In laboratory experiments we are investigating the effect of abiotic and biotic factors on host acceptance of the Asian citrus psyllid (ACP). We previously found that drought stress and time since HLB infection decreased the attraction of ACP toward HLB-infected trees. We started a new experiment to determine if ACP might be attracted by trees that are artificially damaged. The question addressed is whether psyllid attraction to HLB-infected plants results from an active manipulation of the pathogen or is a byproduct of the infection. HLB-negative Valencia orange trees were girdled by excising a 1.5 cm ring of the bark tissue using a laboratory scalpel. This procedure results in the accumulation of starch grains morphologically similar to those formed by leaves affected by HLB. It results in the appearance of leaves with the similar leaf yellowing as in HLB-infected trees. This procedure allows us to imitate HLB-symptoms on a healthy tree. In olfactometer bioassays, we found that psyllids were significantly attracted toward leaves on girdled trees, as compared to, leaves from undamaged branches. Interestingly, psyllids did not show any preference between leaves from girdled branches and the leaves from HLB infected trees. This suggests that the attraction of psyllids toward HLB-infected plants is induced by the damage resulting from the disease, but not from an active manipulation of the host phenotype by the bacteria. In terms of practical application, it also suggests that damaged trees in groves might be particularly attractive to psyllids and particular attention should be taken after a climatic event that results in significant physical damage to trees. We also collected volatiles from girdled plants and we will compare the odor of leaves from girdled trees to the odor of control and HLB-infected trees. We are preparing the next set of experiments. The question that will be addressed is to understand how the different defense pathways of citrus interact with one another and how this interaction may change ACP acceptance of citrus. We will submit citrus to jasmonic acid odor, in order to induce the jasmonic acid defense. It has been reported in other plant models that activation of the jasmonic acid pathway silences the acid salicylate pathway. Because ACP are attracted by methyl salicylate, a byproduct of the acid salicylate pathway that is activated after HLB infection or after psyllid feeding, we hypothesize that a plant with the jasmonic pathway activated might be less attractive than a control plant.
We have completed our analysis of the data collected from the 2014 survey to monitor for insecticide resistance wherein no significant case of insecticide resistance in the Asian citrus psyllid was identified at the LD50 estimate, for any field population for any insecticide tested. However, as previously reported, it does appear that there are significant differences in the insecticidal response between some field populations with regard to dose-response, with some populations showing a much broader response to insecticidal treatment (remaining alive over a wider dose range). To determine the biological basis for this observation and the potential impact on insecticide management will require further investigation. During this last quarter, we responded to two reports of potential insecticide resistance, in one case against chlorpyrifos and the other fenpropathrin. Evaluation revealed equal response to insecticidal treatment of these grove specimens and laboratory strain for both chemicals. Our working hypothesis is that psyllids from surrounding untreated groves re-infest treated areas. Other work in our laboratory demonstrated that psyllids can fly at least 2.4km without aid of wind current. The impact of re-infestation by psyllids on insecticide management, treatment and efficacy warrants further investigation. We have completed the molecular characterization of the pyrethroid target, the voltage-gated sodium channel, to determine the potential for target-site insensitivity against this class of insecticides as observed in other insect species. We found that with sufficient pressure from pyrethroid exposure, insecticide resistance is likely to occur through this mechanism in ACP.
The overall goal of this three-year research project is to efficiently deliver antimicrobial molecules into citrus phloem against Las bacteria. This quarter’s (from Oct 2014 to Dec 2014) research continued to evaluate the effectiveness of our optimized O/W nano-delivery system to reduce HLB bacterial titers in potted citrus plants in the greenhouse and to reduce bacterial titers in the field when chemicals were applied by bark-painting or gravity bag infusion. Thirty-two treatments of 14 compounds in combination or alone at various concentrations were evaluated using our nano-delivery system in the greenhouse. The results, 2 months after initial greenhouse treatment, indicated that Amp and Pen most effectively reduced HLB bacteria titers; Act and Act+Va could partly suppress HLB bacteria, but the Act treatment was phytotoxic to the treated citrus; the other treatments had no significant effect on the HLB bacterium. In field trials, 10 months after initial treatment, the HLB bacterial titer was not significantly different among Pen, SDX and the untreated controls when applied by bark painting. HLB bacteria titers were very low in trees treated with Pen by gravity bag infusion both 2 and 10 months after treatment. Bacterial titers remained high in trees treated with SDX or the control 10 months after treatment by gravity bag infusion. In the next year, we will continue to treat HLB-affected greenhouse citrus using nano formulations of various chemicals, and investigate bacterial titers in potted citrus. In field trials, effectiveness of bark painting and gravity bag infusion to deliver compounds (Penicillin and SDX) will continue to be evaluated by analyzing trees for bacterial titer, growth performance and fruit quality.
