Research results from the first season were successfully completed, the results reported at the 2008 annual Florida State Horticultural Society (FSHS) meetings and at Packinghouse Day and the Indian River Postharvest Workshop in Sept. 2008, a manuscript published in the FSHS proceedings (http://www.fshs.org/; Proc. Fla. State Hort. Soc. 2008. 121:322’325), and results reported in previous FCPRAC progress reports. To summarize, preharvest treatments included up to three foliar potassium (K) sprays (March, October, and January) or withholding irrigation for up to two months before harvest. In one commercial block, foliar mono-potassium phosphate (MKP) was applied at 23.5 lb MKP per acre (0-52-34; 8 lb K2O per acre) with 4 lb per acre low-biuret urea (46-0-0) applied at a total volume of 125 gal per acre. In another block, the grower applied 3 gal per acre of a commercial 3-18-18 formulation at a total volume of 250 gal per acre. Postharvest treatments included holding fruit for three days at 30%, 55%, or 100% (including wetting the fruit) RH before washing and storing at 70F under ambient RH. In the first grapefruit block, preharvest foliar MKP treatments applied 2 months or more before harvest did not significantly reduce peel breakdown. However, fruit set was poor in that block producing very large and misshapen fruit. In the second block, peel breakdown was significantly reduced with foliar 3-18-18 applied 2, 3, or 4 weeks before harvest. Preventing irrigation or rainfall for 49 days before harvest increased peel breakdown, whereas wetting the fruit after harvest and maintaining high postharvest RH reduced peel breakdown compared to exposing fruit to lower RH for 3 d before washing. Funds for the second year of this study were received very late in the season on April 3, 2009. So the bulk of the funds will be used on experiments this coming season. However, we did conduct several additional experiments in 2008-09 before the project’s approval. After three sets of field trials on White grapefruit and Valencia oranges, we found less promising results with foliar K than in the previous season. In one experiment, foliar K even increased peel breakdown. The reason for this is unclear. However, applying a combination of foliar K and Mg or use of an antitranspirant resulted in a significant reduction in peel breakdown during postharvest storage. Application of foliar Mg (Epsom salt) alone showed a trend in reducing peel breakdown that, while not significant, is worth repeating. Four sets of postharvest experiments gave similar results as the previous year. Interestingly, postharvest treatments with Imazalil sporadically reduced peel breakdown as was noted the previous year. The reason for this is unclear but worth investigating further. For the current 2009-10 season we are establishing at least two field plots in two different commercial grapefruit groves to apply preharvest foliar K, antitranspirants, and Mg. We will also test combinations of these materials as well as other treatments that might be suggested by research and/or grower reports.
Repeated low volume (LV) applications of horticultural mineral oil (HMO) were initiated this year on 14 April although funding for this project was released on 27 April. The replicated experiment is being conducted in the same 65 acre block as last year, although we have increased the rate from 1 to 2 gal/ac pure FL-435 oil and are comparing two truck-mounted LV applicators: a modified London Fogger model 18-20 provided by Chemical Containers and the Proptec rotary atomizer P400D. Applications continued through fall flush at 2-4 week intervals depending on pest pressure. Significant differences in the number of Asian citrus psyllid (ACP) adults between the Proptec P400D (0.06’0.06 ACP adults/tap) and the control (0.23’0.08 ACP adults/tap) were observed on 15 June. A similar trend has continued to be observed, although there were no statistically significant treatment effects due to low psyllid populations. Leaf extractions have been made as a first step in the process of quantifying mineral oil deposition on leaf surfaces of citrus following LV ground applications. Results from these studies support those obtained last year indicating the effectiveness of frequent LV applications of HMO, but we still need higher populations to feel confident with this tactic. We also conducted a separate study on a highly infested, 38-acre block of ‘pineapple’ orange in Glades County to compare the London Fogger ‘ applying 2 gal/ac to bed tops only (typical application) with an airblast sprayer treating tops and swales at 116 gal/ac. Delegate WG (spinetoram) @ 4 oz/ac. and Dimethoate 4EC (24 oz/ac.) were both applied with 2 gal/ac. oil on 15 June. Insecticides