This is the second year of a 3-year project. The overall goal is to evaluate the efficacy of 11 compounds individually and in combination for control of the HLB disease based on reducing titers of HLB bacterium in HLB-affected citrus. In this quarter (from June 2014 to October 2014), effectiveness of 11 compounds individually and in combination against HLB bacterial was continued to be evaluated by bark application using our nano-delivery system. After 4 months and 6 months from initial treatment, the HLB bacterial titer was significantly reduced by Act+VA and Amp treatment and no phytotoxicity appeared in treated trees, which was the same as our previous results in the last quarter. The formulation Act+VA will be applied in a field trial next quarter. Samples will be taken every two months for PCR analysis. Six new compounds have been tested by our graft-based test in the greenhouse. All tests will be analyzed next quarter. The temporal phytotoxicity in trees from the trunk-injection treatments have been restored to normal growth in this quarter. They will be treated using lower concentrations of compounds in the next quarter.
This is the first year of a three-year project. The overall objective was focused on determining the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can registered for control of HLB in the field. In this quarter (June 2014- October 2014), eighty-one HLB-affected citrus trees with diameters of 10-15 cm were treated once every one month by chemical treatments (Carv and Pcy) formulated with O/W nanoemulsion using both bark application and trunk-injection by gravity bag infusion, using a randomized block complete design. After 2 months and 4 months from initial treatment, the HLB bacterial titer was still high in the treated citrus trees while no phytotoxicity appeared in the trees. In next quarter, more HLB-affected citrus trees will be treated using the other effective compounds and coupled with heat-treatment and site-fertilization. The agronomic performance of treated trees will be investigated.
The large-scale validation of citrus leafminer (CLM) disruption with the ISCA DCEPT CLM technology has continued. Our group has continued in assisting of collecting data to monitor the efficacy of the product during the project. We have been assisting in monitoring certain areas of the ca. 3,000 acres that were treated at three locations in southeast and southwest Florida. We assisted with collection of efficacy data by trapping CLM males through September with pheromone traps as a surrogate measure of mating disruption. Thus far, it appears that the product has been effective.
For the second year, we compared the densities of Asian citrus psyllid (ACP) on reset trees planted in solid set plantings (all trees planted within the last two years) versus the densities of psyllids on resets (trees planted within the last two years to replace mature trees) present within mature groves. This year, the experiment was conducted in three groves of three different citrus varieties (Hamelin, Grapefruit and Valencia). More psyllids were found in the solid set plantings as compared to the resets within mature groves. These data confirm last year’s results. Overall, this study indicates that solid set re-plantings will require more intense vector management than resets within mature groves. Regarding laboratory experiments, we investigated the effect of abiotic and biotic factors on host acceptance of the ACP. We know that ACP are attracted by HLB-infected citrus, and that this attraction is achieved through the emission of Methyl salicylate (MeSA) by the plant. We now investigated how abiotic factors such as drought stress and time since Clas infection may affect the behavior of ACP and the release of volatiles by citrus plants. We found that HLB-infected plants that were more attractive than non-infected plants during olfactometer assays in control conditions, were not more attractive as compared with non-infected plants if they were submitted to drought stress. Leaf volatile collections and GC-MS analyses revealed that HLB-infected plants under drought stress were characterized by a decrease of MeSA emitted, compared to HLB-infected plants under control conditions. In our experiments, drought stress consisted of decreasing soil moisture from 95% to 35% of the water carrying capacity of the pot. We also found that plants infected with the Clas pathogen for over 18 months (‘old infection’) were no longer attractive in olfactometer assays as compared with control plants; whereas, citrus plants infected for 12 months (‘young infection’) were strongly attractive to ACP as compared to control plants. This was confirmed by settling experiments showing that ACP preferred to settle on infected versus non-infected plants, but also preferred settling on young infected plants rather than on old infected plants. Again, these behavioral results were associated with a reduction in the amount of MeSA emitted by plants infected with the pathogen for more than 18 months. We are currently measuring the gene expression of methyle-transferase in citrus plants depending of the time since infection, to investigate if this reduction in MeSA emission is associated with a reduction of the mRNA abundance of the salicylate methyltranferase gene (CsSAMT) that we have identified in the citrus genome.
