The purpose of this project is to preserve citrus germplasm in Florida that is threatened by loss due to huanglongbing (HLB) and citrus canker. Using input from stakeholders and scientists, a priority list of germplasm for recovery has been made of citrus germplasm in Florida. At USDA ARS USSHL Ft. Pierce the penicillin-streptomycin treatment shown to be effective by Zhang et al. has been widely used in the rescue of budwood in Florida. Vacuum infiltration into buds ready for propagation has been shown to have negligible phytotoxicity compared to non-treated control buds and has resulted in nearly 100% bud take. Sixty four unique hybrid advance selection field trees from the USDA citrus breeding program have been rescued by propagation into the greenhouse. Experiments led by the Yongping Duan lab, USSHL Ft. Pierce, have shown a 50X reduction in Clas from HLB-infected unique hybrid field trees. These trees will be tested as sources of budwood for rescuing further material in combination with antibiotic treatment of budwood and thermotherapy of young plants. Selected cultivars will be subjected to STG in Gainesville and forwarded to Riverside for further indexing. Several accessions have been received by the USDA ARS Repository, Riverside, CA and five are in final stages of therapy/indexing for release from quarantine. The USDA ARS Repository, from other sources of funds, has been able to purchase two growth chambers for thermotherapy use, a better microscope for shoot tip grafting, and to upgrade the greenhouse cooling/heating system to better extend the time period in which cool temperature biological indexing may be performed. This will improve the ability to perform the required biological indexes for release from quarantine following preliminary therapy of the targeted accession in Florida. Research is continuing on the use of antibiotics and/or cool temperature treatments to eliminate HLB from budwood, with citrus stubborn and tomato psyllid yellows being used as model systems in California. In cooperation with USDA ARS, Ft. Collins, CO, the use of cryotherapy to eliminate HLB is being explored.
Website Creation and Development As previously described, the CG-HLB Genome Resources website hosts a genome viewer for the purpose of providing a convenient interface for accessing Ca. Liberibacter genome features and analysis of predicted proteins (http://www.citrusgreening.org/HLB-GBrowse.html). The genome viewer is composed of 10+ “tracks” specifying various predicted features of the genome sequence. Public release of the genome sequence of Ca. Liberibacter solanacearum CLso-CZ1 and a sizable phage-associated region for Ca. Liberibacter asiaticus UF506 has led to incorporation of these additional sequences into the genome viewer along with predictions for the genome features found therein. Addition of new tracks and links to the genome viewer is ongoing as more bioinformatic analyses are performed. In addition, a continuously updated list of research publications on Liberibacter has been added as one of the additional resources provided by the CG-HLB website. Bioinformatic Analyses In conjunction with incorporation of the Lso CLso-CZ1 and Las UF506 phage sequences into the Liberibacter genome browser, more attention is being given to how sequence variation among strains and species might best be represented and used in design of diagnostic primers. Currently, regions of the Las genome determined to be unique relative to the Lso genome (E value cutoff of 1e-5) are shown in a track on the genome viewer. However, we are working on different approaches for displaying variation on a finer scale. This viewer is extendable to displaying variation among multiple strains as they become available. Outreach Information on the CG-HLB Genome Resources web site and associated genome viewer was presented at the 2nd IRCHLB research conference in Orlando Florida and contacts made with researchers sequencing additional Ca. Liberibacter strains. We are also assisting Dr. Wayne Hunter (ARS, Fort Pierce) with metagenome analysis of the Asian citrus psyllid with a particular focus on Wolbachia, the other alpha-proteobacteria associated with this insect. Regions of high similarity between Liberibacter and Wolbachia will be documented in the genome viewer and predicted metabolic capabilities compared between the two bacteria to evaluate the likelihood of complementary metabolism.
