asian citrus psyllid (ACP) populations and Can. Libericacter asiaticus (CLas) titer in plants and psyllids, and impacts in a young citrus block as the result of the main effects and interaction of two treatments: 1) micronutrients + systemic acquired resistance inducers (Micro+SAR), and 2) Psyllid chemical control applications based on scouting. This trial was designed as a replicated (n=4) complete block for a 2×2 factorial experiment on a 12-acre commercial block of now 7-years-old ‘Valencia’ oranges on ‘Swingle’ rootstock that was held back by defoliation for canker control in 2006. Adult ACP populations have been maintained three times higher on the average, and up to 50 times higher on occasion in insecticide-treated plots compared to treated plots throughout both years of the experiment. Insecticides have been applied 6 times during that period, either as a single scheduled dormant spray per year or when adult populations exceeded a threshold of 0.5 per biweekly ‘tap’ sample made by striking a limb while holding a laminated sheet underneath to catch falling psyllids. The Micro+SAR treatment has had no effect on ACP populations. ACP adults and nymphs have been collected 4 times since Nov 2008 for PCR analysis along with a leaf sample from the most symptomatic branch from every 5th tree in the block. Insect samples were sent to our collaborators at the USDA in Riverside, CA for analysis and plant tissue samples were processed at the PCR laboratory at SWFREC. In November 2008 and April 2009, 34% of the plants were positive for HLB over all treatments using a cutoff CT value of 36. This jumped to from 86 to 100% of the plants positive in Aug. 2009 with no significant differences among treatments by ANOVA. However, the two treatments employing insecticidal control had significantly higher Ct values (avg. 28.5) than the two non-insecticide treatments (avg. 25.5) indicating lower CLas titer in plants protected by insecticides. Yield was assessed on the entire block, plot by plot in March 2009. Despite the high percentage of infected trees, we observed that trees receiving both insecticidal and nutritional treatments produced 1.32’0.15 boxes per tree of fruit, a 30+% increase over 0.95-0.99 boxes per tree from the other 3 treatments. Fruit was analyzed by the CREC pilot plant in Lake Alfred and no significant treatment effects on fruit or juice quality were observed. These results could be interpreted to mean that the Micro+SAR package is capable of staving off negative impacts of HLB if CLas titer can be maintained below some threshold level through psyllid management. However, we will not feel comfortable with this conclusion until we see similar results from the March/April 2010 harvest.
Cultural Practices to Prolong productive Life of HLB Infected Trees. Through the summer of 2009 trees receiving the nutritional/SAR trees were vigorous and green with only normal HLB symptoms typical of HLB infected trees. This fall of 2009 at our 100% infected trial site we have observed numerous visual HLB symptoms in multiple sectors of the trees. Although our other two sites with lower infection rates of 50% and 15-20% had increased visual symptoms of HLB this fall and winter typical of greening trees, the symptomatic leaves were not as abundant as in the more infected trees. Fruit color break from green to yellow in Valencia oranges began early (late September) at the site with 100% infected trees and was not observed at the two other sites where fruit remained green as typical in uninfected trees. Fruit drop has been observed this fall only in the 100% infected trial. Data collected show treatments with the complete Maury Boyd cocktail, with or without hydrogen peroxide, and the foliar feed macronutrients in a typical range of 2 to 5% fruit drop. The other treatments ranged from 8 to 17% fruit drop. No abnormal coloring of fruit or fruit drop has been recorded in sites with 40-50% or 15-20% HLB infection. Evaluation of Systemic Acquired Resistance Inducers Combined with Psyllid Control to Manage Greening infected Groves We are monitoring Asian citrus psyllid (ACP) populations and Can. Libericacter asiaticus (CLas) titer in plants and psyllids to evaluate main effects and interactions from a 2×2 factorial experiment with with 4 replications and 4 treatments: (1,2) (micronutrients + systemic acquired resistance inducers (Micro+SAR) with and without insecticidal control (3) insecticidal control alone, and (4) untreated. The trial is being conducted on a 12-acre commercial block of now 7-year-old ‘Valencia’ oranges on ‘Swingle’ rootstock that was held back by defoliation for canker control in 2006. Adult ACP populations have been maintained three times lower on average and up to 50 times lower on occasions in insecticide-treated plots compared to untreated plots throughout both years of the experiment. Insecticides have been applied 6 times during that period, either as a single scheduled