During August and September 2013, three locations (Pico’s Farm, Evan’s Properties, and Blue Goose Groves) were selected for the thermotherapy trial. Roughly 30 HLB-affected Valencia trees were chosen using a random block design at each of the locations. Trees were either subjected to no heat or tented for 96 hours which included 4 full afternoons of summer heat. In late November and December, trees were sampled for 3 months post heat treatment data. Trees were sampled again in March and April 2014 for 6 months post heat treatment data. Trees at Pico’s farm and Blue Goose’s properties were in the process of spring flushing and blossoming. Heated trees had an increase in canopy density. Despite the presence of spring flush, untreated trees were not dramatically thicker. Tree height had no change. Fruit crop was counted. The spring 2014 crop will be compared to spring 2015 crop to understand how thermal therapy affects fruit production, fruit drop, and fruit quality. Leaves were collected and processed to determine the Las titer in specific branches before and after treatment. The trees at Evan’s Properties were 15 years old. Before treatment, the Evan trees had very thin canopies with a large crop of fruit. During the spring sampling period, it was noticed that the heat treated trees contained a dramatic amount of new tender flush. Trees continue to look a bit sparse but there were definite improvement of canopy growth in some regions of the tree canopy observed when comparing photos of trees before and after heat treatment. Evan’s trees were slightly shorter after heat treatment as that the tops of the trees were burnt during treatment. Fruit crop was also counted at this location. Leaves from specific marked branches were collected. At this time, all samples from before treatment and 3 and 6 months later have been processed and analyzed for Las titer. Student t-test and Tukey’s test will be used to determine if changes in Las cells/gram of tissue is statistically less after treatment for each time period, as compared between treated and untreated, and between sites. The nine month sampling begins in mid-May 2014. During Spring 2014, a new heat treatment system was developed incorporating a steam generator. A retractable tent was mounted to the side of a fruit hauling truck (Goat truck). The accordion style tent is hydraulically controlled to ease the process of moving from tree to tree. The steam generator along with the electric generator and water tank were mounted into the bed of the truck. A hose is fitted from the steam generator to the side of the goat trailer so that the hose can be placed in the desired position on the ground before the tent is lowered over the tree. A series of trees have been treated at 55C for 1 to 20 minutes. The main objective of these tests was focused on learning the appropriate treatment time and temperature that will not permanently damage young trees. Preliminary results on young small trees (5-6 feet tall) suggested that 10 minutes or higher treatment duration with steam is too harsh for the tree to recover and will kill the tree. The time range was reduced to between 1 and 4 minutes based on tree size and temperature. The temperature around the tree canopy should not exceed 130 ‘F. It was also noticed that placing the outlet of the hose directly against the base of the tree causes too much damage and was later moved to a position in which the hose outlet was facing the tent instead of the tree.
The program research objective is to develop an effective and sustainable phage and/or tailocin (high molecular weight bacteriocin) based biocontrol system for Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker. During the first year of the project, we purified 39 high titer phage lysates and identified 13 phage groups, based on host range studies and type IV pilus dependency for host infection. Groups 1-3 (21 phages), as well as five of eight phages with unique host range patterns, were determined to be type IV pilus-dependent. Purified lysates of 16 phages have been examined using electron microscopy to determine their morphological classification. Electron microscopy results indicate the purified phages active against Xac are from the three families of the Caudovirales. We have identified 11 podophages (7 groups) with short non-contractile tails that exhibit capsids ranging from 52-65 nm in diameter, three siphophages (2 groups) with long non-contractile tails (122-130 nm) that exhibit capsids of 56-57 nm in diameter, and two myophages (2 groups) with long contractile tails (141-149 nm) and capsids of 96 nm in diameter. We have sequenced and annotated five podophage genomes with different host ranges and determined that they exhibit phage phiKMV-like architecture and a single subunit RNA polymerase. This genomic architecture is indicative of a virulent lifestyle, which is a necessary component for the development of functional and sustainable biocontrol program. We are currently sequencing two myophages and three siphophages. We have also identified three tailocins with activity against Xac. Electron micrographs of purified tailocin XT-1 showed a sheathed phage-tail-like structure that resembles an R-type pyocin, with the tailocin having an average length of 135 nm and a width of 18 nm. XT-1 exhibited broad host range activity, killing 13/13 Xac isolates tested. Tailocins XT-2 and XT-3 each had narrow host ranges (killing 2/13 Xac isolates) that overlapped with XT-1, but not with each other. The host ranges of the three XT tailocins indicate receptor site diversity. We have initiated testing of phage using a citrus detached leaf assay. In preliminary studies, the inoculum control panels showed raised, callus-like lesions typical of canker after 4-5 days, whereas the leaf panels challenged with phage (at a multiplicity of infection of 1) 24h post-Xac-inoculation showed no symptoms. qRT-PCR is being used to determine the distribution and persistence of the phage in citrus vascular tissue. To determine the ability of phage to survive in sunlight in the field, UV-C studies were undertaken. Phage CCP504 had a 0.5-, 2- or 4-fold reduction in plating activity when exposed to 10Joules (J), 20J or 40J, respectively, which makes phage CCP504 more resistant than most well characterized phages. UV-A and UV-B studies as well as UV protectant studies are ongoing. First round efficacy greenhouse protection and therapeutic evaluation of a phage cocktail (in cooperation with Dr. Nian Wang, University of Florida) is planned for the next quarter.