Objective 1. Localization of Liberibacter asiaticus (Las) in the Asian citrus psyllid (ACP). [A] Our Q-PCR study on dissected ACP organs will be published soon in the Annals of Entomological Society of America (2011, Vol. 104, in press). Our results provided the first PCR confirmation of Las in the alimentary canal and salivary glands of ACP and strongly suggested that the salivary glands constitute an important transmission barrier to Las in the psyllid vector. These results also suggested that Las may replicate or accumulate in both the alimentary canal and salivary glands of ACP. [B] We finished testing several fluorescent in situ hybridization (FISH) protocols with three Las-specific probes for the localization of Las in hemolymph smears and dissected organs of ACP that fed on HLB-infected citrus trees in the field or laboratory, and in sections from HLB-infected citrus leaves. Las was detected by FISH in the filter chamber, midgut, Malpighian tubules, hemolymph, salivary glands, ovaries and in muscle and fat tissues of HLB-infected ACP, as well as in the phloem of infected citrus leaves, but not in healthy control leaves or psyllids. FISH results indicated that the proportion of Las-infected salivary glands were significantly lower than that of infected alimentary canals or other organs. Our results showed the near systemic infection of ACP by Las and confirmed our previous PCR results suggesting that the salivary glands constitute a significant barrier to Las infection and/or transmission by ACP. Objective 2. Elucidation of various acquisition and transmission parameters between ACP and Las. [A] We finished two experiments to study the effects of various acquisition access periods on HLB-infected citrus plants, between 1 day and 2 weeks, on both acquisition and transmission of Las by ACP nymphs and adults, as well as on Las replication in both life stages. But we are still analyzing hundreds of individual ACP’s from these experiments with Q-PCR. Results will be discussed in coming reports. [B] We continued to develop and test a new ‘detached-leaf assay’ method that can potentially speed up Las-inoculativity tests on psyllids considerably by shortening this period from 3-12 months to 2-3 weeks. Young adults of ACP, reared as nymphs on HLB-infected citrus plants, were tested for inoculativity by caging them singly or in small groups (5 or 10 per leaf) in 50-ml polypropylene tubes on detached healthy leaves of sweet orange as described by Ammar and Hall (2011, Florida Entomol. 94:340-342). In two trials in which 10, 5 or 1 infected adult ACP per leaf were fed on detached leaves for 7 days, percentages of PCR-positive leaves were 40, 18.8 and 4.4% respectively, using Li primers, and 60, 40.6 and 11.1% respectively, using the more sensitive LJ900 primers. These results on Las-inoculativity of ACP on detached leaves using the more conventional and less sensitive Li primers are largely comparable to those recently reported for Las transmission when whole citrus seedlings are used for inoculation (Pelz-Stelinski et al. 2010, J. Econ. Entomol. 103:1531-1541). Our results suggest that detached citrus leaves can be used to test the inoculativity of single or small groups of ACP and that using more sensitive Las primers can increase the usefulness of this method. [C] Among ACP adults collected from HLB-infected citrus trees in Picos Farm (Fort Pierce, FL) since Feb. 2009, there have been no strong trends observed over time with respect to percentages of Las-infected ACP or titers of the pathogen. However, during late summer 2010, there were eight consecutive weeks in which percentages of ACP testing PCR-positive for the Las bacterium averaged below 10%. Statistical analyses indicated that increased rainfall and excessively high air temperatures during these weeks were significantly correlated with the lower percentages of Las-infected ACP.
Panelist could detect bitterness in HLB juice even though the levels of the bitter compounds were below their reported thresholds in most cases. Use of electronic nose and electronic tongue on processed orange juice samples was conducted on both Hamlin and Valencia juices from the last year. The juice had been frozen and was thawed for testing. The effect of nutritional spray systems (Keyplex, Diamond R and the Maury Boyd cocktail) were also investigated as it seems many growers are going to this system rather than scouting and removing trees. These nutritional programs are reported to reverse HLB symptoms on the tree but it is not known if they reverse symptoms on the fruits. So samples will be collected from healthy and HLB-affected trees from conventionally sprayed groves and those using the above nutritional sprays and analyzed for chemical and sensory differences. The data will be reported in the final report. There are differences in the volatile profile and in sensory perception of odors between healthy and HLB fruit juice, however the differences are much more subtle than the differences in taste. Differences in taste do correlate with limonin and nomilin values and lower sugars in both asymptomatic and also higher acids in symptomatic fruit, but more so overall in symptomatic fruit. The levels of limonin and nomilin, however, are generally below reported thresholds, even in Hamlin juice which has much higher limonin and nomiln levels than does Valencia, and even early in the season when these values are highest for both varieties. Threshold tests were done in several model juices and in Valencia juice for limonin and nomilin separately and together as well as in the presence of added sugar or acid. The values of limonin at which panelists could detect the compound were 2.32 ppm and nomilin 1.73 ppm in orange juice which was half the level of detection in model juices. When the compounds were tested together, their odor threshold was 1.89 ppm, which explains why bitterness can be detected in HLB juice. We were also interested in how much really bad symptomatic HLB juice could be blended with healthy juice before panelists could distinguish a difference. This was tried in both Hamlin and Valencia juices and determined to be around 25% of symptomatic juice in healthy juice for panelists to detect a difference in both varieties. Since the differences in aroma profiles between HLB and healthy juice are significant by not dramatic and the corresponding olfactory experience resulting in only subtle differences,it was not surprising that the enose was not as good as the etongue at separating HLB uice from healthy juice. It did, however, separate the juices, but with some overlap of some samples. Conversely, the etongue did clearly separate all HLB juices from healthy juice, even for HLB and healthy juices from the nutritional programs. The etongue also separated the different nutritional treatments from each other and from the conventional spray treatments. This instrument could be a useful tool for discrimination of juice samples based on quality.
