Large amounts of Imidacloprid (IMD) have been applied over the past several years with the potential for many more applications to young trees now being planted with little known about the movement and environmental fate of this compound under Florida soil conditions. Likewise, the impact of irrigation and rainfall on soil concentrations and plant uptake shortly after application of IMD is not well understood. Thus, the objectives of this study were: 1) documentation of citrus greening (HLB) management by application of IMD, 2) Determine sorption and transformation of IMD in soils, 3) Assess the soil transport and plant uptake of IMD, 4) Characterize root zone water movement using bromide (Br) tracer, 5) Monitor Asian Citrus Psyllid (ACP) populations and incidence of HLB. Three age groups of citrus (‘Hamlin’ sweet orange on Swingle) trees were used in this study to determine the efficacy of currently labeled soil drench rates of IMD on ACP populations and the insecticides soil movement and tree uptake. The age groups were newly reset (T1), six years old (T2), and 16 years old (T3). Irrigation was applied to the same age groups for the same time and ground surface area at three rates; 6 gallons per hour (I1), 10 gallons per hour (I2), and 16 gallons per hour (I3). The soil was representative of Immokalee fine sand series (sandy, siliceous, hyperthermic, Arenic Haplaquods). Two spring and two summer studies (total 4) were conducted over a two year period. ACP adult and nymph populations were monitored 12 weeks after IMD application. Soil IMD concentrations decreased rapidly with time to near zero concentrations in the root zone in T1at 28 days after application indicating an average uptake of greater than 60% over the 28 days. The tracer was completely leached out from the soil profile of 12, 18 and 24 inches for T1, T2 and T3 in about 8, 16 and 20 days after application respectively. These data indicate water movement from irrigation below the root zone within 20 days, only 8 days less than the time IMD persists in the root zone. IMD showed effective systemic control of the ACP two weeks after application and persisted for about 7 weeks even though IMD had leached out of the root zone. However, tissue concentrations remained low in T2 and T3 leaves and ACP populations decreased but were higher than T1 indicating a dilution of IMD in leaf tissue and ineffective control. IMD sorption and persistence was quantified in a soil column studies. Organic matter in the A and Bh horizons increased KD near 1.6 mLg-1 from KD of 0.1 to 0.3 in the E horizon. These results agree with field studies indicating high rate of movement in the soil only persisting only slightly greater than movement of a tracer designed to move with the soil wetting front. Results of these studies also indicate that IMD could persist in non-irrigated areas or non-target application areas between rainfall because of its high half-life (1 to 2.2 years) measured in the laboratory.
1. Efficient methods of statistical inference to estimate epidemiological parameters from maps of emerging epidemic. Using citrus canker as a test system, we have developed and tested Bayesian statistical methods for parameter estimation for epidemiological models of an emerging epidemic. The models are stochastic, taking account of inherent variation in weather-driven, disease spread and uncertainties about epidemiological parameters, typical of emerging epidemics. The statistical methodology and model formulation has been adapted and tested to allow for non-isotropic spread of the pathogen and for sudden changes in transmission rates, typical of severe storms. The modes can also account for progressive changes in transmission rates, for example during the onset of dry periods as occurred with citrus canker in Florida. We have also examined the effectiveness of data collection for estimation of epidemiological parameters: including comparison of different time windows (measured by the initiation and duration of monitoring relative to arrival of the pathogen) for parameter estimation. Parameters were efficiently estimated from relatively short-time windows. We also demonstrated marked consistency in epidemiological parameters for different survey sites. The methodology, which includes Markov chain Monte Carlo (MCMC) and Approximate Bayesian Computation (ABC) techniques is transferable to other diseases and has been tested using data for citrus Huanglongbing (HLB). Papers 1) MCMC for estimation of epidemiological parameters in the Miami area, and 2) HLB epidemiology have been submitted to PLoS One and PNAS. 2. Develop a generic epidemiological model to compare control scenarios and to optimize the probability of controlling and managing high-risk pathogens of agricultural significance. The models were used to compare different control strategies, involving varying removal radii around infected sites. In addition to predicting the relative probabilities of success, by accounting for variability