dormant spray in winter or when adult populations exceeded a threshold of 0.5 per biweekly ‘tap’ sample made by striking a randomly selected limb while holding a laminated sheet underneath to catch fallen psyllids. ACP adults and nymphs have been collected 4 times since Nov 2008 for PCR analysis along with a leaf sample from the most symptomatic branch of every 5th tree. Insect samples were sent to our collaborators at USDA Riverside CA for analysis and plant tissue samples were processed at the PCR laboratory at SWFREC. In November 2008 and April 2009, 34% of the plants were positive for HLB over all treatments using a cutoff Ct value of 36. Incidence of HLB jumped to 85 – 100% positive in Aug. 2009. However, the two treatments employing insecticidal control had significantly higher Ct values (28.5’0.7) than the two non-insecticide treatments (26.4’0.6) indicating lower CLas titer in plants protected by insecticides. All fruit was harvested in Mar 2009 and yield was assessed by plot. Despite the high percentage of infected trees, those receiving both insecticidal and Micro+SAR produced 1.32’0.15 boxes per tree of fruit, a 30+% increase over 0.95 to 0.99 boxes per tree from the other 3 treatments. An analysis by the CREC pilot plant in Lake Alfred found no significant treatment effects on fruit or juice quality. These results could be interpreted to mean that the Micro+SAR package is capable of staving off negative impacts of HLB if CLas titer can be maintained below some threshold level through psyllid management. However, we will not begin to feel comfortable with this conclusion until we see similar encouraging results from the spring 2010 harvest.
During this reporting period Dr. Olga Minenkova of Sigma Tau pharmaceuticals (Rome, Italy) spent 2 weeks in our laboratory with Dr. Yuan and initiated scFv antibody protocols using advanced phage display technologies. This included transfer of the specialized cloning vector and E. coli strains, preparation of competent cells, purification of mRNA, construction of the cDNA library, cloning, and trouble shooting and validation of experimental protocols. As a proof of concept experiment we used Xylella fastidiosa 9a5c (citrus variegated chlorosis) mixed with psyllids to immunize mice. Psyllids were raised and provided by Drs. Brlansky and Damsteegt. Xylella fastidiosa 9a5c was added to psyllids at ~200,000,000 cells per injection and ground in extraction buffer to mimic subsequent injections with psyllids infected with ‘Ca. Liberibacter asiaticus’ from Florida. Mice were sacrificed and mRNA from spleens were used to create the phage library. The library was screened 3 times against X. fastidiosa 9a5c with excellent results. Several antibodies have been identified that react strongly against X. fastidiosa 9a5c but do not react at all against strains of X. fastidosa that cause Pierce’s disease. This is a significant ‘bonus’ result that will benefit both the citrus and grape industries, and demonstrates that our basic approach for making antibodies against ‘Ca. Liberibacter asiaticus’ will be successful. Additional screening will be done by Dr. Nandlal Choudhary in Dr. Brlansky’s lab in Florida using ELISA, western blots, tissue blots and immunogold labeling. While the phage libraries against X. fastidiosa were being screened, mice were injected 3 times over a period of three weeks with psyllid extracts containing ‘Ca. Liberibacter asiaticus’ at ~ 200,000,000 cells per insect. The mice will receive 1-2 more injections and will then be sacrificed for production of both scFv (Beltsville) and monoclonal (Agdia) antibodies. In order to prepare injections of mice, more than 500 psyllids have been individually ground in buffer and sampled by Q-PCR to quantify the amount of ‘Ca. Liberibacter asiaticus’ present in each insect. This is necessary to identify the insects with the highest titer of pathogen (~ top 2% of insects) so that each injection contains enough Ca. Liberibacter asiaticus to induce an antibody response. Symptomatic plants or fruits with the highest titers will be used to screen the phage display library for antibodies against Ca. Liberibacter asiaticus’. HLB plants in our greenhouse were individually assayed for titer of ‘Ca. Liberibacter asiaticus’ to be used in the screening of the phage display libraries. HLB symptomatic fruit have been provided by Dr. Brlansky and will be assayed individually to identify sources of extracts to be used in screening the libraries. In related work the outer membrane protein (OMP) of ‘Ca. Liberibacter asiaticus’ has been expressed and purified in the laboratory. This protein has been provided to Cocalico laboratories and they are currently raising polyclonal antibodies against this protein in rabbits. These rabbit antibodies will be useful to capture ‘Ca. Liberibacter asiaticus’ in future experiments that will use the mouse scFv or monoclonal antibodies for specific detection and quantification.