Because of the natural disease cycle of citrus canker, field trials for citrus canker were initiated before the official start of the proposal to adequately determine efficacy. Trials are well underway in a grapefruit block that is a well established site for canker management trials. As the project was just initiated at the beginning of June, we are in the very early stages of a field trial for HLB. A grove of 3 year old infected Valencia has been identified, scouted for disease rating and sampled for starting Liberibacter titer. The first application has been applied testing multiple application methods compared to untreated controls in a randomized trial.
The canker field trial of Zinkicide compared to industry standard management is ongoing with preliminary disease assessment performed and the data currently being analyzed. Final disease assessment will not be done until all canker-induced fruit drop has occurred. The HLB trial in young Valencia trees continues with regular treatment and disease assessment. The timing of first treatment and the trees flush cycle make it difficult to determine efficacy this soon after initial treatment. Sampling and measurement of the initial titer response to Zinkicide application in these trees is currently underway. The ability to detect any symptom or tree recovery differences are not expected until strong symptom expression in the fall or the spring flush, respectively.
This project terminated 30 August, 2014 and this constitutes the final report for this project. During the course of the project, we developed reliable, replicable and efficient methods for recording from the antennae of live, whole Asian citrus psyllids using a specially-designed microscope stage, an immobilization technique to secure individual psyllids, micromanipulators to place glass capillary electrodes and a Faraday cage to eliminate noise. The use of live whole psyllids is superior to using detached antennae or antennae-head preparations because the intact psyllid remains alive for long periods, sometimes exceeding 24 h and therefore allows for extended use in GC-EAD and EAG studies, and the signal does not degrade over time as commonly occurs in antennae-only preparations as the antenna dies. We then proceeded to characterize the volatile profiles of Citrus and Poncirus genotypes and to test the major and minor organic volatile compounds produced by each. Very few compounds elicited antennal responses. By far, the largest and only consistent antennal responses were obtained from degradation products of E-B-ocimene and citral, namely formic and acetic acids. This serendipitous discovery demonstrated how oxidation of volatile compounds occurs spontaneously in the presence of oxygen resulting in the rapid degradation of ocimene and citral and release of acetic and formic acids. Asian citrus psyllid antennae are highly responsive to these two acids and we were able to demonstrate a behavior response as well. Psyllids more readily probed a wax substrate containing the acids compared with wax substrate alone. Degradation of volatile organic compounds (VOCs) is common in nature and results in the release of acetic and formic acids into the atmosphere. In one study, acetic acid comprised 8.4% of VOCs above a citrus grove. Degradation of VOCs can produce acetic and formic acids at rates 200 to 300% higher than the precursor compounds. In our studies, ACP adults probed a substrate impregnated with formic and acetic acids more frequently compared with blank controls and other citrus VOCs. ACP orientation to host plants may involve acetic and formic acid alone or in blends with other compounds. We will continue to study the behavioral response and orientation of Asian citrus psyllid to these compounds alone and in blends containing other citrus compounds that we have shown to be weakly active by electroantennography.
Feeding behavior of ACP is being studied on P. trifoliata and trifoliate hybrids using an electronic penetration graph (EPG), and choice and no-choice assays to study host selection and probing behavior. We continue to record ACP feeding by EPG and are beginning to describe differences in the number and duration of ingestion waveforms on resistant and susceptible genotypes. We have the capacity to simultaneously record the feeding behavior of 8 ACP using a Giga8 DC monitor and anticipate acquisition of a 4-channel AC-DC monitor that provides improved waveform resolution. A bottleneck to both methods is the time-consuming and onerous analysis of the waveforms. We propose to automate analysis of EPG ingestion waveforms by means of unsupervised learning technique, Hidden Markov Models, to automate temporal pattern recognition of the digital output. In collaboration with PhD candidate Denis Willett in the Stelinski lab at UF, IFAS, Lake Alfred, we are generating EPG digital output files from resistant and susceptible genotypes that have been manually scored for waveform type, a process that typically requires >8h to process one 21-hour run by one psyllid. By comparing automated recognition with manually scored runs, and subsequent confirmation of automated determination by visual inspection of machine-identified waveforms, we will validate the model for multiple psyllids on resistant and susceptible citrus, and for multiple plant species to confirm that the model can function across plant taxa. In addition to contributing to identification of resistant accessions and possible mechanisms of resistant, the addition of rapid, real-time analysis of waveforms will contribute to rapid screening of progenies generated by traditional or novel methods. Of particular interest is the application of EPG to describe the effect of stylet sheath inhibitors on ACP feeding behavior. Greenhouse trials continue to confirm resistance to oviposition. Current tests address the hypothesis that a lack of stimulatory volatiles in resistant P. trifoliata contributes to resistance to oviposition. We will attempt to show that oviposition on resistant P. trifoliata genotypes can be increased in the presence of volatiles from susceptible citrus, e.g., C. macrophylla.