and uncertainty, the models also allow us to predict the risks of failure of different control scenarios. The models are generalized to allow for cryptic infection and to develop risk and hazard maps for county and state-wide spread. Risk maps consider where is the pathogen is likely to arrive; hazard maps address where the rate of spread is likely to be most rapid. We have also built a model that represents the spread of HLB within an individual tree. Typically, models at the grove- or landscape-scale assume that each tree is entirely infected or not (rather than considering the specific level of bacterial/viral titer in each tree). We have adapted the compartmental framework (that classified trees as healthy or infected) to include multiple infectious classes, each with a different potential for transmission of the pathogen. The relative importance of transmission within the vascular system of the tree and between leaves via psyllid vectors was explored. A novel feature of our modeling approach is that it explicitly incorporates the ability of LAS bacteria to survive in the vascular system of the plant. The model can be flexibly used for a variety of purposes: to inform the force of infection of each infected tree in grove-scale models and to compare the efficacy of disease control and mitigation strategies including roguing, insecticides, nutritional treatments and thermotherapy. 3. Develop user friendly model ‘front ends’ for researchers/regulatory agencies. Via the ‘front end’ we can examine different ‘what-if’ scenarios by visualizing outcomes of different control scenarios, importantly allowing for uncertainty in weather and knowledge of the pathogen typical of many emerging pathogens. The model is available on a web-portal (http://www.webidemics.com/). The model is underpinned by the detailed grove- and landscape-scale models. A research paper introducing the user-friendly approach is drafted for publication and compares different scenarios for control.
During this reporting period, citrus engineered to produce dsRNA’s with a sequence matching specific psyllid genes identified as RNAi targets in the INNOCENTIVE RNAi project were produced using the Citrus tristeza virus vector. Adult psyllid feeding on leaves from these plants displayed up to 90% mortality. In the same experiments mortality was linked to a noted drop in psyllid transcript abundance for the targeted gene. Also, in a preliminary choice test where psyllid were placed in cages with these dsRNA producing citrus and citrus not producing dsRNAs, psyllids avoided the dsRNA plants and colonized control citrus almost exclusively. Finally, gene expression (RNAseq) studies were continued to observe the total psyllid transcriptome response to the dsRNA feeding. The RNAseq sequence results have been obtained and bioinformatics analysis is currently being conducted.
During this reporting period, citrus engineered to produce dsRNA’s with a sequence matching specific psyllid genes identified as RNAi targets in the INNOCENTIVE RNAi project were produced using the Citrus tristeza virus vector. Adult psyllid feeding on leaves from these plants displayed up to 90% mortality. In the same experiments mortality was linked to a noted drop in psyllid transcript abundance for the targeted gene. Also, in a preliminary choice test where psyllid were placed in cages with these dsRNA producing citrus and citrus not producing dsRNAs, psyllids avoided the dsRNA plants and colonized control citrus almost exclusively. Finally, gene expression (RNAseq) studies were continued to observe the total psyllid transcriptome response to the dsRNA feeding. The RNAseq sequence results have been obtained and bioinformatics analysis is currently being conducted.
We repeated an experiment comparing Steinernema krauseii to H. bacteriophora and S. riobrave. S. krauseii is a temperate species capable of infecting hosts at temperatures as low as 8 C. As found in the original trial, this temperate species was inferior to H. bacteriophora and S. riobrave in its ability to kill Diaprepes weevil larvae, even at temperatures as low as 17 C (below the recommended temperature for use of S. riobrave). In contrast to an earlier experiment, an isolate of H. bacteriophora from upstate New York was no more effective than the commercial isolate of H. bacteriophora during simulated winter cold events. We investigated whether duration of exposure to cold temperature (24, 48, 72 h) affected susceptibility of weevils to EPN parasitism. S. riobrave was 3.5-fold more effective than Hb in killing weevil larvae. Unlike the results of previous experiments, cold-stressing weevils at 10 C or 14 C did not appreciably increase their susceptibility to Hb, whereas 10 C doubled the susceptibility of lavae to infection by Sr and 14 C increased infection by 50% compared to non-stressed weevils. Duration of exposure to cold temperature between 1-3 days had no effect on weevil susceptibility to infection. The experiment is being repeated.