The ultimate objective of this project is the development of a phage-based biocontrol system for citrus greening. ‘Candidatus Liberibacter asiaticus’ (Las), the causal agent of citrus greening, has not been successfully cultured. However, a member of the same genus, Liberibacter crescens BT-1, has recently been cultured under laboratory conditions. The current focus of our project is to develop a detection system for bacteriophage (phage) and/or phage components (tailocins; high molecular weight bacteriocins) using L. crescens strain BT-1. We have developed an efficient overlay assay that has been used to test 307 individual phage lysates from diverse hosts including Burkholderia, Pseudomonas, Clavibacter and Xanthomonas species, as well as 11 broad host-range tailocins. Utilizing the same system, 41 plant extracts (weed, citrus, alfalfa, rice, fresh papaya pulp and seed), 27 citrus psyllid extracts, and 38 water samples were assayed for the presence of phage/ tailocin active against L. crescens BT-1. No phage or tailocin active against L. crescens BT-1 have been detected to date. Additionally, mitomycin C and/ or UV-induced filtrates from 24 diverse bacterial isolates were tested for phage, tailocin, and/or bacteriocin activity against L. crescens BT-1 with no activity detected. However, during our survey, we identified two bacterial isolates (Microbacterium strain TM-313 and Brevibacillus strain TB-205) that exhibit antimicrobial activity against L. crescens BT-1. We have undertaken preliminary steps to purify and characterize the antimicrobial factor from Microbacterium strain TM-313. To isolate the compound, TM-313 cultures and control medium were extracted with non-polar to polar solvents. The methyl tert-butyl ether extract of the TM-313 medium produced a zone of growth inhibition on L. crescens BT-1 overlay, with no activity observed in the control medium extract. Preliminary characterization of the active compound(s) [AC313] indicates that it is protease (Proteinase K, Trypsin, alpha-chymotrypsin) and heat (100.C/10min) insensitive, as well as being < 10kDa in mass; these are indicative of a non-proteinaceous small molecule. Further characterization is ongoing. Bioinformatic analyses of L. crescens BT-1 prophage regions (LC1 and LC2) and the Las prophage regions (SC1 and SC2) have identified putative tail fiber genes that can be used to modify an existing tailocin in the laboratory. In cooperation with laboratories in Florida we are testing extracts from L. crescens BT-1 and Las infected plants for phage activity. We are continuing to collect and test environmental samples to isolate phage.