This project is in furtherance of the commercial release of a new product for control of the citrus leafminer and associated spread of citrus canker disease based on a deployment device for the sex pheromone of the leafminer, DCEPT CLM™ (ISCA Technologies, Inc.). Under an agreement with the Commercial Product Delivery Committee of the CRDF, funds are provided to ISCA to subsidize two years of production of DCEPT CLM sufficient to treat 3,000 acres of citrus, mostly grapefruit, at three locations in St. Lucie and Charlotte counties in April/May of this year. The remaining cost of the product is provided by the growers. Funds are also provide to ARS and University of Florida to support monitoring and analysis of the experiments at the three locations. Disruption of trap catch is being monitored weekly throughout the growing season at the three sites. At the Emerald Grove location (NW St. Lucie County), adjacent blocks of grapefruit untreated with pheromone to the north, east and south of the treated blocks are being monitored as controls. At the SW St. Lucie County location, the entire grove has been treated. At the Charlotte county location, the effect of skip rows will be evaluated. All evaluations will be done at the end of the season as we are able to collect and collate grower practices along with estimates of mining damage in treated and untreated areas.
Potted plants of Corymbia (Eucalyptus) torreliana (a widely used wind break in citrus) and Pongamia pinnata (a leguminous tree under development for biofuel) were infested with Diaprepes larvae and evaluated after 4 weeks to assess the relative susceptibility of these trees compared with that of a known susceptible host, Citrus macrophylla. Survival on C. macrophylla (7.4 +/- 0.4 larvae recovered from pots (n = 10) infested with 10 larvae each) was significantly greater than that on either Pongamia (5.3 +/- 0.6) or Corymbia (3.7 +/- 0.7). Weight gain of larvae was greatest on C. macrophylla as well. Mean +/- SEM fresh weight of larvae after 4 weeks was 229 +/- 6.3 (n=74) on C. macrophylla, 165 +/- 7.5 (n=53) on Pongamia and 74 +/- 9.0 (n=39) on Corymbia. Root damage was assessed by weighing the root mass of infested plants and expressing root loss as a percent of noninfested plants (n=10). Root loss for C. macrophylla was 71% and significantly greater than either Pongamia (53 +/- 4%) or Corymbia (44 +/- 4%). These results suggest that both P. pinnata and C. torreliana are susceptible to feeding damage by Diaprepes abbreviatus and may serve as complete hosts albeit to a much smaller extent compared with Citrus. We have observed adults feeding, mating and ovipositing on both species in the field near Fort Pierce. We are currently conducting adult feeding assays to complete the assessment of relative susceptibility in these species. A heavily infested planting of young Pongamia is currently being used to study movement of adult Diaprepes and response to known male and putative females-produced pheromones. This will be described in future reports
After discovery, identification and successful synthesis of methyl (.)-3-(2-hydroxyethyl)-4 methyl-2-pentanoate, laboratory behavioral assays confirmed it as a male-produced aggregation pheromone in Diaprepes abbreviatus. Colony-reared DRW used in choice assays confirmed that females are attracted to synthetic pheromone when given a choice between pheromone and clean air. Because the pheromone is developed in the male hind gut and released from frass, validation of the presence and attraction of the pheromone in the frass was tested with frass from locally collected wild-type males in the same type of choice assay using wild-type females. Wild-type females preffered male frass over humidified air. Identification of specific plant volatiles (kairomones) that serve as attractants to DRW and the ability to produce the synthetic pheromone led to a small field trial that attempted to attract DRW to traps baited with log doses of synthetic pheromonne. The baited rubber stoppers failed to attract any DRW. Further validation of the naturally produced male pheromone and kariomones (carvacrol and linalool) in laboratory behavioral assay trials took precedence throughout the remainder of 2013 and much of 2014. Early evidence of colony-reared female attraction to one batch of carvacrol (CAS# 499-75-2) at rates of 0.1.L ‘ 1.0.L elicited interest as a possible attractant. An assay conducted at that time that used 50.L of carvacrol obtained from a newly purchased bottle (also CAS# 499-75-2) deterred DRW in the olfactometer: 83% chose humidified air. Choice trials conducted in 2013 and 2014 focused on locally collected wild-type male and female DRW’starved for 24 hours with each sex separated from the other for the duration of the starvation period’response to previously untested component blends to develop a better understanding of the relationship of the male-produced aggregation pheromone and other naturally produced stimuli. All assays were conducted using frass collected from the colony cage housing either females only or males only. Assays conducted to date that produced response choices with a significant difference (G-test with Yates Correction, .=0.05): 85% of females chose male frass vs. air; 75% of females chose air vs. male frass + ‘new’ carvacrol + linalool; 80 and 90% of males and females, respectively, chose male frass vs. male frass + ‘new’ carvacrol + linalool. In these tests, carvacrol was clearly a repellent to both sexes of DRW.