This is the third 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 bacteria in HLB-affected citrus. In this quarter (Oct 2014 to Dec 2014), several compounds and combinations (such as Amp, Act+Va, Carv, and Pcy) were screened in field trials by gravity bag infusion or bark painting based on the results of bark painting and root drench application in the greenhouse. In field trials, the above molecules were applied to HLB-affected citrus trees by bark painting or gravity bag infusion. After 2 months none of the treatments except the positive control (Amp) have significantly reduced bacterial titers in the field. In the next quarter, we will submit the final report containing all the greenhouse and 6 month field data.
Jan 2015 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. Molecular data is being collected to match with counts. A large field trial was initiated and leaf litter was collected from the plots for evaluation. The evaluations are completed and being compiled. 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. After inoculation of the leaves, G. citricarpa conidia were observed on D1 but not D15 and no isolations were successful either day. There was a significant difference in the amount of leaf decomposition but not the rate with the control have the least decomposition and the bagasse treatment the most. Collection of leaf litter material continues in Florida and is being collected every two weeks from a grove with moderate black spot incidence in the previous season. Some data summary has begun and 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. Phyllosticta capitalensis and G. citricarpa isolates were collected in Australia to look at the competitive interaction of the two fungi. They are also evaluating the relative sensitivity to fungicides of the two species. They did lower germination rates for P. capitalensis than G. citricarpa. Preparations are being made for the leaf litter dynamics studies. The same Valencia block will be used but the second block in a new location to be determined. Also preparing for a second round of germplasm inoculations. A leaf litter degradation trial is in the planning stages that will closely follow the Florida experiments.
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 with the DMIs are nearing completion with assays with a QoI/SDHI on-going. Molecular characterization of CYP51 was initiated. Manuscript preparation has begun. 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 still 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 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 (10 or 13’C) temperatures. The fungicides include 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. Fruit treated with 1,000 mg/l thiabendazole at 25, 50, or 56 significantly inhibited subsequent lesion development. Fruit treated with 1,200 mg/l azoxystrobin at either 50 or 56’C also significantly inhibited lesion development, while treatments at 25’C were not significant. Treatments with SOPP solution (20000 mg/l; 25’C, 50’C, or 56’C) were usually not significant, except for an occasional significant reduction in lesion development in experiment #2. Fruit treated with 1,200 mg/l fludioxonil, 1,000 mg/l pyrimethanil 10,000 mg/l KPhos, or 1,000 mg/l imazalil were not effective at any temperature.
Dec 19, 2014 The objective of this project was to investigate three questions: 1) How long does a leaf needs to be infected by Guignardia citricarpa before ascospore production can be initiated; 2) How does infection and colonization of leaves by Guignardia citricarpa occur and potentially showing how pseudothecia, the sexual spore producing structures, are produced; and 3) what is the interaction between the common twig colonizing pathogen Diaporthe citri and the black spot pathogen Guignardia citricarpa and whether they can co-exist to successfully sporulate on dead twigs. Guignardia mangiferae sequencing is complete and the assembly is continuing. To get better resolution with the G. citricarpa genome, the organism was resequenced, giving far better resolution from the genome data. Analysis of the gene structure is being continued. A manuscript on mating type genes is in preparation. The data has been finalized and a third draft of the materials and methods completed. Introduction, results have a first draft and the discussion has begun. Sample collection for the greenhouse inoculation experiment continues and the samples are being fixed and embedded. Monthly collections continue from the greenhouse. Sectioning has continued at a faster pace. We found that D. citri overwhelmed the population of G. citricarpa on twigs under the conditions we were working with. The twigs that were incubating from the last experiment are being processed and a new experiment has begun to look at the effect of RH conditions on G. citricarpa sporulation on twigs.
Dec 19, 2014 The objective of this project was to investigate three questions: 1) How long does a leaf needs to be infected by Guignardia citricarpa before ascospore production can be initiated; 2) How does infection and colonization of leaves by Guignardia citricarpa occur and potentially showing how pseudothecia, the sexual spore producing structures, are produced; and 3) what is the interaction between the common twig colonizing pathogen Diaporthe citri and the black spot pathogen Guignardia citricarpa and whether they can co-exist to successfully sporulate on dead twigs. Guignardia mangiferae sequencing is complete and the assembly is continuing. To get better resolution with the G. citricarpa genome, the organism was resequenced, giving far better resolution from the genome data. Analysis of the gene structure is being continued. A manuscript on mating type genes is in preparation. It was decided that we needed further confirmatory evidence to strengthen our work so a new experiment was initiated. Sample collection for the greenhouse inoculation experiment continues and the samples are being fixed and embedded. Monthly collections from the greenhouse have begun. Sectioning continues at a slow pace. We found that D. citri overwhelmed the population of G. citricarpa on twigs under the conditions we were working with. We have began an experiment to look at the environmental conditions of G. citricarpa sporulation on twigs. It is incubating.