The primary goal of this project is to develop cryotherapy as a practical, reliable method of eliminating graft transmissible pathogens from Citrus and citrus relatives without inducing juvenility. Permits have been obtained to ship pathogen infected citrus material from Riverside, CA and from the Exotic Disease Quarantine Laboratory, Beltsville, MD to Ft. Collins, CO, and materials from Ft. Collins, CO can be sent under permit to Riverside for lab testing. In the first 18 months of the project, we have developed a cryotherapy protocol which works well with any type of citrus or near citrus relative. Two technicians from Riverside and one technician from Beltsville have been trained in Dr. Volk’s lab on the technique. Initially we concentrated on therapy from citrus viroids and Citrus tatterleaf virus, and were able to eliminate these pathogens, the most difficult to remove by traditional shoot tip grafting (STG) using the cryotherapy protocol. Presently we are testing the protocol for elimination of huanglongbing, Citrus mosaic virus, and citrus variegated chlorosis (in Beltsville), citrus stubborn, Citrus psorosis virus, Citrus leaf blotch virus, Dweet mottle virus, citrus concave gum disease, and Citrus vein enation virus. Efficiency of therapy with cryotherapy is being compared with results obtained by shoot tip grafting with most pathogens. For Citrus tatterleaf virus, we have recovered 32 plants which are clean from 32 recovered plants; viroids 11 clean from 14 recovered plants; Citrus leaf blotch virus 6 clean of 6 recovered plants; Citrus psorosis virus 25 clean of 25 recovered; and Citrus Stubborn 10 clean of 10 recovered. Because we wait at least 12 weeks to conduct lab testing of recovered plants, we have a constant backlog of plants to be tested. In Riverside, we are testing recovered plants after they have been grafted from the test tube to a rootstock, thus representing plants that would be continued through the indexing required for a variety release. The research is continuing.
The primary objective of this research is to identify diverse germplasm which has a tolerance to huanglongbing (HLB) and its psyllid vector, Diaphorina citri. An earlier germplasm screening block containing 87 different accessions of citrus and citrus relatives and planted in the field at Ft. Pierce for four years provided evidence of different types of tolerance to HLB. As a result of the first trial, this is a second trial which includes accessions of Poncirus trifoliata, hybrids with P. trifoliata, sour orange types, and kumquats. For this present trial, 97 different accessions were selected based on the population structure which suggested that the accessions contained genetic material from P. trifoliata, kumquat, or sour orange. Seed was collected from the University of California Citrus Variety Collection; heat and fungicide treated using standard protocols, and shipped to Ft. Pierce where the seedlings were grown under greenhouse conditions. The trees in Riverside used for seed collection were tested for Citrus leaf blotch virus, Citrus psorosis virus, HLB, and Xylella fastidiosa prior to shipment of the seed. Seven seedlings of each accession were inoculated with HLB while in the greenhouse in March 2013, seven seedlings remained un-inoculated. The seedlings were planted in the field in late spring 2013 and the first visual assessment was made in November 2013. Sampling for HLB will continue biannually and incidence of Asian citrus psyllid will be made this summer season.
The purpose of this project is to determine methods to effectively eliminate Candidatus Liberibacter asiaticus (Las), the bacterium associated with huanglongbing (HLB) in Florida, from citrus. Emphasis is being placed on cryotherapy with conventional shoot tip grafting being used for comparison purposes. Application of crytherapy to recover plants free of HLB from budwood approaches 95 percent efficiency. The project also includes determining the effectiveness of using young indicator plants for biological indexing to verify elimination of graft transmissible pathogens. During this past quarter, additional selections of mandarin and sweet orange materials have been forwarded to Ft. Collins for therapy using cryotherapy and shoot tip grafting. Recovered plants are allowed to grow for 12-14 weeks following therapy before testing for the presence of HLB. With pre-treatment and cryotherapy, results indicated the procedure is very effective at eliminating Citrus tatterleaf virus and citrus viroids; pathogens most difficult to eliminate by thermaltherapy and by shoot tip grafting. Additional selections of several varieties of citrus infected with huanglongbing have been forwarded to Ft. Collins for therapy, and will be tested in another 6-8 weeks. In Riverside, a system has been developed using Jiffy pots for the seedling used as indicator plants, allowing for a growout of about 75 days from the seed planting until the young plants can be inoculated and used as indicators. The inoculum bud survival is very high, greater than 98 percent. The entire indexing procedure can be done with the plants in the Jiffy pots. A trial was performed to compare the results obtained by using the mini-plant biological indexing compared with the traditional standard indexing protocol, using indicator plants 10-14 months old, for 15 accessions. The results from the mini-plant index revealed the presence of Citrus vein enation virus in one accession which had been missed using the traditional standard indexing protocol. The advantage of using the mini-plants for biological indexing is that 35 indicator plants may be contained in the space normally occupied by 3 indicator plants; quicker turnover of the small plants for indexing means more accessions may be tested per year; biological indexing for pathogens expressing only under cool temperatures may be indexed for year-round as the smaller plants may be placed close to the cool pads in the greenhouse, and because they occupy less space, the whole trial can be done utilizing the cooler temperatures.
