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.
Under Objective 1 (Survey and confirmation of HLB in seedlings from HLB-affected trees) 200 seeds extracted from mild to severely affected fruit from HLB-affected Pineapple orange and Murcott tangor groves were assayed for HLB detection in emergent seedlings. All seedlings were negative for HLB by PCR assay. Assay of the same seedlings will be repeated. Under Objective 2 (Thermotherapy of HLB-affected seed) 100 seeds of the same seed sources were treated at 125, 130 and 135F to test for the effect of heat treatment on detection of HLB in seedlings. Thermal treatments were lethal to Murcott seed, but not Pineapple seed. The emergent seedlings were PCR tested and all were found to be negative. Under Objective 3 (Follow-up on HLB-positive trees from DPI annual survey), DPI survey of seed source trees in Florida nurseries is conducted every other year (R. Gaskalla, DPI Director, personal communication), so samples from the survey, if any, will not be available for testing until the second year of the project.
Since the last update, we focused on characterizing callose accumulation in the Liberibacter-infected phloem tissues. First, we localized callose in the infected phloem tissues by immunogold labeling. The main purpose of the experiment was to verify whether callose over-accumulation is responsible for the swelling of the plasmodesmata pore units (PPUs). We utilized a monoclonal callose-specific antibody from Dr. Bacic. This antibody has been the favorite callose antibody in the plant cell wall community during the last two decades thanks to its specificity and low background (Meikle et al., 1991). The swollen PPUs were intensely labeled by callose-specific immunogold particles indicating an excessive callose buildup. In addition to the PPUs, immunogold particles were associated with plasmodesmata between the sieve elements and with sieve pores. An interesting observation is that the inner surface of the sieve elements was covered with callose. In uninfected samples, callose was detected only in the plasmodesmata and sieve plates where callose is naturally deposited. However, numbers of associated gold particles were far smaller (less than 30%) than those in the infected samples. Callose accumulation was not seen in non-phloem cells such as mesophyll sponge cells and parenchyma cells in both infected and uninfected samples. These results indicate that sieve element cells synthesize callose in response to Liberibacter infection and the callose often swells plasmodesmata of the cells. Recently it was reported that sieve plate pores are plugged by callose in Liberibacter infected sieve elements (Kim et al., 2009). The sieve pore plugging is the first wound reaction against phloem damages, so that plants do not lose their precious photosynthetate (Ehlers et al., 2000). We examined sieve plates from infected and uninfected mid-vein samples by scanning electron microscopy (Mullendore and Knoblauch., 2009). The advantage of this microscopy technique over TEM is that cell wall structures including sieve plates, plasmodesmata, and callose are seen in three-dimension, facilitating comparison sieve pore size and callose accumulation in the phloem. In the face-on views of sieve plates, diameters of sieve pores in the infected samples were smaller than those of uninfected sieve plate pores. However, we have not found any sieve pores that are completely blocked by callose in the infected phloem samples. The pore sizes in the infected sieve plates were still larger than the width of regular Liberibacter cells. These data suggest that callose synthesis in the sieve plates is not efficient enough for completely sequestering Liberibacters to their infection sites. For the next quarter, we will investigate callose distribution by fluorescence microscopy to combine with what we have learned from high resolution electron microscopy imaging. We will also finish 3D reconstruction of Liberibacter cells and plasmodesmata deformed by callose from serial sections of citrus sieve element samples. (We have collected 3 serial sections from citrus sieve element cells.) Ehlers, K., Knoblauch, M., and van Bel, A.J.E. (2000). Ultrastructural features of well-preserved and injured sieve elements: minute clamps keep the phloem transport conduits free for mass flow. Protoplasma 214, 80-92. Kim, J.S., Sagaram, U.S., Burns, J.K., Li, J.L., and Wang, N. (2009). Response of sweet orange (Citrus sinensis) to ‘Candidatus Liberibacter asiaticus’ infection: microscopy and microarray analyses. Phytopathology 99, 50-57. Meikle, P.J., Bonig, I., Hoogenraad, N.J., Clarke, A.E., and Stone, B.A. (1991). The Location of (1-]3)-Beta-Glucans in the Walls of Pollen Tubes of Nicotiana-Alata Using a (1-]3)-Beta-Glucan-Specific Monoclonal-Antibody. Planta 185, 1-8. Mullendore, D. L. and Knoblauch, M. (2009). A new method to investigate the cell wall of living cells by high-resolution scanning electron microscopy. Annual Meeting of American Society of Plant Biologists Abstract number: P14001.