Xambr’, Paran’, Brazil canker control plots: The experiment was designed to quantify the effect of windbreaks, copper sprays, and insecticide sprays individually and in various conbinations on citrus canker management. The preliminary studies followed by initial plot preparation has continued over a 10-year period. At one point the experiments were ready to start in Minas Geris State, Brazil after 6 years of preparation, but halted due to political posturing between the new State Minister of Agriculture and the Sao Paulo State Experiments Station. Unfortunately, we were forced to abandon these plots and move to the State of Parana to re-establish the experiment, which required additional years. This re-establishment is the portion funded by CRDF. In 2010 replicated field plots were established with natural windbreaks of Casuarina. Trees will finally be inoculated and the experiment started in December 2013, unfortunately, after the grant has ended. However, USDA, ARS, Fundecitrus, and the University of Sao Paulo are committed to this project and will continue on without funding to complete the experiments and publish the results within the next two years. Over the 2012 and 2013 seasons, we also established windbreak plots in Florida commercial groves and collected substantial data that has been integrated into our understanding of wind/rain dynamic of canker and publications. We were able to confirm that the number of wind gusts >11 mph (canker infection inducing) increased with distance from the windbreak whether measured within the north-to-south rows or across the rows from east to west. Consequently, the highest fruit disease recorded in the cv. Valencia orange orchard (10 percent fruit cankered) was in the center of the 11-acre block surrounded by windbreaks and the lowest incidence (<2 percent fruit cankered) was in the east-west row location nearest the windbreak. This has helped us to understand how best to design windbreaks in Florida citrus and to develop mathematical algorithms for maximization of windbreak efficacy. This methodology will be highly beneficial to the Florida grapefruit growers who are attempting to maximize canker control to maintain market share in EU markets under increasing scrutiny of canker infection of imported fruit from Florida. We have also been developing a programmable leaf wetness controller which is a computerized devise that allows us to create, control, and monitor leaf wetness under grove conditions. By doing so we are studying the effects of wetting and drying and their duration and dynamics on surface populations of canker and other bacterial pathogens. This is of heightened concern to understand how to diminish canker and food-born bacterial pathogens on citrus surfaces that affect international marketing and human health. Along the way we have developed a new leaf wetness sensor (an adequate sensor to accurately measure leaf wetness has not previously existed). Potentially, this will have a profound effect on future bacterial population work on citrus and other crops. Concurrently, eight studies on the effect of wind on citrus canker and canker populations on fruit have been completed and published and additional publications are in preparation. Thus, the project has been quite successful and has moved our knowledge base of canker distribution via wind and rain and disease control forward substantially and additional valuable results will continue to come as outgrowths from this grant for a few more years.
We tested the effect of cold temperature stress to the weevil on the subsequent infectivity by Steinernema riobrave at various soil temperatures. The aim was to determine the lower temperature limit at which weevil stress increases nematode infectivity. Weevils were pre-exposed to 10, 12, 27oC for 72 hours after which nematodes were added to soil and incubated at 13, 15, 17 and 27oC. All weevils were killed at 27oC post-exposure. At 17oC post-exposure, 58% of weevils were killed when pre-exposed to 10oC and 12oC, and 20% were killed if preexposed to 27oC. At either 15oC or 13oC post-exposure, more than twice as many weevils were killed at the two lowest pre-exposure temperatures compared to 27oC pre-exposure, but fewer than 30% of the weevils were killed. Therefore, the cold temperature stress appears to reduce the lower temperature limit at which Sr effectively kills weevils by approximately 4oC, making it much more likely that EPNs applied during a freeze event will subsequently experience suitable temperatures for infection. A second experiment to measure the duration of ‘super-susceptibility’ of weevils post stress was unsuccessful for unknown reasons (no significant effect of low vs high pre-exposure temperatures). It is being repeated. We conducted two experiments in the field during cold events in January. However, the average minimum soil temperature for the winter months (December-February) this year were the warmest in more than 10 years, 3oC warmer than the average minimum for the previous decade. The coldest event (12.2) did not approach the threshold (10oC) for weevil stress measured in the laboratory assays. Nevertheless, two trials were conducted in which caged sentinel larvae were buried (15 cm) beneath citrus trees on 18 and 22 January. Lowest average temperatures attained were 12oC and highest temperatures during 10 days following burial was 16oC. Fewer than 10% of larvae treated with S. riobrave or H. bacteriophora were killed during the trial. It is interesting that whereas low temperature stress did not affect susceptibility of weevil larvae to S. krauseii (a species effective at low temperature) in the laboratory assays, 40% of the insects treated in the field in January with this EPN species died (although the nematode did not reproduce in the cadavers incubated in the laboratory). Because of the elevated soil temperatures experienced this winter, we shall repeat this work in the coming winter not only in Lake Alfred, but in fields at the extreme northerly range of citrus in Florida, in order to at least test proof of concept.
