Current HLB management strategy involves protection of citrus plants from HLB transmitting vector, Asian Citrus Psyllid (ACP) and adaptation of a good nutrition program for preventing root loss as there is no cure available to date. The objective of this research project is to develop and study a new non-phytotoxic, environmentally friendly film-forming ACP repellent material, called hereafter Organo-Silica Composite Film (OSCF). This material form a film barrier on plant surface upon spray application. This barrier is expected to change ACP feeding behavior. Previously we reported synthesis of six new OSCF formulations (OS-SG6 through OS-SG10) using different polymer and cross-linker ingredients. Phytotoxicity testing on Vinca sp and Persian Lime showed no plant tissue damage from any of these formulations. We also looked into potential heat trapping by OSCF material. By using an infra-red (IR) temperature sensor, we monitored the temperature the OSCF coated plant surface (including leaves and stem) at different time points post application of the OSCF materials. As a control, Surround’, a recommended pest deterrent based on Kaolin Clay was included in our studies. Four of the six candidate OSCF materials showed plant temperature fluctuations comparable to that of Surround’ after application on our testing citrus plant, Persian Lime. From the four candidate OSCF formulations that did not exhibit phytotoxicity, OS-SG6 and OS-SG10 were chosen as the most promising candidates. This selection was based on formulation stability at room temperature and minimal change of plant surface temperature before and after spray application (i.e. minimal heat trapping when compared to the Surround’ control). These two OSCF formulations were delivered to UF-CREC collaborators for efficacy testing which will be conducted in a Psyllid house. Detailed materials characterization results on the OS-SG6 and OS-SG10 formulations will be reported in future reports.
Successful area-wide management of the Asian citrus psyllid (ACP) and Huanglongbing (HLB) requires effective control of the psyllid in abandoned citrus groves. Since ACP adults are highly mobile, they can disperse from abandoned to productive citrus groves and spread HLB. ACP is susceptible to Isaria fumosorosea (Ifr), a native entomopathogenic fungi. ‘Autodissemination’ is insect dispersal of a pathogen to members of its own population. Our project goals are to develop and field test an ‘autodissemination’ system for inoculating ACP with Ifr and use these infected psyllids to instigate epizootics that will decimate ACP populations in abandoned citrus groves and; thereby, reduce movement of both ACP and HLB to nearby managed groves. The Ifr formulation is being provided by Dr. Mark Jackson (USDA-ARS, Peoria, IL) and is based on a south Texas isolate (Ifr 3581). During the first year of our project, we field-tested the durability and efficacy of different cardboard and plastic autodisseminator components fabricated to our specifications by AlphaScent Inc. By September of 2014, we determined which components were most suitable for Texas conditions and ordered sufficient quantities from AlphaScent for fall and winter trials (September to December). Due to a delay in obtaining the raw plastic materials required, AlphaScent was not able to deliver the components until the second week of October 2014. During the fall and early winter (October-December) of 2014, we collaborated with Paramount Citrus to repeat field trials in Hildago County with one of their managed groves and an adjacent abandoned grove. The site was a managed Valencia Orange grove next to an abandoned Rio Red Grapefruit grove. The managed grove was treated with insecticides for ACP earlier in the fall and flood-irrigated before our field trial. The abandoned grove was not treated with insecticides or irrigated. At this site, we evaluated the impact of autodisseminators on ACP movement and infestation in plots of abandoned citrus trees and managed citrus trees. For treatment plots, we hung pairs of autodisseminators coated with Ifr-spore formulation and equipped with citrus-blend lures in trees on the edge rows of the abandoned groves. For control plots, we hung autodisseminators with only citrus-blend lures. One week before the start of our trial, we hung ACP sticky traps on the managed trees directly across from the abandoned trees to assess ACP movement from the abandoned grove to the managed grove prior to deployment of autodisseminators. To facilitate monitoring of ACP movement during our trial, we captured and released up to 4800 ACP adults marked with fluorescent powder on abandoned trees every week for three consecutive weeks. We used different fluorescent colors to distinguish psyllids released in treatment plots from psyllids released in control plots. Every seven days during the first to fourth weeks of the trials, we inspected and replaced dispensers and ACP traps. During the fifth week, we also ‘tap sampled’ managed trees and abandoned trees. Movement of ACP from abandoned trees to managed trees was greatly reduced by the deployment of autodisseminators coated with Ifr-spore formulation. Prior to autodisseminator deployment, the numbers of ACP adults trapped on managed trees in treatment plots and control plots were not significantly different. After autodisseminator deployment, weekly trap counts of ACP adults were 28-50 % less for treatment plots compared to control plots. Our findings showed that deployment of Ifr-autodisseminators at the end of the active growing season for commercial citrus in Texas can reduce the numbers of ACP adults moving from abandoned groves to managed groves. We plan to repeat this trial during the spring (March-May) of 2015 to determine if our autodisseminators can reduce movement of ACP from abandoned groves to managed groves at the start of the active growing season in Texas. Deployment of Ifr-autodisseminators at the end and / or start of the growing season should minimize intraguild interactions between Ifr and Tamarixa radiata.