The project entitled ‘Further characterization of HLB resistant clones of selected citrus varieties’ (project no. 758) is aimed at conducting experiments to understand the basis of HLB tolerance in Aurantioideae with genera sexually compatible with citrus like Microcitrus, Eremocitrus and Poncirus. Research conducted this quarter (from Jan, 2014 to March, 2014) consisted of: a). Second batch of pollinations conducted in Riverside (Citrus Variety Collection) using HLB tolerant and susceptible accessions. This year we have utilized certain mandarin cultivars known to be more tolerant to HLB than others based on field trials in Fort Pierce by collaborators. The crosses between these ‘tolerant’ mandarins and ‘resistant’ citrus relatives (as determined by our previous trial) is likely to yield useful resistance/tolerance to HLB. We have now performed over 1100 crosses using mandarin and pummelo as seed parents. Based on previous year’s results, we were able to select seed parents that are likely to set fruit when crossed with the pollen from HLB resistant citrus relatives. b). Seeds collected from previous year’s pollinations were germinated in the greenhouses in Riverside for confirmation of genotypes. These experiments are in progress. A part of the seeds will be sent to collaborators in Fort Pierce in the next few weeks for field evaluation of resistance. c). One batch of selected HLB tolerant and susceptible seedlings that were psyllid challenged in Fort Pierce (Hall lab) were used for making RNA extractions in Fort Pierce. The samples are now being processed and analyzed. The experiment continues in Fort Pierce and sampling will be done periodically according to our original plan. We are on track as per our proposed milestones.
The goal of this study is to understand the role of biofilm formation and quorum sensing (QS) in X. citri ssp. citri infection of citrus fruit and to prevent its infection by interfering with biofilm formation and QS. Three compounds exhibited a significant reduction in biofilm formation both on polystyrene surface and in glass tubes compared to the untreated control, where the level of biofilm formation were reduced to 50% and 60% of control, respectively. Plant test in greenhouse showed that treatment with the three compounds prior to infection could reduce biofilm formation of Xac on leaf surface, reduce the formation of canker lesions on spray-inoculated grapefruit leaves with the wild-type strain. Effects of the three compounds on Xac on detached immature citrus fruit were also tested using spray inoculation. Preliminary results showed that these small molecules affected Xac 306 infection of unwounded and wounded citrus fruits at sub-inhibitory concentrations. We have completed testing the effect of those compounds in different combinations with copper based bactericides in controlling Xac infection of grapefruit plants in the greenhouse. The sensitivity of biofilm and planktonic cells of Xac 306 to copper (copper sulfate) were evaluated by measuring the MICs. Biofilms are less susceptible to copper than planktonic cells. Effect of the selected compounds on sensitivity of Xac planktonic cells and biofilm cells to copper sulfate was also investigated. In the NB medium, planktonic cells exhibited a MIC of 0.50 mM CuSO4 without biofilm inhibitor. In the presence biofilm inhibitors at sub-MIC concentrations , the MICs of CuSO4 against Xac 306 planktonic cells were decreased to 0.25 mM. In a in vitro biofilm system test, the combined use of copper sulfate and the compounds individual or both resulted in significantly increased killing compared to killing by copper sulfate alone. The results have been published by Phytopathology in a manuscript entitled: Foliar application of biofilm formation-inhibiting compounds enhances control of citrus canker caused by Xanthomonas citri subsp. citri. One patent is filed based on the results. We also identified multiple new biofilm inhibitors. The effect of those biofilm inhibitors to control citrus canker is being investigated. We tested the survival of both biofilm deficient and QS mutants on fruit surface. Effects of biofilm formation inhibitors on Xac infection on detached immature citrus fruit were tested using spray inoculation. The inhibitors affected the infection of Xac on both unwounded and wounded citrus fruits. We are testing more potential biofilm inhibitors. We continue characterizing how quorum sensing and biofilm formation contribute to Xac infection of citrus fruit. Multiple virulence genes involved in quorum sensing and biofilm formation are being investigated. The field trial is ongoing to test the effect of the identified biofilm inhibitors to control citrus canker. One new compound is able to inhibit QS at a concentration of 100 .M based on observation of bacterial phenotype of aggregate formation. Plant test in greenhouse showed that the QS inhibitor (100 .M) treatment could reduce the formation of canker lesions and bacterial population on spray-inoculated grapefruit leaves simultaneously with the canker bacterium Xcc 306. Repeated experiment in greenhouse is underway.