The goal of this study is to introduce broad resistance in citrus against HLB and other diseases via manipulating salicylic acid (SA)-mediated defense signaling. Specifically, we proposed to identify SA-related genes in citrus and manipulate expression of these genes genetically to elevate SA levels and/or signaling in citrus and subsequently test the transgenic citrus plants for resistance against HLB. Towards this goal, we have three specific objectives and have made the following progresses since last report. Objective 1: Identify genes positively regulating SA-mediated defense in citrus In last report, we described the identification of citrus SA homologs via bioinformatics analysis and the cloning of the citrus NPR1 (ctNPR1) gene. Now we have cloned three additional full-length citrus SA genes, ctEDS5, ctNDR1 and ctPAD4, using combination of RT-PCR and 5Õ and 3Õ RACE approaches. These three new SA genes were cloned in the pGEM vector. To provide a more detailed analysis of expression of citrus SA regulators, we infected eight-month-old trees with ÔValenciaÕ orange (Citrus sinensis (L.) Osbeck) scion on Cleopatra mandarin (C. reticulata Blanco) rootstock with Ca. L. asiaticus and are in the process of collecting tissue for RNA extraction followed by RT-PCR. At five-week post inoculation, 14 out of 30 inoculated plants were PCR-positive for Ca. L. asiaticus but no plant showed any leaf symptoms of HLB. At 11-week post inoculation, 24 out of 30 inoculated plants were PCR-positive for Ca. L. asiaticus, among which 17 plants displayed disease symptoms. Control plants were always PCR-negative and did not display any leaf symptoms. Additional time point, about 14-week post inoculation, will be used to collect leaf tissue from these plants. Objectives 2: Complement Arabidopsis SA mutants with corresponding citrus homologues We cloned ctNPR1 in the binary vector pBINplus/ARS and already transformed Arabidopsis mutant npr1-1 and the wild type control Columbia with the ctNPR1/pBINplus/ARS construct. The T0 seeds will be harvested in about a month followed by selecting transgenic plants and possibly also testing disease resistance in the next generation. In addition, we are currently in the process of cloning ctEDS5, ctNDR1 and ctPAD4 into pBINplus/ARS for a complementation test of Arabidopsis eds5-1, ndr1-1, and pad4-1 mutants, respectively. Objectives 3: Assess the roles of SA regulators in controlling disease resistance in citrus While we are waiting for the result of a complementation test for ctNPR1 in Arabidopsis, we have also placed the ctNPR1/pBINplus/ARS construct in the pipeline of transforming citrus.
The new spectrophotometer, a Nanodrop 8000, has just arrived. The software will be mastered and the task of adjusting the nucleic acid content of samples prior to running qPCR will begin. This is very important to the testing component of providing clean Shoot-tip grafts of new cultivars. Samples vary in nucleic acid content. Too much nucleic acids can cause inhibition, whereas too little can reduce the chance of detecting the pathogen. The greatest differences are in dry field trees compared to young tender greenhouse tissue. The first of the two microscopes, a Leica teaching microscope arrived, but during installation, it was discovered that the light source had a European plug, the light source did not fit on one of the objectives and neither objective could fit the body of the microscope (which was a newer model than the objectives). These errors should be rectified within two weeks. Meanwhile, we now are sure of the dimensions of this large microscope and plans are under way to purchase a hood to use the microscope in instead of just using it on a table. Teaching will take place in a sterile environment and the shoot-tip grafts (STGs) produced will be kept and evaluated so that the success rate of the work done can be determined. With an additional microscope and hood space, the STGing capability could also be increased when needed. During this quarter, the number of selections being cleaned up is down to 93. Four new selections have been added to the list and 22 selections were released. Four hundred seventy six STGs were set up. These represented 12 varieties including two rootstocks, two breederÕs selections, two private growersÕ selections, 2 difficult varieties, two with mild isolates of Citrus tristeza virus (CTV), and two with severe isolates of CTV. During this same time period, 36 successful STGs were grafted onto rootstocks in the greenhouse. These represented 16 varieties (which included two rootstocks, four breederÕs selections, three private growersÕ selections, two difficult varieties, two Florida Citrus Arboretum varieties, one which had a mild isolate of CTV and two which had severe isolates of CTV). For testing, over 58 STGs and parents samples were extracted and 252 real-time PCR tests were performed on STGs that grew to sufficient size in the greenhouse. One hundred forty trees were budded for increase from tested original STGs to either be planted in the Citrus Budwood Foundation at Chiefland and/or to be given back to the breeder/owner. Eighty-nine propagations were planted at Chiefland, 85 of these were breedersÕ selections. The Citrus tatter leaf virus SYBR green real-time PCR assay has been found to be robust and has been incorporated into the comprehensive qPCR testing for STGs and parents.