Work continues with a susceptible CLM colony towards estimation of LD50s and LD80s for some commonly used insecticides. Larvae and adults from the susceptible colony are exposed for 48 h to different doses (0; 0.01; 0.03; 0.1; 0.3; 1; 3; 10; 30; 50; 100, 300, 600 and 1000 ppm) of: Actara 25WG, Agri-Mek 0.15EC, Cyazypyr, Danitol, Delegate WG, Dimethoate, Intrepid 2F and Micromite 80WGS. For CLM larvae: Actara (thiamethoxam) LD50=4.18 (CL95: 1.86-8.51) ppm, LD80=524.12 (CL95 209.70-1827) (n = 1047; .2 = 9.54; d.f = 10; Heterogeneity = 0.95); Agri-Mek (abamectin): LD50= 0.314 ppm (CL95: 0.13-0.69), LD80=16.68 ppm (CL95: 6.14-78.56) (n = 1249; .2 = 13.17; d.f = 8; Heterogeneity = 1.65); Cyazypyr (cyantraniliprole): LD50=43.36 ppm (CL95: 15.80-131.55), LD80=49,413 (CL95: 0.19E+07) ppm, (n = 1,196; .2 = 7.64; d.f = 8; Heterogeneity = 0.96); Danitol (zeta-cypermethrin): LD50=381.78 (CL95: 86.33-11,771), LD80= 37, 220 (CL95: 31.70-53761.1) ppm (n = 561; .2 = 5.29; d.f = 9; Heterogeneity = 0.59); Delegate (spinetoram): LD50=2.67 (CL95: 0.87-6.42), LD80= 93.75 (CL95: 33.89-489.58) ppm (n = 938; .2 = 10.69; d.f = 7; Heterogeneity = 1.53); Dimethoate (dimethoate) LD50=1.56 ppm (CL95: 0.15-56.31) and LD80=497.45 ppm (CL95: 22.27-0.37E+09) (n = 546; .2 = 13.07; d.f = 6; Heterogeneity = 2.18); and Micromite (diflubenzuron): LD50=74.30 (CL95: 13.63-285.15), LD80=5,723 ppm (CL95: 994-0.129E+07), (n = 816; .2 = 19.92; d.f = 9; Heterogeneity = 2.21). For adults: Micromite LD50=387.40 (CL95: 143-2817), LD80= 9392 (CL95: 0.192E+07) ppm have been estimated but further replicates are required. Contact toxicity for Intrepid LD50 and LD80 values remain inestimable at the concentrations tested (up to 10 ppm). We will continue tests of higher concentrations. Delegate (spinetoram) LD50=2.84 (CL95: 0.77-73.74), LD80= 183.52 (CL95: 16.47-0.44E+07) ppm (n = 430; .2 = 6.30; d.f = 4; Heterogeneity = 1.58). Additional tests of Actara 25WG, Agri-Mek 0.15EC, Cyazypyr, Danitol, and Dimethoate are slated for the coming weeks. Work also continues on the insect growth regulator, methoxyfenozide (Intrepid). This insecticide acts as an ecdysone agonist, interfering with molting, and exhibiting low contact toxicity as indicated above. Therefore, we have initiated larval bioassays on caged branches infested with CLM in the laboratory. Applications of doses of 0; 0.01; 0.03; 0.1; 0.3; 1; 3; 10; 30; 50; 100, 300, 600 and 1000 ppm are applied when infested leaves contain 3-4th instar larvae. Evaluations of mortality will be conducted 14d after application. We have also initiated work to develop a bioassay method using artificial diet and two diet recipes and are being tested. We are developing a method to collect large numbers of CLM eggs from the established laboratory colony. Eggs will be transferred to cups containing insecticide-spiked artificial diet to determine diagnostic doses for this compound against larval CLM. Field-collected larvae are currently being evaluated for their responses to these insecticides. Although cases of field resistance have thus far not been observed, populations of CLM are currently high in groves in SW Florida and subjected to frequent insecticide applications for control of ACP, CLM and other pests.