This proposal aims to continue improvement to a novel psyllid trap and to use the trap to gather new information on the behavior, biology, population dynamics and biological control of ACP/Candidatus Liberibacter asiaticus. Lab and field testing was and continues to be conducted to increase trap efficiency by exploiting unique vector behaviors in response to traps and behaviorally active components. 570 – Grant Obj. 1: We continued to conduct field and laboratory studies toward obtaining an understanding of ACP trap response behavior by manipulations of visual cues as well as trap physical components and component orientation. This within Florida research component is continuing and we now added several new locations in Puerto Rico to exploit the significantly higher ACP populations there that make the research easier. Doing this quarter we have successfully tested, winnowed out inferior traps and retested trap prototype configurations in Puerto Rico and Florida in cooperation with Dr. David Jenkins and have now eliminated a number of configurations that are inferior as well as identified the most successful trap under field conditions. We have had no success with trap additions related to odor cues used as reported to be potential attractants in the literature. We are continuing this research with new ideas but now have a trap identified a “standard trap” that can be used to capture and preserve the DNA of the psyllids and their associates in situ. We also initiated a sampling regime to try to delineate the northern most established populations of ACP. Our sample locations toward this end have been Gainesville, Live Oak, Quincy and Marianna, FL. To date we have not detected ACP at any sampling location except in Gainesville. Captured ACP (n=100) from the Gainesville captured monthly were analyzed for the causal CLas bacteria of greening. Interestingly, to date none of the psyllids analyzed have been infected with CLas. Obj. 2: We have initiated the areawide psyllid sampling objective to detect and develop new biological controls for use against ACP. We are continuing the sampling in the northern most citrus populations in Alachua County and around Ft. Pierce within Florida and in Puerto Rico. The standard trap is being used for this work and performs well enough to complete this effort, i.e., where ACP occur, the trap captures and preserves them in proportion to their populations. We also begun to supplement the trapping effort by also collecting psyllids in “out-of-the -way” places that may hold relic citrus trees and relatively isolated ACP populations. So far several new pathogens have been identified. We have obtained cooperation with other USDA-ARS research personnel with expertise in the identification and rearing of entomopathogenic fungi and they have evaluated samples toward this end from Florida and Puerto Rico. This effort will be continued. Project Enhancement Grant: given the investment by CRDF and the shortened period of time to execute the objectives of the enhancement grant with the objective of developing and testing sentinel plants, this research was also continued in conjunction with other trapping improvement efforts. As a result of these studies we have found that sentinel citrus plants that are flushing are just as attractive as established flushing plants and highly attractive to ACP when nearby established citrus plants are not flushing. This work will be continued.
The CATP13 Proposal #860 titled, ‘Optical and physical deterrent for preventing ACP vector attack on citrus’was selected for funding earlier this year. However as one of the co-PIs moved to another University a revision to the project with subcontract in budget was submitted. The sub-contract was cleared by June and project started in July. No experiments could be performed before the official start of the project.