The two primary objectives of the current proposal are: (1) determine and compare the metagenomes of healthy and infected citrus phloem, and (2) continue to develop and apply AthenaBio’s cultivation technologies towards producing a pure culture of Ca. Liberibacter. Objective 1 Discussion. Laser cutting microdissection (LCM) will be developed to purify phloem-containing sieve cells from both healthy and infected citrus samples. DNA extracted from the purified cells will be analyzed by 16S rRNA sequencing to evaluate the contained microbial diversity. The most diverse DNA samples will be selected for large-scale metagenomic analysis. All DNA extraction and sequencing work will be performed by Dr. Ravel’s group (U. of Maryland) Objective 2 Discussion. AthenaBio will continue to develop and apply its cultivation technologies to isolate a pure cultures of Ca. L. species. In addition to Ca. Liberibacter species, we aim to cultivate other phloem endophytes to provide reference genomes for metagenomic analysis. Two approaches will be employed to disrupt quorum sensing (QS) and thereby enhance growth: (1) physical methods (i.e. a ‘flow cell’) will be used to wash QS signals away from cells, and (2) enzymes will be used to inactivate QS signals through chemical modification. The advantage of the flow cell approach is that knowledge of the chemical structure of the QS signal is not required, and this approach is therefore expected to work on a broad variety of QS systems. Both approaches (flow cell and enzyme) will be combined with various media formulations and incubation conditions. Before work could begin, a research agreement was formulated between the FCPRAC and Athena Biotechnologies, Inc. This agreement was finalized in May 2009 and work has begun to develop a method for LCM of citrus phloem cells. Healthy leaf tissue was dissected into small pieces (~5mm), immediately placed in tissue freezing medium and placed on dry ice. Sample sections were achieved using a Leica CM3050 S Cryostat and sections were cut using a Laser Capture Microdissection Microscope (LCM). We are currently optimizing section thickness and staining procedures to obtain ideal conditions for LCM processing. Once an optimum protocol has been developed, we will begin processing samples for DNA analysis.
Differential mobility spectrometer sensor for HLB-biomarker detection: Sampling protocol to collect volatile organic compounds (VOCs) from citrus leaves in open-air environment (field conditions) and analysis protocol (for GC-MS and differential mobility spectrometer-GC/MS and GC/DMS) have been developed jointly in Dr. Davis’s lab, UC, Davis and Dr. Rouseff’s lab ,UF, CREC . Sampling and analytical protocols have been exchanged between the two labs. A special positioning devise has been developed to hold the solid phase microextraction fiber for field volatile sampling. Optimal sampling time of day has been determined. Two different fiber types have been evaluated and major leaf volatiles identified. The major leaf volatiles have been identified as terpenes, aldehydes and alcohols which have been previously reported in citrus. In one sample set, the GC/MS chromatograms showed certain distinguishable patterns between healthy and HLB samples. An auto-regression model with an order of 100 was applied to extract features (auto-regression coefficients) from the chromatogram in Dr. Davis’s lab. The algorithm was able to classify the healthy from HLB-infected samples, and asymptomatic-HLB from symptomatic-HLB infected samples. In addition to these tools, other sophisticated machine learning and feature extraction (biomarker detection) approaches such as wavelet analysis, genetic algorithms, and ant colony algorithms have been developed. Portable fluorescence spectrometry for HLB detection: A laser-induced fluorescence spectrometer system was developed by Embrapa Agricultural Instrumentation for diagnosis of Brazilian citrus disease. Some preliminary experiments were conducted using the biophotonic system in the laboratory to classify healthy citrus leaves (from Valencia grafted on Swingle rootstock) from that of HLB infected leaves. The study was carried-out to calibrate the instrument. Preliminary analysis showed that the system was able to classify HLB-infected leaves with 90% classification accuracy, while healthy leaves were classified with an accuracy of 70%. The results were promising. A partnership has been set-up with Fisher Group (one of largest Brazilian producers of oranges and juice) for data collection in Citricola Farm. Hundred trees in two blocks of 1000 trees (one with low and high HLB incidence each) will be monitored using the biophotonic system each month, with data collection on weather, geo-reference, and