Mid Florida Citrus Foundation (MFCF) currently supports the research efforts of scientists from the University of Florida, USDA and private industry. The majority of research projects are conducted by UF scientists at the approximately 112 acre citrus grove. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. The management of this grove requires extra financial commitment as grove care cost tend to be higher than a commercial grove due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. Currently MFCF is in the processes of laying irrigation for an additional 35 acres of citrus groves, which will support seven new research projects. This expansion was made possible due to this grant in conjunction with an EQIP grant from the NRCS. The NRCS grant provides for the leveraging of CRDF funds; that is for every dollar spent on expansion projects the NRCS cost shares approximately 50% of the cost. The completion of the establishment of new grove is scheduled for the beginning of May. Continued support is needed for the care and maintenance of these newly established groves into 2011
Management of citrus Huanglongbing (HLB), which is associated with Candidatus Liberibacter asiaticus, could be achieved by application of antimicrobials and stopping the spread of HLB pathogen. Curing Ca. L. asiaticus infected citrus trees is one attractive goal due to the high value of citrus trees and the high cost of citrus tree removal and replanting. Wide spread of HLB throughout Florida renders curing Ca. L. asiaticus infected citrus trees necessary. Treatment of Ca. L. asiaticus infected citrus could be pursued by applying antimicrobials to infected trees. The most common targets for antimicrobial agents include receptors, proteins and enzymes, DNA, RNA and ribosomal targets. Among them, proteins have become the major target due to their druggable characteristics. In this study, we presented our research on screening small molecule inhibitors against SecA. SecA is one essential component of the Sec machinery which provides a major pathway of protein translocation from the cytosol across or into the cytoplasmic membrane. The Sec pathway was also shown to be required for virulence of Ca. L. asiaticus in our study. SecA is the protein translocase ATPase subunit, which involves in pre-protein translocation across and integration into the cellular membrane in bacteria. First we filtered the structures based on their physico-chemical properties, e.g. Molecular Weight, H-Bond Donor, H-Bond Acceptor & Rotatable bonds and structurally similar to adenine moiety. Approximately 5000 structures were retrieved from ~5 million structures of commercially available databases. The identified data set was used for virtual screening by molecular docking method. Based on the dock scores we eliminated about 4500 low scored structures and selected ~500 (10%) for further molecular docking & minimization to evaluate the scoring functions (Dock glide scores, Hydrophilic, Hydrophobic e.t.c). Based on scoring functions, structural diversity, and our chemical intuition we have chosen forty structures for biological activity studies against purified SecA protein. For that purpose, SecA of Ca. L. asiaticus was expressed in E.coli using the pE-SUMO vector and purified. The inhibition assays of the 40 compounds against SecA was done as described previously by Denis et al. 1978. The IC50 ranged from 0.3’M to 100’M. At 20 ‘m seventeen compounds showed >50% inhibition and four compounds had more than 65% inhibition. One potent compound 18 was found with IC50 value of 2.5 ‘m. The differences between the high & low activity structures could be partially explained by the ‘CN groups which are only present in low activity compounds and 1, 3 Di-Methoxy groups which are present in the high activity compounds. The compound 18 is being further tested on Sinorhizobium and in planta. We are further screening the small molecules for activities in nm level against SecA.