Sept 2014 The objectives of this proposal are 1) to determine if a) leaf litter biodegradation treatments reduce Guignardia spp. pseudothecia and improve control afforded by routine fungicide applications; b) if biodegradation is affected by the current fungicide application practices; and c) whether the biodegradation treatments will affect current citrus best management practices (BMP); 2) to determine the seasonal dynamics of leaf litter inoculum load in varying management regime intensities and how environment affects pseudothecia production in the leaf litter; 3.) to test if the resistance to black spot in the leaves and fruit in sour orange is correlated and under simple genetic control through laboratory and field testing of progeny of sour orange crosses in both Florida and Australia. The small plot work of the leaf litter was completed and the data processing has begun. The field site for the large trial with 3 treatments was mapped for disease incidence and laid out. To look at the effect of bagasse, a controlled experiment portion was conducted twice. It involved an in vitro study of the decomposition of citrus leaves and inactivation of G. citricarpa by various amendments including bagasse including the microbial consortium of fungal and bacterial strains to aid in the decomposition of bagasse and of citrus leaves. Leaves and other components were collected at time zero and at 10 day intervals. At each collection time, leaves and other components were oven dried and weighed. Soil weight, leaf weight, and then the total weight of each box were recorded. Leaf infection by G. citricarpa was verified by plating and qPCR will also be used. Data collection and analysis is ongoing. Collection of leaf litter material has begun in Florida and is being collected every two weeks from a grove with moderate black spot incidence in the previous season. it is too soon to summarize the data but as predicted we are finding much lower structure numbers and incidence in the leaf litter. This is likely because the disease pressure is much lower and more scattered in Florida than it has been in Australia historically. We expect to find the same trends but since our climate is very wet, there may be differences in leaf litter cycle. Work on characterizing the G. citricarpa collection continues. Analysis of the 2 leaf litter collection site found that the Imperial site had too few ascospres to make solid conclusions but the Valencia site gave good results with a clear trend that pseudothecia proceeded ascospore production by approx. 6 weeks. The relationship between pycnidia and conidia was a little more chaotic. We did need to modify the spore collection somewhat because we were finding more ascospores in our conidia collection than expect but we believe we have solve the problem. Many inoculated fruit prematurely abscised but there were atypical symptoms on several of the remaining fruit. When isolations were done, Guignardia spp. colonies were produced. Work is on-going and many fruit are incubating to see if symptom development progresses in high light and warm temperature conditions.
September 2014 The objectives of this proposal are 1) Determine the base line level of Guignardia citricarpa sensitivity to fungicides registered for disease control in citrus and evaluate new products for efficacy against G. citricarpa in vitro; 2) Conduct and improve implementation of spray trials for efficacy of registered products for citrus and to evaluate novel compounds in the field; 3) Optimize field evaluation of control measures through analysis of the spatiotemporal disease progress utilizing past and current field data of the outbreaks to gain knowledge on the incidence, severity and rate of the epidemic and assess the fungal population to increase the likelihood of successful field research and 4) Evaluate products and treatment conditions for postharvest control of citrus black spot. This quarter we accomplished: Objective 1: Tests with the DMIs are continuing and two compounds look promising. Preliminary data analysis underway Objective 2: Trials have been initiated and treatments are on going Objective 3: Preliminary analysis has been carried out on Groves I through III. Where Grove I shows total infectivity of the rows scouted clustering analysis cannot be done. In Grove with emerging disease (Grove II) and intermediate grove (Grove III) both show clustering according to spatial analysis in R (Ripley’s K, Binomial distribution etc’). We are waiting for color change in fruits to re-map Grove II and III for assessment of spread, and severity. Objective 4: Two sets of in vivo experiments were conducted using large numbers of naturally infected fruit. Fresh Valencia oranges from a block infected with citrus black spot (Guignardia citricarpa) were harvested and evaluated for presence/absence of CBS lesions and equal numbers of randomized fruit were distributed among the different treatments. Fruit were treated with one of seven fungicides at ambient or heated (50 or 56F) temperatures. The fungicides included azoxystrobin, fludioxonil, pyrimethanil, phosphorous acid, Imazalil, thiabendazole, and sodium-o-phenylphenate. Each of these have formulations that are registered for postharvest use on citrus. Control fruit were either left dry or dipped in water alone. After treatments, the fruits were air dried and kept at 25 C, 70% relative humidity, 3 ppm ethylene, and continuous light condition to promote CBS lesion development. The fruit are being assessed for disease incidence and severity but data is incomplete.
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