A major objective of this proposal is developing a rapid method of detection of HLB bacterium (Las) in citrus trees using non-radioactive Las specific nucleic acid probes. Towards this end, we developed specific PCR probes corresponding to the outer membrane protein (OMP), RNA polymerase beta subunit, DNA polymerase region, the r-DNA region, and the 23S and 16S ribosomal RNA inter-genic regions of the HLB pathogen. Citrus plants from the green house infected with HLB pathogen were used as the source plants for obtaining the needed tissue for the blots and to isolate nucleic acid template necessary for the amplification of Las specific DNA. In the initial tissue blot experiments we did not observe hybridization signals specific for HLB. We used healthy plants grown under similar conditions as controls. The reason probably is the low titer of pathogen and/or the non uniform distribution of the pathogen in the infected tissues. It is also possible that the PCR probes were not of sufficient specific activity so as to detect low titer of the pathogen. Therefore, the amplified regions of Las were also cloned in the transcription vector, and digoxigenin labeled strand specific RNA probes were generated by transcription. However, use of the high specific activity RNA probes did not improve hybridization of the tissue blot and occasionally non specific hybridization was also observed in healthy tissue blots. This suggested less than optimal hybridization regimen. We have subsequently focused on optimizing the hybridization conditions and use of multiple probes in an effort to increase the extent of signal. These changes have substantially improved detection of Las in citrus tissues. In addition to tissue blots of the stem sections, we have used the midrib and petiole region imprints from the leaves of infected citrus on nylon membranes. The hybridization observed with the midrib imprints showed much clear signals compared to stem imprints. In forthcoming experiments we will use imprints of the leaf midrib, petiole and stem from new flushes of infected citrus since the psyllids preferentially feed and acquire the pathogen from such tissues. We also imprint on the membrane the inside surface of the bark which contains phloem tissue in which the HLB pathogen is located. Although this procedure is not optimized, we believe this to be very useful and the distribution of Las in infected citrus tissue could be easily documented. The second area of our focus is on the detection of Las in psyllid vector by tissue blots (squash blot) on nylon membranes. At the outset, a procedure for isolating the nucleic acid from single psyllid was optimized, and we have been able to amplify Las specific amplicons from single infected psyllids using pairs of Las specific primers. Conditions of amplifications were optimized with different primer pairs and now we have been able to amplify HLB specific amplicons without non-specific bands in PCR. In dot blot hybridizations using the known amount of Las specific DNA from a psyllids we observed a linear relationship with input DNA and the degree of hybridization with probes from OMP and r-DNA. In initial studies of whole psyllid tissue blots, hybridization signal was also observed with healthy psyllids (psyllid colony from the healthy psyllid containment facility). However use of specific primer pair corresponding to the EFTU gene of Las has been promising and we will use the probe generated for this gene in tissue blots of psyllids henceforth. The observed background was probably due to the extraneous tissues of the psyllids like wings that attach to the nylon membranes during psyllid squash and very hard to exclude during membrane wash. Therefore we intend to separate the head and the abdomen regions of the psyllids in tissue blots and exclude the wings region to reduce the background signal. We will use the results of this technology to test the progression of Las spread in citrus and infection ratios in populations of psyllids in the field.
Transmission of a pure culture of ‘Candidatus Liberibacter asiaticus’ (Las) by citrus psyllids into susceptible sweet orange plants is the initial step in completing Koch’s Postulates. This project uses two mechanisms to introduce Las into the psyllids, membrane feeding and microinjection. We have done a number of experiments where Asian citrus psyllids were fed on sachets containing media with pure culture Las. The success rate of these experiments has been very low, but we report that we have successfully transmitted pure culture Las into sweet orange using psyllids. The infected trees are positive by real-time PCR, three months after inoculation, but do not show significant symptom development at this point. We are currently working to confirm the presence of Las in the inoculated trees using multiple methods including sequencing, but are reasonably confident that this represents the first success in the alternative confirmation of Koch’s postulates via transmission by vector. In addition, we have begun conducting microinjections, beginning with control experiments where healthy psyllids were injected with hemocoel from Las infected psyllids. The microinjections were successful at multiple levels, as the injected psyllids survived, tested positive for Las, and were able to transmit the Las to healthy sweet orange seedlings. We plan to move forward with micro-injection of pure cultured Las, but at this point we have great difficulty obtaining adequate pure cultures and we have begun efforts to grow our own live, pure cultures of Las using various techniques.