Citrus canker is a serious disease of most commercial citrus cultivars in Florida.The goal of the proposed research is to identify and characterize novel and critical genes involved in pathogenicity and copper resistance present in X. axonopodis pv. citri (Xac) and related strains. Identification of critical virulence factors is a crucial step toward a comprehensive understanding of bacterial pathogenesis, host-species specificity, and invasion of different tissues thus to design new management strategies for long term control. Treatment of citrus with copper-based bactericides is one of the most common practices used for control. However, there is potential for horizontal gene transfer of copper resistance genes from other closely and distantly related bacterial strains, which will drastically reduce the efficacy of copper bactericides. Currently, copper resistant strains of other xanthomonads, including X. axonopodis. pv. citrumelo, the citrus bacterial spot pathogen, have been isolated from fields in Florida.Understanding the potential mechanisms of copper resistance in Xac and potential horizontal gene transfer of this resistance to Xac is also important for the long-term management of citrus canker.Currently, five Xac related strains are being sequenced with FL-1195 completed, XacAw close in completion (chromosome completed, plasmids in progress), while three more genomes are in the gap closing stage. Genome sequence of Xac strain Aw was completed. It is currently being checked for any problematic regions. The GenePrimp analysis of the annotation showed 1728 anomalies. Manual curation is currently in progress. Multiple gaps still exists for the two plasmids. Primer walking,cloning and sequencing are being used to close those gaps.The genome sequence has been compared to the reference strain XAC A 306 using MAUVE and global rearrangements were observed. FL-1195 was sequenced using 454-pyrosequening, Illumina (Solexa) sequencing and Opgen optical mapping. The finished sequence of XACM (4,967,469bp) was annotated and curated. Our analysis revealed that XACM lacked plasmids, although they are commonly associated with other strains of xanthomonas. Phylogenetic analysis based on housekeeping genes revealed a close relatedness of XACM to Xanthomonas axonopodis pv. citri str. 306 (XAC) causing citrus canker and Xanthomonas campestris pv. vesicatoria str. 85-10 (XCV) causing bacterial spot in tomato and and pepper. Whole genome comparison revealed a gene order similar to both XAC and XCV. Several genome rearrangements and insertion/deletion regions indicating genome plasticity were found. An all against all BLASTP of the complete proteomes revealed a total of 410 coding sequences unique to XACM. Several type III secretion system effector genes of XACM uniquely shared with the citrus or the tomato pathogen were found. Comparative genomics also showed various other changes in genes encoding cell wall-degrading enzymes, lipopolysaccharides, etc.
In Florida, the use of low volume sprayers has been adopted as a cost effective way to reduce Asian citrus psyllid populations. Low volume sprayers are calibrated to deliver 2 to 5 gallons per acre with a volume mean diameter droplet size of 100 microns. This relative measure of droplet size describes the range of droplet sizes, where 50% are smaller than 100 microns and 50% are larger. Smaller droplets are more susceptible to environmental conditions and drift at the time of application. A review of the literature on drift and droplet size identified a number of environmental conditions that could affect the deposition of these small droplets. This study monitored these environmental conditions in a Florida citrus grove from June to December 2010 and provides some insight into the prevalence of the favorable weather conditions for low volume spray applications. A number of environment conditions were monitored during this study. These measured conditions were then used to develop an estimate for the relative favorability for applying a low volume spray application on a given night. The study evaluated wind speed inside and outside a grove, an air dryness factor developed from the difference between dew point and air temperature measurements and the presence of inversion conditions. The trigger values used in the study were when average wind speeds were 0.0 mph or greater than 10 mph, a difference between the air temperature and calculated wet bulb temperature of greater than 140F and wind speeds less than 2 mph with air temperatures higher at the 25 foot level compared to the five foot level (used to determine the potential for a temperature inversion). From the collected weather measurements, the greatest potential threat to low volume spray applications in Florida could be conditions conducive to the development of temperature inversions. During the study period, the potential for temperature inversion existed on 16 to 32% of the nights. It appears in Florida that there is very little potential for evaporation to volatilize low volume spray droplets based on the established criteria. Wind speed conditions were taken from inside the grove at two locations and compared to observations outside the grove. Typically wind speeds fell into the 0.0 to 10 mph range from 82 to 99% of the time. The average wind speed recorded above the tree canopy at a 20 foot level was 8.75 to 20.5 times greater than the wind speed recorded in the tree row within the grove at a level of five-feet. The wind speed above the tree canopy most closely resembled the average wind speed measurements recorded from outside the grove at five and 25 foot levels. Mature hedgerow citrus groves in Florida appear to act as effective windbreaks, thereby significantly reducing wind speed within the block or grove as compared to outside grove observations. Additional activities were also conducted to offer low volume calibration events to Florida citrus growers with 3 events being conducted during the grant period. These events were conducted in conjunction with USDA ARS. These calibrations events assisted growers in adjusting sprayers to provide droplet sizes to be in the 100 micron range. The ability to effectively calibrate low volume spray equipment is not a task that can easily be accomplished without sophisticated and expensive equipment.