sprayed with the airblast showed significantly fewer adults compared to the control from week 1 to week 4. LV sprays showed significantly lower populations than the control only for weeks 3 and 4. This experiment demonstrated that LV application to bed tops with these two insecticides did not provide adequate control. Growers should evaluate such applications carefully. In a third experiment, we evaluated the effectiveness of aerial versus ground application of two selective insecticides: Delegate WG (spinetoram) @ 4 oz/ac, and Movento (spirotetramat) @ 10oz./ac., and a broad-spectrum insecticide Imidan 70W phosmet (Imidan) @ 1Lb/ac, all with 2gal/ac of 435 mineral oil. The trial was conducted on thirty 24-acre blocks of mature orange trees. Imidan by ground (0.1’0.1 ACP/tap) was the only treatment that significantly reduced ACP populations compared to the untreated control (2.6’0.5 ACP/tap) 2 weeks after application. At week 4, ground applications resulted in lower ACP numbers when compared with aerial applications. We also saw fewer psyllids on trees treated with Movento (by air: 0.7’0.1 ACP/tap, by ground: 1.1’0.3 ACP/tap), and Imidan by ground (0.2’0.2 ACP/tap) compared with the control (2.1’0.5 ACP/tap). The effect of these treatments on rust mites was evaluated on 6 August by counting the number in 2 lens fields (14x) per fruit, 4 fruits per tree, 10 trees per plot . Ground applications of Delegate (0.13’0.03 mites/fruit), Movento (0.12’0.02 mites/fruit), and Imidan by ground (0.28’0.08 mites/fruit) and by air (0.16’0.07 mites/fruit) had significant lower populations when compared with the untreated control (0.42’0.13 mites/fruit) and oil only (0.40’0.08 mites/fruit). Results from these trials have been presented at the Florida State Horticultural Society 2009 annual meeting and will be published in their proceedings, and also be presented at the Entomological Society of America December 2009. In an area wide spray during the 2008/09 dormant season, 80,000 acres were treated by air and more by ground. A field survey coordinated by SWFREC-Entomology and DPI-CHRP found that 5 months after the application groves treated by air (0.1’0.02 ACP/tap) and by ground (0.3’0.1 ACP/tap) had significantly fewer ACP than the untreated which had 2.8 ACP/tap, a 2400% increase. We are working with Golf Citrus Growers Association to organize two area wide sprays this season.
Data still have been collected from this field experiment. From June/09 to September/09 the disease incidence increased from 15.4 to 20.3% in plots with psyllid control program and from 34.1 to 44.9% in plots without vector control. Like observed in past years, the seasonality of HLB symptoms was repeated this year. The incidence of symptomatic trees in each assessment started to increase after February (end of summer) reaching high values in April to June (autumn), and started to decrease after July, reaching low values in September (beginning of spring). Until now, there was no significant relationship between the frequency of local inoculum reduction and all assessed variables. Even the local vector control program significantly reduced the adult psyllid population in 79% and the psyllid eggs and nymphs in 95% in treated trees; psyllid control significantly reduced the cumulative number of HLB-symptomatic trees in 55%. No significant difference was observed on the delay of the beginning of epidemics and on disease progress rates. This preliminary result shows that the tested vector control program did not completely avoid HLB contamination from external source of inoculum (20.3% of HLB-symptomatic trees 40 months after planting). After the first harvest in September/09, the average yield on plots without vector control (40.4 box/acre) was 56% smaller than the average yield on plots with vector control (91.6 box/acre). Additionally, the yield per plant was 38% less in non-treated trees (0.33 box/plant) than in insecticide treated trees (0.54 box/plant). Trees without systematic insecticide sprays had higher attack of other pest such as citrus leafminer and aphids that reduced the normal vegetative flux development. Until September/09, US$ 7.79 per plant was spent on psyllid vector control program. Additional data for economic analysis have been collected. Annual maps of HLB-symptomatic trees are being prepared for spatial analysis using stochastic models (MCMC) to verify the effects of each treatment on primary and secondary spread of HLB. Also, all psyllids captured on yellow stick traps since the winter/07 are being prepared to be tested for the presence of Candidatus Liberibacter spp. by PCR. The assessments on this experiment will continue to allow more detailed temporal and spatial analysis and get better conclusions.