Plasmid vector and helper DNA construct microinjections into ACP embryos have continued, though the survival of G0 nymphs has decreased from 25% to 15%. This is due in part to decreased numbers of fertilized eggs and embryonic viability which may be the result of seasonal changes and/or ACP colony inbreeding. The survival of hatched nymphs from injected eggs continues to be challenging with less than 20% surviving the transfer to flush (with most becoming stuck to double-stick tape), and less than 20% of these G0 nymphs surviving to adulthood. From 19 G0 adults, 8 were fertile and none yielded putative fluorescent transformed progeny. Plant health and flush quality have been variable affecting ACP viability, and improvements in both remain a high priority. To improve the number of surviving nymphs after hatching, alternatives to double-stick tape (used to hold eggs in place for injection) are being tested, in addition to setting up eggs on filter paper (that can be moistened) versus glass slides. Initial tests with two types of glue (E6000 and Tacky glue) have resulted in 60-80% nymphal hatching from uninjected eggs on either slides or filter paper. Once reaffirmed, survival of nymphs from glued injected eggs will be tested with and without moistened filter paper. While we expect the transformation marker gene to be expressed in ACP, this must be confirmed, and RT-PCR tests for transient DsRed fluorescent protein gene expression regulated by the IE1 and polyubiquitin promoters have been initiated. ACP transformation tests (since 10/1/2014) ‘ 3,680 eggs injected ‘ 550 G0 nymphs hatched (~15%) ‘ 99 transferred to flush ‘ 19 G0 adults ‘ 8 fertile G0 adults ‘ 40 G1 progeny screened – none fluorescent In addition to the above, assays are continuing to confirm passage of transinfected Wolbachia cell lines from injected parents (P1) to offspring (F1 generation). The survival of injected psyllids is high, with around 80% survival. Horizontal, or transovarial, passage of injected Wolbachia appears to be possible for at least one Wolbachia strain, however the frequency of passage to offspring appears to occur at a low frequency. This may be due to limitations of the injection procedure (e.g. developmental stage of psyllid, duration of infection with foreign Wolbachia, titer of foreign Wolbachia in P1 psyllids). Alternatively, injected Wolbachia strains may not be colonizing reproductive tissues sufficiently to promote transovarial passage to F1 psyllids. Concurrently with these studies, psyllids injected and naturally infected with Wolbachia strains of interests are being evaluated in bioassays to determine their capacity for induction of cytoplasmic incompatability and pathogen interference.
The large-scale validation of citrus leafminer (CLM) disruption with the ISCA DCEPT CLM technology has continued. During this past quarter we have continued to assess efficacy data, although many of the experiments were suspended during the dormant winter season. Our role continues to be with assisting in collection of efficacy data by trapping CLM males with pheromone traps as a surrogate measure of mating disruption. We have been analyzing our data and also modeling the economic return of using this product. Specifically, we have been investigating the need for two pheromone components as the active ingredient versus only a single component, which would make the product more cost effective. Cost contour plots of our data indicated that there was an interaction between the amount of pheromone released and the number of trees treated with pheromone. However, the difference in costs between a 3:1 blend (two component-more expensive) and triene only (one component-less expensive) application were insubstantial. This economic analysis suggests that choosing a triene application at a relatively low release rate with a point source density of approximately 89% (40 treated trees out of 45 total trees per plot) should be economically more desirable to growers than the 3:1 blend at the same rate of application. Although damage by CLM was not eliminated in these trials, there appears to be significant and fairly consistent reduction in damage. Planning for 2015 field trials will begin with a meeting at the Florida citrus show in Fort Pierce in late January.