Our project aims to determine which species and varieties of citrus and citrus relatives are being smuggled into California, with the consequent risk of transmitting exotic pests and diseases; to acquire germplasm of these genotypes if they are not now legally present in California; to make sure this germplasm is pathogen-tested; to make budwood or propagating materials available for distribution through the Citrus Clonal Projection Program; and if feasible and advisable, encourage California cultivation of these commodities, so as to undercut the incentive for their smuggling. In November 2010 we received updated information gathered by the USDA/APHIS/PPQ Smuggling Interdiction and Trade Compliance program listing seizures of citrus and citrus relatives being smuggled from foreign countries, including the plant parts concerned, quantities, countries of origin, and the states where the items were seized. We will use this data to help set our priorities as to which commodities most urgently need to be addressed by our program. The information has prompted further questions regarding its interpretation which we are pursuing in consultation with both USDA and other sources. In November 2010 team member David Karp gave a PowerPoint talk before the California Citrus Nursery Society, explaining the program. He discussed individually with members their perceptions of the horticultural and commercial potential for local cultivation of the various smuggled species. In January 2011 Mr. Karp attended the Huanglongbing Conference held in Orlando, Florida, where he gathered information about recent scientific discoveries germane to the project, and also about Florida sources for, cultivation of, and trade in commonly smuggled citrus and citrus relatives. He then traveled from Orlando to Homestead visiting botanical and germplasm collections, ethnic and specialty farmers, extension agents, backyard farmers, and ethnic stores, to observe, photograph and discuss the germplasm resources and cultivation of these items. Most of the relevant commodities, including curry leaf (Bergera koenigii), bael (Aegle marmelos), and woodapple (Limonia acidissima), grow and fruit more rapidly than in California in the generally warmer and more humid Florida environment. Mr. Karp has also continued to meet with aspiring growers of citron (Citrus medica) for Jewish ritual purposes, in order to guide them to fulfill legal and regulatory requirements in introducing seeds and cuttings of this species into California. We are currently consulting with the Citrus Research Board to determine if a consensus exists favoring distribution of curry leaf seed, in order to help facilitate the growth of California cultivation of this commodity.
The overall goal of this project is to characterize the virulence mechanisms of Candidatus Liberibacter asiaticus, 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 Ca. L. asiaticus with the advice and permission from FCPRAC. The tile has been changed to the following to better suit the goal: Understand the virulence mechanism of Ca. L. asiaticus by genome sequencing and functional genomics approaches. Major achievements: To understand how Ca. L. asiaticus make a living including nutrients acquisition and overcoming of plant defense in citrus, we first analyzed its metabolic capability and nutrient requirement by conducting curated metabolic reconstruction. Some novel insights were acquired regarding its respiration, amino acids synthesis, Co-factors and nutrient transportation. Our data indicated that Ca. L. asiaticus could rely only on aerobic respiration rather than on anaerobic respiration as reported previously (Duan et al. 2010). Ca. L. asiaticus could not synthesize 12 amino acids and needs external folate, quinines, biotin, thiamine, pantothenate, and pyridoxine for its growth. In addition, genome comparison of Ca. L. asiaticus against Mycoplasma genitalium, one of the smallest bacterial genomes, has identified more than 200 homologs. Those shared genes might be close to the minimal set required for viability of bacteria and good candidates for screening antimicrobial small molecules. Several potential targets including SecA are being used to screen potential antimicrobial small molecules to control HLB. In order to identify the potential virulence genes, real-time quantitative PCR assays using total RNA isolated from infected plants and psyllids, were conducted to test the expression profile of Ca. L. asiaticus. Gene specific primers were used to check the expression of more than 500 genes in Ca. L. asiaticus. The genes showing a differential expression of two fold or more in either the plant or psyllid were selected, and categorized into COG (Clusters of Orthologous Groups of proteins) functional categories. Selected genes that were overexpressed in planta were further studied by expression or screened on Nicotiana benthamiana plants for symptom expression, using transient assays. Totally 28 potential virulence genes were cloned into TMV30bGFP viral vector for transient expression on Nicotiana benthamiana plants. Out of the 28 genes cloned, 13 were successfully assayed on tobacco plants for symptom expression. Three out of the 13 showed interesting symptoms, and hence were selected for further characterization. The following symptoms were observed: LasA1 showed vein clearing after about a week after inoculation, and subsequent wilting and death of the whole plant within 2-3 weeks. LasA2 showed phyllody, stunting and very clear growth defects. The symptoms were significantly different from the infection using the empty vector (TMV 30BGFP). The expression of the genes in planta were confirmed using RT-PCR. Further study is under way to further understand the roles of those two potential virulence factors.