We repeated an experiment measuring the effect of duration (24, 48, 72 h) of cold temperature stress on susceptibility of weevil larvae to entomopathogenic nematodes. Results for Steinernema riobrave were essentially as reported previously ‘ exposure to low temperatures (10C and 14C) for more than 24 h did not increase susceptibility. In contrast to a previous experiment, H. bacteriophora killed more weevils exposed to low temperatures than weevils maintained at 27C, but there was a pronounced but non-significant trend for greater efficacy with longer exposure to low temperature. This should be confirmed, because cold temperature events in Florida are frequently of short duration. We initiated trials to 1) determine the time required by weevils to recover from cold stress when exposed to various higher temperatures, 2) determine the relationship between cold stress and EPN efficacy in a range of temperatures below the recommended low-temperature threshold (20C) and 3) determine if nematodes adapt to stressful low temperature by becoming less susceptible to EPNs with repeated exposure to low temperature.
Entomopathogenic nematodes: Amending soils to increase biological control of insect pests We repeated an experiment at the Bartow site in which we buried caged weevil larvae in all plots and evaluated the mortality rate after 7 days in the soil. The previous trial revealed no significant effect of sulfur amendments (to reduce soil pH) on infection of buried weevils by S. diaprepesi. We obtained similar results in the second trial. S. diaprepesi infected weevils were 23% more abundant in low pH plots and H. indica were 44% more abundant in high pH plots. The trends were the same in both trials, but treatment effects were not significant in either. We assayed soils from the two treatments (sulfur amended and unamended) by adding S. diaprepesi encumbered with spores of Paenibacillus sp. After seven days spore-free nematodes were more abundant in sulfur amended, compared to non-amended soil, but the differences were not great (12% vs 5%; P=0.05) and most nematodes remained encumbered by spores. Soil pH of sulfur amended soil was 6.4 vs 7.1 in non-amended soil. The pH difference was less than observed earlier in the year and at a level approaching ineffective for removing spores. Consequently, soil in the field experiment was again treated with Tiger 90 sulfur and assays using caged weevil larvae will be reinitiated when soil pH in amended plots becomes ‘6.0. Because soil phosphorus was positively related to S. diaprepesi abundance in several field trials, we bioassayed Paenibacillus spore adherence to S. diaprepesi in solutions of 10, 50, 100 molar phosphate buffer, each adjusted to a range of pH between 5.0-8.0. As shown previously, spores detached from the nematode cuticle in inverse proportion to solution pH, but phosphate levels had no effect on spore adherence. We initiated bioassays to study effects of soil water potential on survival of the 4 most commonly encountered EPN species. Soil from the conventionally managed and advanced production system treatments (Schumann study) were oven dried and moistened to 2% (dry), 6% (
The long-term goal of this project was to develop a framework for sustainable nutrient management in South Texas Citrus orchards to ensure healthy productive trees with improved fruit quality. Specific Project Objectives for 2013 were to develop a baseline picture of native soil fertility across citrus orchards in the Rio Grande Valley, TX, and to determine which nutrients are most limiting. Procedures: Soil and leaf tissue samples were collected from thirty-six (36) orchard locations across the Valley and analyzed for mineral nutrient contents. Different soil analytical procedures were evaluated in several soil testing labs. Findings: While critical mineral nutrients such as potassium and phosphorus were found to be adequate according to the widely-used Mehlich-3 soil extraction method, they were low or marginal based on the CO2/H2O method. Although calcium, magnesium and potassium are abundant in calcareous soils in south Texas, these minerals may not be readily available for tree uptake. Potassium is readily fixed in clay minerals and rendered unavailable for uptake. Adequate calcium uptake may be limited by low transpiration since this mineral relies on a steady transpiration stream for uptake. Competitive uptake interactions among these minerals could also influence uptake. Soil phosphorus was consistently below adequacy levels, whereas boron levels seem to be excessive. It is now known that HLB and phytophtora infection severely limit root growth and uptake activity. Low soil P levels may contribute to this observation. Earlier observations from Florida indicated that Ca may play a major role in tree tolerance to HLB infection. More recent observations indicate that other nutrients, especially micro nutrients such as zinc and manganese may also be important. In the current study, zinc and manganese concentrations in grapefruit leaf tissue were consistently below sufficiency levels. There are currently no nutritional guidelines for citrus production based on local soil and weather conditions in south Texas. Fertilization guidelines adopted from other production regions are not optimized for fruit quality (solids content, color, etc.) or tolerance to pests and diseases. Nutrients are continually mined/removed from orchards each year with harvested fruits with little replacement. Higher yields are expected to remove larger quantities of nutrients, thus necessitating replacement. Future research as a continuation of this project will estimate nutrient removal amounts in order to determine which nutrients are being removed in the largest quantities and the implications for fruit quality and tolerance/resistance to biotic and abiotic stresses. Future research should also determine possible mechanisms and strategies to enhance nutrient solubilization and availability for tree uptake. In summary, while soils in south Texas seem to contain adequate levels of critical macro and micro-nutrients, most these nutrients are fixed and unavailable for tree uptake. Orchard-specific nutrient budgeting programs based on yield expectations are needed for optimal tree growth, fruit yield/quality, and resistance to biotic and abiotic stresses.