Funds for this project were released on 9 June 2009. Objectives and corresponding results are listed below. 1) Evaluate and refine the use of the tap sample to monitor adult psyllids and methods of assessing flush density, and infestation rates in blocks of growing and dormant citrus trees of different ages and varieties planted at experimental and commercial groves. Tap sampling, sticky traps, and sweep nets were compared for sampling low and high density populations of psyllid adults as maintained in several insecticide treated and untreated blocks. A tap sample consists of 3 consecutive strikes on a randomly chosen branch using the hand or a PVC pipe, and counting fallen psyllids on a laminated white sheet of paper held 1 ft below the foliage. At low density populations, adult psyllids per trap per tree on ‘Yellow Corn Rootworm Trap’ (YCRW), and ‘ACP Trap’ hung within the canopies of citrus trees in a commercial grove over a two week period averaged 1.8 ‘ 0.3 and 1.1 ‘ 0.02, respectively, compared to 0.08 ‘ 0.04 per tap sample made once per tree at the end of the two week period. Averages of 9.3 ‘ 1.2, 4.8 ‘ 0.6, and 0.5 ‘ 0.1 were obtained for YCRW trap, ACP trap, and tap sample, respectively, using the above procedure at the high density. Sticky traps have a high cost (approx. $ 1 per trap), provide data after at least a 1-week delay and require an average 7 minutes to deploy and collect data from one trap compared to an average of 3 sec for a tap sample that provide instant information. Sampling conducted at the experimental grove of Southwest Florida Research and Extension Center used two tap samples and two sweeps on the same tree. Significantly more adults were detected using tap sample compared to the sweep net at low density with means of 0.1 ‘ 0.02 and 0.06 ‘ 0.01 per one tap or sweep sample per tree, respectively. Means of 0.3 ‘ 0.1 per tap and 0.4 ‘ 0.1 per sweep sample per tree were obtained at high density and were not significantly different. Time to conduct one sweep (3.1 ‘ 0.04 sec) was significantly more than to conduct one tap sample (2.9 ‘ 0.04 sec) at low density but did not differ between the two methods at high density and averaged 3.4 ‘ 0.1 sec. 2) Test the influence of these parameters on precision of estimated means and the distribution of the population within blocks using the appropriate aggregation and regression models Regression analysis based on bootstrapping of the first set of low density data indicated that to obtain an SEM:Mean ratio of 0.1, considered necessary for research purposes, numbers of YCRW traps, ACP traps, and tap samples needed would be 130, 219, and 1929, respectively, compared to 75, 74, and 66, respectively, for the high density data. For a ratio of 0.25, considered necessary for routine monitoring, required numbers of YCRW traps, ACP traps, and tap samples were 16, 43, and 267, respectively, at low density, and 12, 13, and 8, respectively, at high density. For the second data set at low density, the required numbers of tap or sweep net samples were 554 and 1281, respectively, for a SEM:Mean ratio of 0.1 and 98 and 158, respectively, for a ratio of 0.25. Therefore, the sweep net could take less time to provide the same data but could also involve more work and difficulty in counting at very high densities and could spread citrus canker. 3) Evaluate and integrate methods for assessing these parameters into a user friendly system accessible to consultants and managers. We made several presentations and published to reach consultants and managers. Several of our presentations, publications, and details of tap sampling procedure are available at our website: http://www.imok.ufl.edu/entlab/. A survey of 27 groves conducted by UF-IFAS and Multi-County Cooperative Extension reported 95% growers use tap sample along with flush observations (76%), sticky traps (14%), and sweep net (9%), with 85% sampling > 3 times a year.