The overall objective of this project is to investigate the feeding deterrence provided by reflective colored kaolin clays on citrus plants from ACP. Previously we reported the development of colored kaolin clays using select dyes, including FDA certified colorants approved for agricultural use by the EPA (list ‘180.920), which covers the range of visible spectrum for coating clays. During this reporting period a scale up synthesis to produce colored clays was carried out for ACP deterrence assays and further characterization. Ten different colored clays were developed with reflectance in the visible region with dyes e.g. FD&C Blue 1, FD&C Red 40, Basic Blue 54, Crystal Violet, and FD&C Yellow 6, D&C Violet 2 etc. ACP greenhouse bioassays are underway to determine reflective wavelength deterrence efficiency. The characterization of the colored clays has been carried out to determine their optical and physical properties. The optical properties of the clays have been determined by total reflectance measurements using an integrating sphere (Lambda 800 UV/VIS Spectrometer). Measurements of all modified clay materials were made using unmodified kaolin clay as a reference. Reflection measurements showed that each material has an altered absorption peak and reflectance not exhibited by the unmodified clay. For example modified clays showed high reflectance in 350- 490 nm (D&C Violet 2), 350- 550nm (FD&C Blue 1), 575-750nm (FD&C Red 40), 505-750nm (FD&C Yellow 5), and 625 -750nm (Basic Violet 16), covering the visible spectrum range. Future experiments are being planned to investigate the change in the reflectance of citrus leaves with developed colored clay materials. The dispersion of the clay particles in suspension, the ability to apply as foliar spray, and eventual adhesion to the leaves is dependent upon the surface charge of the particles. Zeta Potential experiments were performed for each colored clay material to estimate the surface charge. While the unmodified kaolin clay zeta potential was found to be negative, the surface modifications and doping of clay material with the dyes, led to significant changes in the zeta potential. These results will help the selection of appropriate adjuvants necessary to provide dispersion of clay particles. Tests are also being conducted on modified kaolin formulations to increase rain-fastness.
Issue date for this grant was 13 July 2012. Objectives are: 1. Assess effects of abiotic factors (light quality, photoperiod, air flow, temperature fluctuations) on psyllid movement, 2. Evaluate physiological limits and biotic factors effecting of movement including feeding, egg load, infection status, and population density, 3. Evaluate techniques for tracking psyllid movement in the field for mark recapture studies, 4. Characterize seasonal patterns of ACP distribution and movement at different scales in the field, 5. Develop strategies to protect young trees from colonization by ACP utilizing UV reflection for repellency and insecticide treated trap crops (such as Bergera koenigii) to attract and kill. Objective 1. Bioasssays using a wind tunnel to determine ACP response to different stimuli have shown that ACP movement stopped at temperatures below 67 F, and in the absence of light. Wind speeds above 4.25 mph reduced movement as did humidity over 70%. ACP tended to move with the wind when lighting is uniform but also moved into the wind. Increased light intensity did not increase ACP movement and lighting type with varying spectrum of light intensity did not impact ACP behavior. Objective 2. Dr. Stelinski’s group recently analyzed results from three field experiments where citrus trees were sprayed with egg and milk proteins. Subsequently, sticky traps were deposited at various distances from the sprayed area. Psyllids collected were submitted to ELISA for detection of milk and egg proteins. Therefore, we knew that each psyllid positive for milk protein as indicated by ELISA had dispersed from the sprayed area. The result of these field experiments demonstrated that: 1) psyllid movement from abandoned citrus groves to adjacent managed citrus groves was greatest during the spring and summer months and decreased significantly during the colder winter months; 2) Psyllids were able to traverse geographic barriers such as roads and fallow fields; 3) Psyllids were able to disperse at least 2 km within 12 days; 4) wind direction was not correlated with the number of marked psyllids captured; 5) grove management and presence of flush were the most important factors that increased the capture of marked psyllids. The results further confirm that abandoned citrus groves in Florida serve as reservoirs for ACP which can disperse over long distances despite geographical barriers. These results are in accordance with our laboratory results obtained with a flight mill. Objective 3. Sticky cards were placed in a field of young citrus placed between trees on tomato stakes and monitored hourly for 15 different 24 hour periods. Wind speed, air temperature, and solar radiation were found to be good indicators of ACP movement while barometric pressure was not. Rainfall and time of day were marginally correlated to ACP movement. No ACP were observed to land on sticky cards at night. Objective 4. Two-sided clear and yellow sticky cards were placed in several groves to compare directional responses in an attempt to determine whether ACP adults flying in one direction may turn back and be captured on the opposite side of the yellow card. The results of the clear vs. yellow sticky cards thus far are inconclusive due to low ACP movement in the test areas. Additional attempts will be made to determine differences in ACP response to the two types of sticky cards when movement and populations increase in the spring. Objective 5. A paper reporting positive results using UV reflective mulch to repel ACP the first 2 years after planting has been published in the refereed journal Pest Management Science. Tap sampling has reveled the mulch still works to reduce the ACP populations on trees 2.5 years old. In another trial, repellency was demonstrated by the result that fewer ACP were captured on sticky traps set at 1 m in reflective mulch compared to 2 m, where as more ACP were captured more at 1m than 2m over white mulch or no mulch (grass). Manscripts are being prepared on environmental factors influencing psyllid behavior and movement and dispersal patterns of the Asian citrus psyllid.
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.
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