The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. In the enhencement project, we are expanding the field test to include two more field trials in two more locations, larger scale of field test, test more beneficial microbes, and test different approaches to enhance the survival of the beneficial microbes in the soil. The field trial site were identified. For one site, six applications were conducted. For another site, the first application was conducted in February, 2014. We conducted the background survey regarding HLB disease incidence, severity, Las titers, Phytophthora, nematodes, root (root density and health status), and microbial diversity right now. The following up study is ongoing.
The goal is to develop short term approaches to control citrus Huanglongbing (HLB) using antibacterial-producing bacteria. Recent studies indicate that HLB severely damaged citrus roots. The destructive effect of HLB on roots partly results from Candidatus Liberibacter asiaticus (Las) infection of the roots. In this study, we will conduct the following objectives: Screen bacteria that can produce antimicrobial compounds against Liberibacter crescens and related Rhizobiaceae bacteria; purify and characterize antimicrobial compounds produced by the screened bacteria; illustrate the regulation of antimicrobial production by producing bacteria at different environmental conditions and with different inducers; investigate the growth of the producing bacteria in different conditions; and control HLB by delivering antimicrobial compounds using the producing bacteria in the soil. We have isolated 84 strains of Streptomyces spp., Bacillus spp., Paenibacillus spp., and Pseudomonas spp. producing antibacterials from Florida citrus groves. We have also acquired 28 different species of Streptomyces spp., Bacillus spp., Paenibacillus spp., and Pseudomonas spp. strain producing different antibacterials. Field trials are being conducted.
The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. Currently, we are setting up the experiments to test different Microbe Products in management of HLB. Assay for compatibility between isolates using antagonistic survival tests showed that all the selected beneficial bacteria are compatible with each other. Plant growth promoting activity of six selected isolates was evaluated using the model plant Arabidopsis grown in vitro. The results suggested that three isolates could promote plant growth. The plant growth promoting activity of these six isolates was tested using citrus (grapefruit) seedlings in greenhouse. Greenhouse assays suggested that a consortium of three Bacillus and relative isolates (AY16, PT6 and PT26A) may delay the development of both HLB symptoms and pathogen population on citrus leaves after root inoculation. The potential of the consortium to recover the tree decline from HLB infection is being evaluated in greenhouse. The growth conditions of the three strains were optimized using a small fermenter. Three antifoam agents, A204, PPG200 or M-Oil did not affect the growth of the three bacterial strains. The initial neutral to alkaline pH values (7.0 ~ 8.0) favor growth of the three bacteria in LB, while acidic pH (5.0 ~ 6.0) suppress bacterial growth. The optimal cultural temperature was determined to be around 30C with average bacterial population of 109-1010 cfu/ml after 20-hour incubation, although the bacteria may grow slowly under room temperature (~ 23C). The shelf life of three different formulations of the bacterial culture is being evaluated under room temperature. In a six-month time course, the bacterial populations in LB broth, OPB broth and tape water are comparatively stable with initial and final both at ~ 108cfu/ml. Four field trials are being conducted including more beneficial bacteria. For one of the field trial, six applications have been performed. We are evaluating the survival of the beneficial bacteria in the soil. The application method has been changed during application to improve the survival of microbes in the soil. For two trials, four applications were conducted. For another field trial, the application started in February, 2014. We have surveyed the HLB disease incidence, and Las population. We are investigating the survival of the applied microbes in the soil.