The main focus thus far has been to improve the current media to increase growth of the group of bacteria known as Candidatus Liberibacter. Our main focus for improving the growth has focused on understanding the phloem chemistry of citrus. A phloem analysis of sweet orange was completed by UC Davis MetaCore facility using GC/MS. Along with the genomic metabolic analysis of the available Ca. L. asiaticus genome, several key growth factors were indicated for further testing. Several nitrogen sources were tested as additions to the published media; ethanolamine has shown to improve initial growth. The seven micronutrients were tested with only the addition of zinc showing any improved growth. None of the amino acids or carbohydrates tested thus far have shown any improved growth, but several remain to be added to the media. Our current focus is to combine the different metabolites that show improved growth to optimize the media. In addition to these metabolites, sterilized fresh juice from uninfected plants was added to media in place of the citrus vein extract. We found that bacteria isolated from sour plants (lemons, limes) grew better on plates with sour juice compared to sweet juice and vice versa. From this information, we plan to do phloem analyses on multiple species and cultivars of citrus to identify commonalities and differences in the phloem chemistries. In collaboration with Ft. Pierce, FL, the testing of Koch’s postulates under field conditions began this quarter. A Florida-derived culture of Ca. L. asiaticus was initiated at Ft. Pierce last quarter. Inoculations using original isolations and transfers occurred this quarter. Symptoms are not expected for several months, but we will use RT-PCR assays to determine if Ca. L. asiaticus is present in the next quarter. The second focus has been sequencing the genomes of the three Liberibacter species from pure culture. In cooperation with the National Center for Genome Resources (NCGR), we used Illumina sequencing to sequence the strain used in the published pathogenicity trials (China1 Ca. L. asiaticus). The current genome is in the alignment phase and we plan to release the sequence in the third quarter. The second strain used in the pathogenicity trials (Brazil1 Ca. L. americanus) is ready for shipment to the NCGR and sequencing will begin once the alignment of China1 is complete. Currently we are ensuring the quality and purity of the representative strain of Ca. L. africanus (Africa2).
This work will determine if certain alternative hosts 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 HLB infection once again was achieved by graft transmission from HLB (Ca. Las) infected citrus to Severinia buxifolia (orange boxwood) and to C. jambhiri. Quantitative real time PCR (qPCR) was done on both hosts and showed that they were very good hosts for the Las bacterium. Psyllid transmission tests from Severinia to sweet orange were repeated this quarter and will be PCR tested after the appropriate incubation period. Additional infections were achieved with rootstocks Carrizo citrange, Swingle citrumelo and Kinkojii. At the Texas A&M Citrus Center, Weslaco psyllid feeding tests continued on the rutaceous plants that are established there. Esenbeckia berlandieri (jopoy), Amyris madrensis (torchwood), Choisya ternata and C. arizonica all were found to be feeding hosts for the psyllid. Egg laying was found on torchwood and egg laying and nymphal development were found on C. ternata. This work is part graduate student Jose Sandoval’s researchwith Dr. da Graca. Plant materials of Severinia buxifolia and Murraya paniculata were established at the USDA, ARS, Beltsville quarantine greenhouse and inoculations are pending with exotic Ca. Las, Ca. Lam and Ca. Laf isolates. Work with dodder as an alternative host to study the plant infection process and for its use with plants that are not graft compatible with citrus was concluded and a manuscript was submitted for publication. Dodder becomes infected and phloem necrosis occurs similar to that found in citrus. At the USDA, ARS, FDWSRU, Ft. Detrick, MD a manuscript was completed and submitted for publication on the work of infection of Murraya paniculata and the psyllid transmission to sweet orange. A presentation will be made at the HLB-Zebra Chip workshop in Weslaco in November.