A major objective of this project is to develop an understanding of how the HLB bacterium (Las) interacts with citrus genotypes to cause disease. After finding that different citrus genotypes respond differently to Las from extremely sensitive (sweet orange and grapefruit) to tolerance with minor symptoms, we have focused on the one citrus genotype that is most resistant to citrus. Las is restricted to very low levels in Poncirus trifoliata. Most plants remain PCR negative, but a few have barely detectable levels of Las. We are determining whether this is due to plant genetics, Las variation, or randomness. Some Poncirus hybrids are more susceptible than others suggesting that resistance to Las is segregating. We are beginning experiments to map citrus genes that provide Las resistance. Las also appears to have difficulty spreading in Poncirus. We are examining the value of using Poncirus rootstocks and interstocks to reduce or prevent spread of the disease in sweet orange or grapefruit. We have developed a containment plant growth room to examine natural infection of citrus trees by psyllid inoculation. We already have made several significant observations: First, we have found that the time period between when plants first become exposed to infected psyllids and the time that new psyllids can acquire Las for those plants can be as little as 6 weeks. We are examining this process in more detail now. Second, when we allowed the infected psyllids a choice of different citrus genotypes, there was a large difference in the time and number of plants that were inoculated by the psyllids: (Citrus macrophylla >> Swingle citrumelo >> Volkamer lemon = Duncan grapefruit > Madam Vinous sweet orange >> Carrizo citrange). Most of the Citrus macrophylla plants became infected with only 2 months of exposure in the epidemic room, whereas only a few of the sweet orange and grapefruit became infected after 4 months. Since there was such a clear preference, we are now investigating its cause ‘ whether the preference is related to genotype, growth habit, flushing, or other possible differences. It is clear that psyllids reproduce on new flush, but feed on older leaves. We are examining whether and how well the psyllid can transmit the disease in the absence of flush. Third, these results have led to the development of methods to greatly speed up results of field tests for transgenic or other citrus trees or trees being protected by the CTV vector plus antibacterial or antipsyllid genes. In order to interpret results of a field test, most control trees need to become diseased. Under natural field pressure in areas in which USDA APHIS will allow field tests, this level of infection could take 2-3 years. By allowing the trees to become adequately inoculated by infected psyllids in a containment facility, we can create the level of inoculation that would naturally occur in the field within 2-3 years in 2-5 months in the containment room, after which the trees are moved to the field test site. Another objective is to provide knowledge and resources to support and foster research in other laboratories. A substantial number of funded projects in other labs are based on our research and reagents. We supply infected psyllids to Mike Davis’s lab for culturing of Las and Kirsten Pelz-Stelinski’s lab for psyllid transmission experiments. Among the plants being screened for resistance or tolerance to HLB for other labs are: 1) a series of elite lines for the citrus improvement group; 2) a series of transgenic plants designed to examine the relationship of pectin production to disease development for Jude Grosser, Gene Albrigo, and Nian Wang; 3) we are testing a series of transgenic plants that we made in collaboration with Zhonglin Mou to have increased disease resistance. The trees, which have high resistance to citrus canker, are presently being tested against HLB; and, 4) a series of lemonine trees reported to be resistant to HLB for Gene Albrigo.
This is a continuing project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in two ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. We think that this approach could be used beginning 2-3 years from now and until probably 15 years from now when resistant trees should be available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We have completed several large screenings of antibacterial peptides against Las in sweet orange trees. About 40 different antibacterial peptides have been tested in trees. We initially found three peptides that allow much better growth of trees that were grafted with HLB-infected buds. Some trees had no symptoms and no detectable Las, some trees had no symptoms and low levels of Las, and other trees had leaf symptoms but continued growth of the trees with normal levels of Las. Another result is that we found that leader peptides for the export of the peptide from the CTV-infected cell is not needed for HLB but is needed for citrus canker. Because we were concerned that graft inoculation of HLB into the trunks of small trees is a too severe challenge that might cause peptides that could work in the field to be missed, we developed a system that only allows inoculation by infected psyllids. We have established a containment plant growth room in which psyllids inoculate the plants expressing the peptides. These experiments are on-going, but we appear to have several more peptides that are protecting sweet orange. To speed up the search for effective anti-HLB genes, Falk (UC Davis) has developed a tobacco-tomato psyllid/liberibacter model to screen for effective genes against the similar bacterium. This system is working and screening is on-going. We also are improving the CTV-based vector to be able to produce 2-5 peptides at the same time. This will allow expression of genes against both HLB and canker or multiple of genes against HLB. We have developed a vector that can be re-added to trees if the anti-Las gene is lost or a better gene becomes available. A major objective that we are pursuing is to make a vector that cannot be transmitted by aphids. Another major goal is to do a field test of the CTV vector with antibacterial peptides, which is an initial step in obtaining EPA and FDA approval for use in the field. We have received permission from USDA APHIS for the field test, but were delayed by EPA. We are now submitting a revised application to USDA APHIS to include EPA requirements and are expecting to establish the field test this spring. In addition, we are screening a series of transgenic sweet orange and grapefruit expressing antibacterial genes for Erik Mirkov of Texas A&M and Mike Irey of Southern Gardens.