Management of phloem-limited bacterial diseases is very challenging. These bacteria employ unusual and sometimes unique strategies by which to optimize their niche occupation and obtain their nourishment from the host plant. Their location within the living (sieve tubes) plant cells, rather than in the intercellular spaces, offers different challenges and opportunities for them to avoid the host plant’s defense system. Phloem is also difficult for any bactericides to reach to control the pathogen population. Among the phloem-limited bacterial diseases, citrus Huanglongbing (HLB, greening) is one of the most devastating diseases. The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, the current management practices have not been able to control HLB and stop spreading of Candidatus Liberibacter asiaticus (Las). The goal of the proposed study is to develop HLB management strategies which boost plant defense to protect citrus from HLB by exploiting the interaction between Las and citrus and understanding how Las manipulates plant defense. Recently, we compared the gene expression of PR1, PR2 and PR5 in healthy trees and Las infected citrus plants. The expression of PR1, PR2 and PR5 was significantly reduced in HLB diseased grapefruit as compared to healthy grapefruit after inoculation with Xac AW. We also tested whether infection by Las can make citrus more susceptible to infection by Xanthomonas citri subsp. citri. We also sprayed four times with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB in one grove. Multiple compounds showed control effect. To further test those compounds, we have selected two more groves to expand the field test. The disease index of the two groves have been investigated and treatments already started. The follow up investigations are ongoing, including monitoring the HLB symptoms, disease incidence and Las titer in leaves. We compared the SA levels in HLB infected and healthy grapefruit after the inoculation with Xac AW. We also compared the SA levels in HLB infected and healthy Valencia citrus. We are continuing to evaluate the effect of different compounds on management of HLB both in greenhouse and in citrus grove. We have applied different compounds at three separate field trials. Four compounds were shown to have positive effect on controlling HLB based on two year field test results. We are also testing the mechanism of those compounds showing positive effect on HLB control. For example, the analysis of gene expression of pathogenicity related genes and callose synthase CalS1 in treated plants and nontreated plants is underway. We have investigated the effect of those compounds on disease severity, yield, juice content and quality. We will repeat those treatments for one more year. Currently, the treatments are being conducted.
The goal of the research is to control citrus HLB using small molecules which target essential proteins of Candidatus Liberibacter asiaticus (Las). In our previous study, structure-based virtual screening has been used successfully to identify five lead antimicrobial compounds against Las by targeting SecA. SecA is one essential component of the Sec machinery. Those compounds showed promising antimicrobial activity. However, further work is needed to apply the compounds. We will evaluate the important characteristics of our antimicrobial compounds including solvents and adjuvants, phytotoxicity, antimicrobial activities against multiple Rhizobia, antimicrobial activity against Las, application approaches, and control of HLB. Those information are critical to for the practical application of those antimicrobial compounds in controlling HLB. We also propose to further optimize the five lead compounds. In addition, we propose to develop antimicrobial compounds against lipid A of Las. The lipid A substructure of the lipopolysaccharides (LPS) of Sinorhizobium meliloti, which is closely related to Las, suppresses the plant defense response. Las contains the complete genetic pathway for synthesis of lipid A. We hypothesized that Las uses lipid A to suppress plant defense. Thus, targeting lipid A could activate plant defense response. Lipid A is also an ideal target and has been targeted for screening antimicrobial compounds for multiple pathogenic bacteria. We are optimizing the compounds in collaboration with IBM. Two compounds with slightly higher binding affinity than C16 were identified. Currently, we are evaluating the best range of composition ratio among each component (%weight) of AIs, solvents and surfactants. The following characteristics are being evaluated: 1) emulsion stability and ease of emulsion; 2) stability of diluted concentrate; 3) freeze-thaw stability; and 4) phytotoxicity to citrus species. We have successfully identified one formulation suitable for all five compounds without phytotoxicity. Using the formulation, we have tested all five compounds against eight different bacterial species including Liberibacter crescens. We are repeating the results for one more time. Field test is being conducted. We have sprayed once. Greenhouse trial is being conducted.