The goal of the proposed research is to understand how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease on citrus. Citrus HLB is the most devastating disease on citrus. There are very few options for management of the disease due to the lack of understanding of the pathogen and citrus interaction. Understanding the citrus and citrus HLB pathogen interaction is needed in order to provide knowledge to develop sustainable and economically viable control measures. We proposed to expand our current research to different citrus varieties which are either susceptible or tolerant to HLB and a more facile model system tobacco (Nicotiana tabacum Xanthi). The specific objectives of this proposal are: transcriptional and microscopic analyses of citrus varieties which are either susceptible or tolerant to Ca. L. asiaticus infection at different infection stages in greenhouse and citrus grove; and transcriptional and microscopic analyses of host response to Ca. L. asiaticus infection with a model system tobacco. Microarray analysis and/or suppressive subtractive hybridization libraries approaches will be used to study the host response to the HLB pathogen infection followed by confirmation with Northern blot or quantitative reverse transcriptional PCR. Anatomical study will be performed with light microscopy using different staining methods. The following objectives related to this project were pursued: (A) An initial microarray analysis of host response of sweet orange to Las infection in greenhouse and in field; and (B) A preliminary study of the effects of HLB on phloem at the microscopic level and phloem transport of sweet orange. The results of these experiments are detailed below. Major achievements: 1. Microarray analysis of host response of sweet orange to Las infection in greenhouse. The results have been published in the following paper: Kim, J., Sagaram, U.S., Burns, J. K., and Wang N*. 2009 Response of sweet orange (Citrus sinensis) to Candidatus Liberibacter asiaticus infection: microscopy and microarray analyses. Phytopathology 2009 99:50-7. 2. To further expand our current understanding of Las-host interaction, we are currently comparing two susceptible and two resistant hybrid/cultivars in greenhouse. Currently, we have collected samples for more than 4 months for the two susceptible varieties including grapefruit and Madam Venus. The two resistant/tolerant varieties have not been inoculated successfully. 3. Leaf samples from different varieties including grapefruit, Murcott, and Hamlin were collected from Florida citrus groves. Both healthy and infected trees were sampled. It is expected gene expression of those trees in the citrus grove will reveal more information when compare with the gene expression profile in greenhouse. Those samples will be used for gene expression analysis using microarray or SSH approaches. 4. Gene expression of Valencia in citrus grove have been done. The data is under analysis.
Citrus Huanglongbing (HLB) is one of the most destructive diseases on citrus. Very little is known about the causal pathogen and the microbiome associated with the infected trees. The goal of the proposed research is to characterize the bacteria (endophytes and plant pathogens) associated with HLB positive citrus, the Asian citrus psyllid, dodder, and periwinkle. The specific objectives of this proposal are: (1) Comparison of the microbiomes in leaf midribs and roots of HLB pathogen positive and free citrus of different varieties; (2) Characterization of the microbiomes that can be transferred among citrus, psyllid, dodder, and periwinkle; and (3) Understanding how Candidatus Liberibacter asiaticus colonizes and moves inside the phloem. Comparison of the microbiomes associated with HLB pathogen positive and negative citrus will illuminate the causal agent of citrus greening. Potential beneficial endophytic microorganisms could be identified from escape plants which survived in heavily infected citrus groove with HLB. Beneficial microorganisms have been shown in previous studies to have the capacity to control plant diseases by accelerating seedling emergence, promoting plant growth and development, and preventing the invasion of plant pathogens. The investigation of the microbiomes associated with different hosts will help understand the transmission of microorganisms between different hosts. Major achievements: 1. A comprehensive study of the bacterial diversity associated with healthy and HLB diseased citrus indicated that Candidatus Liberibacter asiaticus as the pathogen responsible for HLB disease in Florida. Phytoplasma was not found in any of the samples collected from Florida. The result has been published in the following publication: Uma Shankar Sagaram, Kristen M. DeAngelis, Pankaj Trivedi, Gary L. Andersen, Shien Lu, and Nian Wang 2009 Bacterial diversity analysis of Huanglongbing pathogen-infected citrus using PhyloChips and 16S rDNA clone library sequencing. Applied and Environmental Microbiology 2009 75: 1566-1574. 