Objective 1: Sprays of copper (Cu) formulations, containing copper hydroxide (Kocide, Champ, Kentan, Badge) or copper sulfate (Cuprofix), were moderately to highly effective for control of canker on fruit of susceptible Ruby red grapefruit and Hamlin orange in the lack of high disease pressure due to early and late season wind-blown rain storms in 2009. A chelated Cu (Magna-Bon, copper pentahydrate) at a 50% lower rate of Cu per application than standard Cu formulations performed as well for reducing fruit disease incidence for grapefruit or canker-induced fruit drop for Hamlin. Early season infection and fruit drop of grapefruit and Hamlin was minimal because April was relatively dry and Cu treatments were initiated before significant rainfall occurred in May. Cumulative fruit drop due to early season infection of untreated Hamlin amounted to about 5%. Five sprays of Cu formulations reduced the incidence fruit drop due to canker by about 40-50% to 2.5% cumulative fruit loss. Objective 2: In Marsh grapefruit, canker control increased with number of Cu sprays from 3 to 11 (April to October), canker infection and copper burn occurred after rains commenced in July. In August, fruit were growing most rapidly which would produce a thinning of fruit cuticle and an increase in the rate that new stomates open due to more rapid expansion of the fruit surface. Season-long copper spray also gave the best control of late season scab and melanose on fruit. In Hamlin, sprays beyond mid-July provided additional canker control of fruit drop confirming that late season lesions do not cause fruit drop like early season lesions. Objective 3: In two grapefruit trials, Firewall (streptomycin[Sm]) applied alone or in combination with a reduced rate of Kocide 3000 in July and early August gave equivalent control on grapefruit to Kocide alone. The adjuvant, Polymer Delivery System (PDS) did not increase the residual activity of Cu on grapefruit or control efficacy of Cu. The residual activity of Cu on fruit was not affected by Kocide rate but decreased with time after application due to increase in fruit surface area over 21 days. This result supports the recommendation for use of 21 day interval Cu sprays for adequate canker control and explains the reduced efficacy of 28-day interval sprays. Objective 4: The Cu resistance gene was identified as CopL on a plasmid from a resistant Xanthomonas citri subsp. citri (Xcc) strain from Argentina that was exposed long-term to Cu for canker control. The identical resistance gene sequence was found in Xanthomonas spp. causing bacterial spot in tomato and pepper. Primers constructed based on the gene sequence were used to screen the remaining Cu resistant strains of Xcc from Argentina and Cu resistant strains of X. alfalfae. pv citrumelonis from Florida citrus nurseries with citrus bacterial spot. All strains screened thus far contain the CopL resistance gene. In addition, a non-pathogenic strain of Xanthomonas isolated from a citrus grove was found to be Cu resistant and may represent a pre-existing source of risk in citrus groves for horizontal transfer of CopL into Xcc. Cu and Sm resistance were monitored in Xcc and epiphytic bacterial populations on grapefruit trees sprayed with Cu or Sm every 21 days for two growing seasons (22 sprays total). Each season Cu and Sm sprays increased the ratio of epiphytic bacterial population with tolerance to these chemicals. Overall, the Sm resistant bacterial populations were proportionally lower than Cu tolerant bacterial population. No resistance to either Cu or Sm was verified in Xcc or epiphytic populations after two years of season long sprays. Objective 5: In 2009, canker management talks were given at county extension and other meetings. Updated 2010 canker management recommendations have been published in the Florida Citrus Pest Management Guide and Citrus Industry Magazine. Oral presentations have been scheduled for the Florida Citrus Production Managers and Florida Citrus Show.
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