Comparison of the microbiomes associated with HLB pathogen positive and negative citrus will illuminate the causal agent of citrus greening. Potential beneficial microorganisms could be identified from healthy 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. Characterization of the beneficial microorganisms associated with citrus in the local environment might identify beneficial bacteria for practical use to promote citrus growth and control HLB. Major achievements: This research has resulted in four publications. 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 (Sagaram et al. 2009 AEM). 2. We characterized the effect of HLB on the bacterial community associated with citrus roots. This research has been summarized in the following publication ‘Huanglongbing, a systemic disease, restructures the bacterial community associated with citrus roots’ (Trivedi et al. 2010 AEM). 3. Developed a method to quantify viable Candidatus Liberibacter asiaticus with the aid of ethidium monoazide (EMA) which can differentiate live from dead cells (Trivedi et al. 2010 EJPP). 4.Isolation of plant growth promoting bacteria from healthy citrus. Isolation of bacteria with the potential of plant growth promoting and biological control potential might reveal innovative ways of controlling the HLB disease. We specifically focused on delineation of the cultivated endophytic bacterial isolates and characterization of their salient metabolic features. A total of 227 and 125 morphologically distinct colonies were isolated and characterized from HLB asymptomatic and symptomatic trees, respectively. We observed that the frequency of bacterial isolates possessing various plant beneficial properties was significantly higher in the asymptomatic samples. A total of 39 bacterial isolates showing a minimum of 5 beneficial traits related to mineral nutrition [Phosphate (P) solubilization, siderophore production, Nitrogen (N) fixation], development [Indole acetic acid (IAA) synthesis], health [production of antibiotic and lytic enzymes (chitinase)], induction of systemic resistance [salicylic acid (SA) production], stress relief [production of 1-amino-cyclopropane-1-carboxylate (ACC) deaminase] and production of quorum sensing [N-Acyl Homoserine Lactones (AHL)] signals were characterized. A bioassay using EMA-qPCR was developed to select bacteria antagonistic to ‘ Candidatus Liberibacter asiaticus’. Using the modified EMA-qPCR assay, we found 6 bacterial isolates showing maximum similarity to Paenibacillus validus, Lysinibacillus fusiformis, Bacillus licheniformis, Pseudomonas putida, Microbacterium oleivorans and Serratia plymutica could significantly reduce the population of viable ‘Ca. L asiaticus’ in HLB symptomatic leaf samples. In conclusion, we have isolated and characterized multiple beneficial bacterial strains from citrus roots which have the potential to enhance plant growth and/or suppress diseases. Further study is needed to develop appropriate formula to utilize those beneficial bacteria.