2. Samples (leaf and root) from different varieties including grapefruit, Murcott, and Hamlin were collected from Florida. Both healthy and infected trees were sampled. Bacteria associated with the root system will be studied comprehensively. 3. Samples (leaf and psyllid) from three different locations were collected. The bacterial diversity study of the HLB infected trees and the psyllids in those groves will reveal potential competitive microorganisms for the control purpose. 4. Isolation of bacteria with the potential of plant growth promoting and biological control potential might reveal innovative ways controlling the HLB disease. Fifty-four morphologically distinct isolates were obtained from surface sterilized roots of symptomatic and asymptomatic (potential escape trees) citrus plants from a citrus grove with a HLB infection rate of more than 60% and an infection history of approximate five years. Qualitative screening showed that for all of these criteria, asymptomatic plants harbor a significant greater diversity of potentially beneficial bacterial strains. 5. Development of a proper in vitro screening system that provides repeatable and reliable results in shorter periods of time is an important step for isolation of efficient bacterial antagonists. We have developed a method to screen antagonistic bacteria against unculturable HLB pathogen. The method uses the discrimination of live-dead cells by EMA and speed and sensitivity of QPCR. Six bacterial isolates were found to reduce the number of viable Las cells.
The research activities during this period were working on objectives; (1) Identification of Simple Sequence Repeat (SSR) loci and validating SSR markers designed from genome sequences of “Candidatus Liberibacter asiaticus” (Las). (2) Analyses of global genetic diversity of HLB Las strains in American and Asian continents (U.S. Florida, Brazil, China and India) using new multilocus SSR markers. Followings are the summary of research activities and accomplishments during this period. 1. Extensive genome wide sequence search resulted in identifying 58 new loci containing perfect, imperfect or compound SSR repeats that can be potentially useful for designing SSR markers. Most of these loci are either located in or next to the Open Reading Frames (ORFs). Molecular Beacon software v7.0 was used to design SSR primers with the same criteria reported earlier. 40 new SSR primers were evaluated by PCR experiments. Those primers produced single clean bands with expected amplicon sizes were further evaluated their polymorphic power by using HLB asiaticus strains collected from U.S. Florida and other geographic regions. Amplified products were separated by the high resolution of 5% polyacrylamide sequencing gels. Primers that passed the test were synthesized with 5Õ-labeled fluorescent dyes (FAM, NET, VIC) and run by an ABI 3130 Genetic Analyzer. 2. Genetic diversity analyses were conducted for HLB Las strains collected from Florida, Brazil, China and India. These included 43 samples from 14 counties in Florida, 35 samples from 15 cities in Brazil, 30 samples from 8 areas in northern and southern India and 58 samples from five southern provinces in China. Five SSR primers were selected for this set of data, Preliminary data showed that polymorphic allele types ranged from 3 to 18 in five SSR loci among total 166 isolates with average of polymorphism 34% in Florida, 32% in Brazil, 52% in China and 57% in India, respectively. Relative lower genetic diversities detected in Florida and Brazil (34% and 32%) reflect recent introduction of HLB in south and north American continent while higher genetic diversities in India and China (57% and 52%) account for the long history of the evolutionary process resulting in differentiation of these populations. These results also demonstrated that the multi-locus SSR molecular markers developed in this project are a useful tool for detection and population genetic analysis for HLB Las.