Report for period ending 6/30/14 Acitivites of the CHMA program assistant (4/1/14 – 6/30/14): During this reporting period, the CHMA assistant provided support to the CHMA program by attending various local CHMA meetings as well as statewide and multi state meetings. Specifically, Mr. Page attended 12 meetings including the following: Growers institute meeting (4/8/14), Volusia CHMA Meeting (4/11/14), Gulf CHMAs meeting (4/14/14), Conserve Field day presention (4/22/14), Hardee CHMA meeting (5/5/14), meeting with Noel Troxclair from texas citrus mutual (5/12/14), CHMA working group meeting (5/23/14), presentation for Hillbillies meeting (5/27/14), Florida State Horticulture Society Annual Meeting (6/1-6/2), Mountain Lake / Dundee CHMA meeting (6/3), Florida Citrus Mutual Conference (6/10-6/12), and attended meeting at SWFREC (6/25/14). In addition to attending meetings, Mr. Page continued to spend time working with CHMA captains and other interested growers on a daily basis identifying trouble areas, planning programs, and updating website information.
Report for period ending 9/30/14 Acitivites of the CHMA program assistant (7/1/14 – 9/30/14): During this reporting period, the CHMA assistant provided support to the CHMA program by attending various local CHMA meetings as well as statewide and multi state meetings. Specifically, Mr. Page attended 12 meetings including the following: FFA Meeting (7/2-7/3), American Sociey for Horticultural Sciences meeting (7/25-7/29), Small farms Conference (8/1-8/2), Citrus Expo (8/12-8/14), Gulf CHMA meeting (8/28/14), Grower meeting in Sebring (9/3/14), Hardee CHMA meeting (9/5/14), meeting with county agents (9/12/14), county agent Oj break (9/24/14), Landscape tradeshow (9/25-9/26), FDACS CHMA planning meeting (9/29/14), Hardee county CHMA roundtable (9/30/14). In addition to attending meetings, Mr. Page continued to spend time working with CHMA captains and other interested growers on a daily basis identifying trouble areas, planning programs, and updating website information. Mr. Page also worte an article for Citrus Industry Magazine to update growers on the success of CHMAs statewide and also began working with the new UF/IFAS Citrus Extension Economist to gather data from the CHMA program to conduct an economic analysis of the benefits of CHMA participation.
Report for period ending 6/30/14 Previously we reported on greenhouse -based trials examining the ability of soil applied insecticides to disrupt psyllid feeding such that incoulation of the plant with the HLB bacterium is prevented. In that work, treated plants were challenged with approximately 50 ACP (the majoirty of which tested postivie for Las) and then held for several months to test the plants for Las. Thus far after more than 9 months, none of the plants, including the untreated controls, have tested positive. These results demonstrate the difficulties in conducting such transmission studies with larger plants and suggest that perhaps developmental stage of leaves and variety used may play a role in successful transmission studies. We examined the effects of leaf struture to determine how this may affect psyllid feeding and ultimately pathogen transmission. Using an electrical penetration feeding graph (EPG) monitor, we compared psyllid feeding on leaves of different ages and varieties. Across treatments we found significant differences in the amount of time required to access both phloem and xylelm for psyllid feeding. We then conducted a histological examination of leaf tissues from these treatments, examining the pathway of the stylets. Here we found a certain defensive structure within certain leaf tissues that was present in leaf samples where psyllids had difficulty in accessing both phloeom and xylem. The presence of these structures will likely delay both inoculation and acquisiton of the Las pathogen by psyllids. These findings will not only be of use in designing future insecticide feeding studies, but may also be a potential factor for apparent host-plant resistance to ACP we have previously reported in other plants such as Cleopatra mandarin.