At the request of the CRB, Grafton-Cardwell and Morse merged their core entomology research efforts under a single project, 5500-501 (Morse’s portion of the project is 5500-501b). We have always coordinated our research efforts but this arrangement formalizes the situation. This report summarizes the Morse lab’s recent research under this coordinated project (all arthropod research except ACP which is a separate project, i.e. 5500-189). Our major effort this last year has focused on helping the industry to deal with Fuller rose beetle (FRB) in relation to citrus exports to Korea. For the last several years, Korea has put increasing pressure on the CA industry to reduce egg mass levels on export fruit and there is a concern that in 2014-15, loads found to be infested with viable egg masses may be denied entry into that country. Morse and Dr. Andy Cline of CDFA conducted 6 training sessions for county agricultural inspectors in different areas of California during Nov. 2013 — at the prompting of APHIS, county inspectors have agreed that all loads of citrus shipped to Korea would be sampled to determine the percent of fruit infested with unhatched FRB egg masses. Field FRB control trials were run at Lindcove by Grafton-Cardwell (15 treatments evaluated) and in Pauma Valley by Morse (10 treatments evaluated). None of the treatments results in 100% control but it appears that 2 treatments are relatively effective — either 2 ground sprays of bifenthrin, 2 trunk sprays of bifenthrin, or 2 foliar sprays of combinations of carbaryl, cryolite, and/or thiamethoxam. A method of analyzing bifenthrin residues on trunks was developed in collaboration with Dr. Jay Gan (UCR Dept. of Environmental Sciences). Studies were conducted on 3 dates to determine bifenthrin levels on trunks necessary to cause beetle paralysis after they walk over the trunk spray (“LC”50 of 0.04874 ug/cm2). The persistence of 5 formulations of bifenthrin trunk sprays were evaluated in Riverside by taking residue samples 6, 12, and 18 weeks after trunk application. Citrus thrips resistance to Delegate has been confirmed in the San Joaquin Valley. Two products nearing registration on citrus that will be useful in control of citrus thrips (as well as other pests) are Bexar and Closer. To better study Delegate resistance, we started a greenhouse colony from an area reporting poor control and have confirmed that the population is resistant to Delegate (very flat dose-response line). So that we can study the mechanism and genetics of resistance, we will select for higher levels of resistance. We are examining contaminants of export citrus using 10 randomly selected cartons per load from a variety of citrus packing houses. To date, we have processed 29 such loads with a diversity of commercial varieties examined. Genetic identification of insects appears fairly routine but we will have to continue to work on methods for mite identification — initial extractions have been problematic.
At the request of the CRB, Grafton-Cardwell and Morse merged their core entomology research efforts under a single project, 5500-501 (Morse’s portion of the project is 5500-501b). We have always coordinated our research efforts but this arrangement formalizes the situation. This report summarizes the Morse lab’s recent research under this coordinated project (all arthropod research except ACP which is a separate project, i.e. 5500-189). Our major effort this last year has focused on helping the industry to deal with Fuller rose beetle (FRB) in relation to citrus exports to Korea. For the last several years, Korea has put increasing pressure on the CA industry to reduce egg mass levels on export fruit and there is a concern that in 2014-15, loads found to be infested with viable egg masses may be denied entry into that country. Morse and Dr. Andy Cline of CDFA conducted 6 training sessions for county agricultural inspectors in different areas of California during Nov. 2013 — at the prompting of APHIS, county inspectors have agreed that all loads of citrus shipped to Korea would be sampled to determine the percent of fruit infested with unhatched FRB egg masses. Field FRB control trials were run at Lindcove by Grafton-Cardwell (15 treatments evaluated) and in Pauma Valley by Morse (10 treatments evaluated). None of the treatments results in 100% control but it appears that 2 treatments are relatively effective — either 2 ground sprays of bifenthrin, 2 trunk sprays of bifenthrin, or 2 foliar sprays of combinations of carbaryl, cryolite, and/or thiamethoxam. A method of analyzing bifenthrin residues on trunks was developed in collaboration with Dr. Jay Gan (UCR Dept. of Environmental Sciences). Studies were conducted on 3 dates to determine bifenthrin levels on trunks necessary to cause beetle paralysis after they walk over the trunk spray (“LC”50 of 0.04874 ug/cm2). The persistence of 5 formulations of bifenthrin trunk sprays were evaluated in Riverside by taking residue samples 6, 12, and 18 weeks after trunk application. Citrus thrips resistance to Delegate has been confirmed in the San Joaquin Valley. Two products nearing registration on citrus that will be useful in control of citrus thrips (as well as other pests) are Bexar and Closer. To better study Delegate resistance, we started a greenhouse colony from an area reporting poor control and have confirmed that the population is resistant to Delegate (very flat dose-response line). So that we can study the mechanism and genetics of resistance, we will select for higher levels of resistance. We are examining contaminants of export citrus using 10 randomly selected cartons per load from a variety of citrus packing houses. To date, we have processed 29 such loads with a diversity of commercial varieties examined. Genetic identification of insects appears fairly routine but we will have to continue to work on methods for mite identification — initial extractions have been problematic.
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