PCR results. Data collection is expected to start on November, lasting upto a year. Hyperspectral sensors for HLB-infected trees detection: Different imaging sites having various degrees of HLB infection have been evaluated for HLB detection through aerial hyperspectral imaging. The sites selected were from citrus groves in Southweat Florida Research and Education Center (SWFREC) in Immokalee, Consolidates Citrus, and Evans Property. The hyperspectral imagery will be collected from November, 2009 with the help from Dr. Yang and imaging group at US Department of Agriculture-Agricultural Research Service, Weslaco, Texas. A photographic camera with high resolution, a multispectral sensor, and hyperspectral sensor will be used to collect the data. Greenhouse experiments are being conducted at Citrus Research and Education Center, Lake Alfred, FL to evaluate the performances of the spectroradiometer and hyperspectral imaging based sensors for their ability to detect HLB infected trees in their preliminary stages. The data collection is ongoing since last year. Analysis protocols are being developed to evaluate the classification accuracies of the sensors. Preliminary results indicated the requirement of large dataset to classify HLB-infected trees with high accuracies. Further data will be collected to evaluate the applicability of the sensor techniques.
The objectives of this project were to evaluate the effects on flush management strategies on the population densities of the Asian citrus psyllid (ACP; Diaphorina citri Kuwayama). Two hedging dates (one early in mid February and one late in mid April) and two nitrogen fertilization regimes (one application of 100 lb/acre in February and two applications of 50 lb/ac each in mid February and in mid June) were tested in a factorial design in a mature ÔMarrsÕ sweet orange block near Weslaco, South Texas. Non-hedged and non-fertilized plots served as controls for each treatment factor. Weekly counts of new flush shoot growth and ACP densities were made. Both hedging dates and application of nitrogen significantly affected ACP infestation and densities on citrus flushes. Hedging significantly altered the phenology and intensity of new flush shoot production on trees. In the non-hedged blocks, the spring flush was only followed by a very light flush shoot production in early June. In contrast, both the early and late hedging dates of trees stimulated profuse flush shoot production in June. In addition, in the late hedging treatment of April, new flush growth started in early May and lasted until late June. This alteration in flush shoot availability also impacted the population dynamics of ACP. Significantly higher ACP infestation levels and densities were recorded in the late hedging date compared to the other treatments. Although more new flush shoots were produced in the early hedging treatment relative to the non-hedged treatment, ACP populations were comparable in these two treatments. These results clearly demonstrate that early hedging should be encouraged as it prevents severe outbreaks of ACP populations, while providing the intended physiological benefit of the practice. By contrast, late pruning in spring i.e., during the active ACP growth period, will likely lead to ACP outbreaks in citrus orchards. Application of nitrogen also affected the abundance of new flush growth. Although no alteration of flush cycles resulted from N application, densities of new flush growth were higher in fertilized plots than in the non-fertilized control blocks. The effect was more dramatic in blocks where N was applied in a single dose (100 lb N/ac) compared to blocks receiving split N applications. In the one-time N application treatments, significantly more new flush shoots were produced which resulted in higher densities of ACP eggs and nymphs for most of the sampling dates. ACP densities in the split application and non-fertilized control were similar throughout the sampling period. The interaction of N fertilization and hedging dates was significant for ACP immatures, but not for ACP adults. This indicates that changes in flush shoot production can directly affect the densities of ACP eggs and nymphs. Late pruning, combined with the one-time N application treatment resulted in some of the highest ACP population counts recorded. In summary, N management, and in particular, split N fertilization and early hedging/pruning of trees were associated with lower ACP population densities on sweet orange trees. These preliminary findings from ongoing studies have provided valuable information that orchard managers can use to implement strategies to lower the risk of ACP outbreaks in citrus orchards.
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