Asian Citrus Psyllid (ACP) was first detected in San Diego County, August 29, 2008 (in lemon), and has been slowly moving north. The largest populations of ACP have been discovered in Los Angeles County. The quarantine containment perimeter in Riverside County has remained unchanged since November, 2009; but recently discoveries of ACP have been made in Upland and Redlands. Thus all of the research conducted on the Asian Citrus Psyllid, and its natural enemies, must still be contained within UC Riverside’s certified quarantine facility. Three months into this new project, and we have established good ACP production numbers under the quarantine space and rearing constraints (300 to 400 ACP offspring/0.023 m3 cage). All ACP material must be double caged and all personnel working with ACP production must wear hooded jump-suits. These procedures ensure that no ACP leave the rearing cages or rearing room in quarantine. We communicate to researcher routinely to prioritize utilization of ACP material. Weekly vials of dead adults are provided to the Citrus Research Board (CRB) lab for scout train (e.g. trap card identification of ACP). Other adult specimens (ca. 1,000) were also provided to the CRB lab for special plastic mount curation. We have established quarantine approved methods of removing ACP DNA material for USDA researchers to use as controls for their disease detection research, which they are conducting in collaboration with Florida citrus researchers. This research will commence in April of this year. We continue to provide all of the adult material for the ACP electrophysiology project (Drs. Anand Ray & Robert Luck) and also the associated pheromone work being done in collaboration with Dr. Jocelyn Millar (CRB project). For this same project, we recently provided the material to obtain ACP brains (for brain stain analysis) and developed a collaborative effort to establish protocols that allows the ACP brain material to be removed from quarantine to complete the analysis. We continue to assist CRB and CDFA projects in evaluating the host specificity of Tamarixia radiata, a parasitoid of ACP, by providing adults psyllids and rearing protocols upon request. Our project has also been approached by other researches that may attempt to conduct various pesticide trials in the quarantine facility (e.g., residue & efficacy trials). These projects are currently working on developing protocols that will allow these trials to be conducted in quarantine. There are very strict guidelines for working with pesticides in quarantine. Some of the work may commence this month, for which we will provide the adults. We expect that the demand for ACP material will increase as the year progresses, and will continue to anticipate researchers’ needs so that their research goals can be met in a timely manner under the Federal and State quarantine constraints.
The goals of this ongoing project are to eliminate the incentive, economic and cultural, for smuggling of citrus fruits and plant parts by establishing legal sources of supply for these commodities in California and to provide education about the availability of registered pathogen-tested sources. On July 7, 2011 we received authorization from the California Citrus Research Board to proceed with our proposal to distribute seeds of Bergera koenigii, the curry leaf tree, to California citrus nurseries. This approval was granted after we provided a written description of the seed distribution protocol, which was submitted to CDFA nursery program authorities for comment and approval. Staff at the National Clonal Germplasm Repository for Citrus and Dates (NCGRCD), under the direction of Dr. Richard Lee, are currently testing curry leaf seed source trees at the UC-Riverside Citrus Variety Collection for seed-transmissible pathogens, using real-time PCR tests to test for huanglongbing (HLB), and conventional PCR for citrus leaf blotch virus, psorosis, and xylella (CVC). Approximately 1,000 seeds were previously collected by staff at the NCGRCD, and assuming that the trees test free of seed-transmissible pathogens, the seeds, both stored and freshly collected, will be distributed to several qualified nurseries which have expressed interest in the project. The immediate goal is to facilitate the propagation and cultivation of trees of curry leaf, in order to provide a local source for the leaves, which are in considerable demand from South Asian communities for culinary use. Previously the commercial demand for this commodity was satisfied by growers in Florida and Hawaii, but federal quarantine restrictions now prohibit shipments of curry leaf from Florida, and Hawaii shippers can send to California only product that has been treated with methyl bromide, which drastically curtails shelf life. Because of the unsatisfied demand, curry leaf is being smuggled into California, as attested by official interdictions and media reports; since these leaves are a preferred host for the Asian citrus psyllid, the vector of HLB, such smuggling presents a grave risk of spreading the insect and introducing the disease to this state. It is our intention to facilitate the development of local cultivation of curry leaf that would undercut the demand for smuggled product.