Citrus Huanglongbing (HLB) is the biggest threat to the Florida citrus industry. Florida accounted for 70% of the total U.S. production in 2006-07. HLB, known as citrus greening, can debilitate the productive capacity of citrus trees with losses of 30-100% reported. HLB was found in Florida in 2005 and has spread throughout thirty of the citrus producing counties in Florida by January 2008. The overall goal of this proposed work is to characterize the virulence mechanisms of Candidatus Liberibacter asiaticus (Las), the citrus Huanglongbing (HLB) pathogen, thus to come up with new management strategies by genome sequencing and functional genomics approaches. The original goal of the proposed research is to further complete the genome sequencing of Candidatus Liberibacter asiaticus, for which a draft sequence is available. The goal was modified to meet the current progress in genome sequencing of Candidatus Liberibacter asiaticus with the advice and permission from program manager of FCPRAC. The tile has been changed to the following to better suit the goal: Understand the virulence mechanism of Candidatus Liberibacter asiaticus by genome sequencing and functional genomics approaches. Comprehensive metabolic reconstruction is being used to further understand the biology of Liberibacter asiaticus and identify potential genes for targeting using small molecules and other chemicals. Bioinformatics analysis was performed to identify potential virulence factors. The SignalP v3.0 program was used to predict the presence of signal peptide within the proteins. The secretomeP 2.0 program was used to predict the non-classical secretion proteins without signal peptide. ORF containing transmembrane domains was predicted by TMHMM2.0 program. Smart and other programs were used to further mine the genome sequence of Candidatus Liberibacter asiaticus. The genes CLIBASIA_03170 and CLIBASIA_02180 were cloned into the vector TMV30bGFP by PCR amplification and digestion with the restriction enzymes Pac1 and Xho1. The insertion of the two genes in the TMV vector were confirmed by PCR amplification using gene specific primers, followed by sequencing. CLIBASIA_01555, CLIBASIA_00965, CLIBASIA_02215, CLIBASIA_03975, CLIBASIA_02975, CLIBASIA_00880, CLIBASIA_00470, CLIBASIA_05460, CLIBASIA_05315, and CLIBASIA_02120 were cloned into pGEMT-easy vector. The insertion was confirmed using PCR with gene specific primers, and will be sequenced for further confirmation. In vitro transcription and plant inoculation: CLIBASIA_03170 was inserted into TMV30bGFP, and the vector control TMV30bGFP were linearized using kpn1 and in vitro transcription was done using T7 RNA polymerase. The transcript was then used for inoculation of 4-5 week old Nicotiana benthamiana plants. The plants were observed for symptom expression about 7 days after inoculation. The symptoms shown by the empty vector and the vector containing CLIBASIA_03170 were being monitored.
Administrative arrangements for funding the research and recruitment activities for staff were completed in the previous reporting period. During this reporting period Dr. Yuan Qing arrived from China August 10th, and began to work on the project. Citrus psyllids were obtained from collaborators at the University of Florida and at Ft Detrick, MD. These were both known to be infected with Ca. Liberibacter and field collected insects. These insects were used to work out details of the extraction protocol to be used to inject mice with Ca. Liberibacter asiaticus obtained from citrus psyllids. Two methods of grinding the psyllids to obtain Ca. Liberibacter were compared: Liquid nitrogen with ceramic mortar and pestle with resuspension in buffer or plastic microfuge tubes with plastic pestle in extraction buffer. The latter method gave better results as measured by q-PCR. These results were improved by the addition of a small amount of sand to the extraction buffer, which resulted in a finer grind of the insects. We also compared low speed centrifugation to simply letting sediments settle out by sitting on the bench in ice on the yield of Liberibacter in these extracts. Allowing sediments to settle under gravity gave better results than centrifugation at only 100 RPM. Therefore the protocol to obtain Ca. Liberibacter asiaticus from infected psyllids will be: grind in extraction buffer with sand in microfuge tubes, followed by allowing sediments to settle under gravity. Permits were obtained to use an animal care facility to maintain the mice needed for the immunization experiments. Mice were also obtained and injected to produce antibodies for this project. For