Report for period ending 9/30/14 Field trials were established to compare the amount of imidacloprid, thiamethoxam and clothianidin in leaf tissues based on tree size and rate of product applied. Plots were established at the CREC where no other soil-applied insecticides were being applied. Three rates of product were applied to trees in one initial size class. On a weekly basis, leaf samples were collected and taken back to the lab and processed for residue analysis and then placed into the freezer. Concurrently, work on validating the residue analysis methods being used in our lab is underway (see project 616). Upon completeion of that work, these samples will be analyzed to compare concentration of residues in leaf tissue over time at each rate applied. Additionally, we examined psyllid feeding behavior in the lab by removing branches from treated trees and determining if psyllid woulf still feed (and thus possibly be able to inoculate those plants with Las). Residue analysis of this leaf tissue will be conducted to compare levels of insecticide present and the lieklihood that psyllids fed on the plant. This work is preliminary but will provide a better starting point for work whcih will be conducted in the following reporting period wherein we plan to begin more controlled studies to determine what level of insecticide is needed in the leaf tissues to prevent psyllid feeding behavior.
Report for period ending 6/30/14 During the period of 4/1/14 to 6/30/14 we finished the analysis of nectar samples collected during the bloom of 2014. More than 300 samples were collected, albeit not all were able to be analyzed due to variation in quantity of nectar collected. It was observed that trees diseased with HLB produced far less nectar than healthier trees whcih complicated the standardization of samples. Samples were collected from 110 plots treated with different soil applied insecticides on varying dates up to 4 months prior to bloom. For each of the plots, bloom samples were collected on 3 separate dates to ensure rigourous sampling. The analysis of nectar from one date was reported in a previous reporting cycle. During the current reporting period, we completed the nectar analysis from the remaining samples. The remaining samples provided results similar to those rpeviously reported. Samples collected from trees treated more than 2 months (or more) prior to actual bloom did not contain appreciable levels of inseciticide in the plant nectar.These results support the hypothesis that soil-applied insecticide applications made 2 months or more prior to bloom will result in residue levels in nectar belwo the level of concern for pollinators. Work will now shift to focusing on analysis of insecticide residue levels in leaf tissues.
Report for period ending 9/30/14 During the period of 7/1/14 to 9/30/14 we shifted focus from analyzing citrus nectar to analysis of citrus leaf tissue for the presence of neonicotinoid insecticides and thier associated metabolites. Shifting from nectar to leaf tissue requires changing columns and solvents used for the analysis. THus, a considerable amount of time was spent calibrating the equipment and then validating the new methods used for leaf tissue analysis. By late August / early september, we completed the validation process and thus are now able to accurately quantify imidacloprid, thiamethoxam, clothianidin and their associated metabolites in leaf tissue. Because (accurate) information is not available on the anticipated levels of these compounds in citrus leaf tissue following application to trees in the field, we began sampling trees from commercial groves to understand what our detection range should be. Once complete, the next steps will be to begin analysis of leaf samples from field and lab studies we have planned.
This research seeks to determine whether young trees infected with CLas and displaying typical HLB symptoms can be brought to maturity and produce an economically viable yield. This will be achieved by managing a 58 acre grove of 3-year-old ‘Valencia’ / Kuharske Carrizo trees using a combination of three different foliar and three different ground applied nutritional programs. Factorial AxB treatments consist of A) ground-applied: 1) Liquid/dry+Ca (BHG standard), 2) Liquid+Ca, 3) Liquid/dry-Ca B) foliar-applied: 1) BHG standard-Ca, 2) BHG standard+Ca, 3) “Prescription”(+Ca). The prescription treatment was designed to be dynamic, customized for optimization, with feedback based on frequent leaf tissue analyses, visual symptoms, and the growth of the tree canopies and yield. There are six replications of treatments, with two being pure replications. The grove continues to grow well and also look normal, despite the nearly 100% HLB incidence. Foliation of canopies is dense, and leaf color in the summer and fall was a healthy green. Tree heights, canopy sizes and chlorophyll indices were measured in Fall, 2014, and analyzed. There were no significant treatment differences for measured variates, and selected data are summarized below: Table 1. Tree canopy height (feet) STD-Ca STD+Ca Prescrip SOIL FERT Liq/Dry 8.46 8.02 8.45 Liq 7.91 7.47 8.42 Liq/Dry-Ca 8.13 8.05 7.36 Table 2. Tree canopy volume (cu.ft/tree) STD-Ca STD+Ca Prescrip SOIL FERT Liq/Dry 521 388 495 Liq 413 367 485 Liq/Dry-Ca 454 419 348 Table 3. Tree canopy leaf chlorophyll index (SPAD index) STD-Ca STD+Ca Prescrip SOIL FERT Liq/Dry 72.60 72.20 75.45 Liq 73.50 75.20 74.95 Liq/Dry-Ca 77.05 75.35 74.30
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