During this quarter, the young ‘Hamlin’ orange trees in the Ridge experiment were harvested in December 2010, by which time they were exactly two years old. Large significant differences in fruit yield and external and internal fruit quality were measured at that time between the conventional practice treatment and the six advanced production (ACPS) treatments. Notably the percentage orange peel color was more than doubled , brix/acid ratio was 33% higher, fruit yield per tree was up to 186% higher, fruit yield per acre was up to 375% higher, and soluble solids production per acre was 364% higher in ACPS than in conventional plots. The highest average fruit yield at two years age reached 38.5 boxes per acre. The yield in the microsprinkler fertigated treatment was not significantly different from the drip fertigated treatment on the same Swingle rootstock, but was significantly higher than yield of the conventional production treatment. Although this yield is above average for such early production in Florida, there is still scope for improvement by stimulating more rapid canopy production in the early years. In the first year of this experiment, the tree canopy’s young emerging flush was repeatedly damaged by citrus leaf miner larvae, which restricted the canopy development and fruit setting capacity in year two. Better pest control is essential for the success of the ACPS. Additional gains could also be made through more refined nutrient application timing, dosing, and composition. The first four HLB-positive trees were identified during this quarter. The first of those trees has been protected with a psyllid-proof screened enclosure for continued observation and study of the disease, but the other three trees were removed and subsequently infected trees will also be eradicated. The difficulties experienced with pest control in the first year were thought to have contributed to this early appearance of HLB, but in reality there are infected backyard trees receiving minimal to no care within 300 feet of the experiment. Summary points: 1. Fertigate ‘daily’ at 100 to 150 ppm N concentration in final water volume; use calcium nitrate to provide ample Ca (see recipe on WWW) 2. Inject the actual fertilizer as a pulse and follow with water; Inject from tanks 1 and 2 sequentially 3. Fertigate to keep the top 0-6 inches of soil at about field capacity. Monitor moisture at 18 inches depth to avoid excess. Use sensors. 4. Fertigate in the morning after sunrise when photosynthesis and transpiration are at their peak 5. During long dry periods in the first year, flush salts from the root zone with plain irrigation (3 to 4x normal irrigation amount) every 2 weeks For all the latest up-to-date information on the ACPS research, especially for photos and calculator which cannot be displayed here, browse to http://128.227.177.113/ACPS/index.html The water calculator is on the Data/Reports page.
As we approach the end of our third year of evaluating the Boyd cocktail of macro-nutrients, micro-nutrients, phosphite, SARs, and hydrogen peroxide we are collecting data that is showing the importance of macro and micro-nutrients, and the phosphite. The ‘Hamlin’ trial was harvested in January and yield results will be available for the next report. The two ‘Valencia’ trials will be harvested in April. The rehabilitation trial begun this year to evaluate the value of buckhorning existing HLB declining trees to rebalance the shoot/root ration and applying foliar nutrients to the regrowth has gotten a lot of interest from growers. The idea of of salvaging existing trees over removal, replanting, and bringing a new planted tree into production has a lot of advantages. One advantage is the cost and another is having the tree out of production only one or two years. Another advantage is having a tree with vigorous regrowth and fully productive canopy. Growth measurements of shoot length and leaf size of the regrowth have shown shoot length to be twice the length of unpruned trees receiving the same nutrients on every flush during the first year. The pruned trees have reached more than half their original height and tree volume in one growing season. Leaf nutrient analysis data has been collected to monitor the status of the vigorous growing trees. The trees in both the Boyd cocktail trials and the rehabilitation trial all look good considering the leaf drop experienced this past October statewide in groves. In addition, we have experienced similar leaf and fruit drop from the freezes in December that other growers have had. We have documented greater leaf and fruit drop from HLB infected trees where the added stress makes the trees more susceptible. HLB makes the citrus tree less tolerant to any stress factor like drought, poor nutrition, freezes, disease, insects, etc., and can result in leaf and fruit drop. We are in the third year of a replicated experiment in a 12-acre experiment commercial block of 8-year-old ‘Valencia’ oranges on ‘Swingle’ to test effects of micro-nutrients + systemic acquired resistance inducers, and Asian citrus psyllid (ACP) chemical control on ACP populations on Can. Libericacter asiaticus (CLas) titer, and plant yield. Since our last report we have conducted two dormant sprays on the treated plots. We are conducting preparation for the third harvest in these plots possibly towards the end of March beginning or April 2011. This third harvest is considered to be key to confirm the observed tendencies from years past. In our last harvest, trees in plots where ACP was managed using insecticides produced significantly more Lb-solid per tree (5.76’0.36 Lb-solid/tree) than trees in untreated plots (4.17’0.65 Lb-solid/tree), continuing a trend seen last year. Also, trees treated with Micro+SAR produced more (5.26’0.55 Lb-solid/tree) compared with untreated trees (4.55’0.64 Lb-solid/tree), following trends seen from the first harvest in the 2008-2009 season. Neither the insecticide treatment nor the nutrients have slowd down the spread of HLB (now virtually 100%), Treatments where ACP is being managed have had consistently lower titer over the two year study, except in our May 2010 where the CT values were not different Treated (24.0’0.3), Untreated (24.1’0.2). In January we are conducting the plant samples for HLB detection. Simultaneously, we are analyzing the data from the last plant sample collection.
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