our first experiment we used Xylella fastidiosa (CVC strain) instead of Ca. Liberibacter. The X. fastidiosa was added to psyllids and treated as above, then injected into mice. This will allow us to work out the complex process of antibody library creation and screening with a cultured, Ôpositive controlÕ, before we begin to work with the non cultured Ca. Liberibacter. Antibodies against the CVC strain of X. fastidiosa would constitute a ÔbonusÕ from the grant. All of the reagents and primers needed to construct the antibody library have been obtained. Antibody libraries will be created early in the next reporting period. USDA ARS at Beltsville has also accepted from the contractor (9/25/09)a new Insect Quarantine Facility which we will use to raise psyllids for use in these experiments. Permit from APHIS to operate the facility is pending approval. We also obtained cloned genes encoding the outer membrane protein (omp) of Ca. Liberibacter asiaticus from Duanping Yuan. The OMP protein has been over expressed in E. coli and has been purified. We will raise antibodies against it to use them to capture Ca. Liberibacter cells from psyllid extracts if our initial methods are not successful. Dr. Yuan also will receive antibodies against this protein from us when available.
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. 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 outplanted 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. 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. 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 to start the experiment within the next 1-2 months. 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. Thus data collection is currently ongoing.
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 canÕt 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. 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 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 againse actual data to ensure correct estimation of disease dynamics.
The Objectives are to: 1) Test the effect of soil applied imidacloprid as an inducer of SAR on HLB disease incidence in newly planted citrus trees subjected to psyllid mediated infection; and 2) test the effect of imidacloprid on HLB disease progress in newly planted citrus trees that are graft-inoculated with budwood from HLB infected field trees. The following treatments were set up in the USDA-ARS, Picos Farm in Ft. Pierce FL: 1) untreated check, 2) foliar insecticide to control psyllids, 3) soil applied imidacloprid/thiamethoxam to induce SAR, 4) soil applied imidacloprid/thiamethoxam plus the foliar insecticide to induce SAR and control psyllids, 5) HLB bud graft-inoculated untreated check, 6) HLB bud graft-inoculated with soil applied imidacloprid/thiamethoxam to induce SAR. The experimental design is a completely randomized block design with 50 trees per treatment (5 blocks of 10 trees per block). Tree and insecticide treatments commenced in May and HLB graft inoculations occurred in July. HLB-SAR experiments of similar design will be set up in Parana, Brazil in October (spring for southern hemisphere).
Under Objective 1 (evaluate the potential for soil application of the neonicotinoids, imidacloprid and thiamethoxam, and acibenzolar-s-methyl, to provide long-lived SAR control of canker in young trees) two trials in 1 and 3 yr old grapefruit in Ft. Pierce, FL have been set up with 14 treatments in each location. Soil applied SAR treatments have significantly reduced foliar disease incidence compared to the untreated check. A trial with complementary soil and trunk applied treatments in Brazil has produced similar canker control effects. Under Objective 2 (integration of SAR with foliar applications of reduced rates and frequency of copper sprays for control of canker on the most susceptible cultivar in Florida, grapefruit) a preliminary trial in young fruiting trees was set up to compare trunk and soil applications of imidacloprid at 2x the label rate to compensate for the larger tree volume. SAR control of canker on foliage was equal to that of 21 day sprays of Kocide 3000, but control on fruit was less effective than the protective effect of copper. This suggests that SAR and copper can be used in an integrated program for canker control in young fruiting trees. Under Objective 3 (evaluation of the complementation of the use of neonicotinoids thiamethoxam and imidacloprid to increase and/or extend insect and disease control) are being addressed within the treatments of the two trials of non-bearing trees set up in Florida.
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