Imidacloprid (IMD) is a systemic pesticide soil-drenched to control the citrus greening primary vector Diaphorina citri Kuwayama, or Asian Citrus Psyllid (ACP). Imidacloprid fate and transport in Immokalee Fine Sand (IFS) soils was studied in SW Florida Flatwoods during citrus greening (CG) management. Soil and leaf samples have been collected in spring (dry) and summer (rainy) seasons for the past two years. Imidacloprid rates have been applied at 1X, 2X and 4X lable rates on transplants (1-2 yrs), moderate age (5-7 yrs), and mature trees (>10 yrs) each season. The 2X and 4X applications typically have proportionally greater soil concentrations in the 0-15 cm depth and decrease to less than 1 .g per mg soil. Deeper soil depths typically never increase above 0.5 .g per mg soil. These results would indicate that Imidacloprid moves with the wetting front with low soil residual concentrations. Sorption and degradation studies showed a weakly-sorbed (log Koc, 1.1-2.4) and persistent chemical (half-lives 0.9-2.3 years) with high potential for leaching. We also established field experiments in young Hamlin trees with micro-sprinkler irrigation, during 4 growing seasons between 2011 and 2013. IMD was soil-drench applied in the root zone (PA), and in a control zone not affected by roots (NPA). IMD concentrations (.g g soil-1) were higher for the NPA than PA as a function of time due to uptake by the citrus trees. IMD leached out of the root zone about 3 to 4 weeks after application in the summer, and about 6 to 8 weeks after application during spring. Nonetheless, there was effective systemic control of the CG vector Diaphorina citri Kuwayama (ACP) at about 2 weeks after application, where treated trees showed consistently lower ACP adults and immatures infestation, continuing for at least 8 weeks. A method to analyze IMD from citrus tissue (ng g-1) was developed using HPLC-MS/MS detection, and preliminary data are in close agreement with our findings in soils and insect counts on trees. Close irrigation and rainfall monitoring during IMD application is of the utmost importance to avoid leaching problems in groundwater resources of SW Florida, and to ensure systemic control of the ACP.
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. Obj. 1: We continue to conduct field and laboratory studies toward obtaining an understanding of ACP trap response behavior by manipulating psyllid behavior around the trap (i.e., increase trap active distance). We have a number of positive results from our latest bioassays and continue to tweak the trap structural components to increase trap efficiency through field testing. We also have continued testing of semiochemical lures that supposedly attract psyllids provided by other scientists as well as a number of possible lures known to be behaviorally-active to other insects that we selected for testing . This work is ongoing and more experiments are being conducted but none of the lures we have tested have provided any increase in trap catch. Results from these studies and similar results from bioassays on other insects (some new research by ARS personnel notwithstanding) lead one to conclude that it is unlikely that a strong lure for ACP exists, can be made or discovered. This research is continuing and we expect to make much more rapid progress in the next quarter by exploiting the significantly higher ACP populations in Puerto Rico. Obj. 2: We have initiated the areawide psyllid sampling objective to detect and develop new biological controls for use against ACP. We have begun sampling in the northern most citrus populations in Alachua County and around Ft. Pierce with the intention of systematically sampling by working south from the northern region and outward in all direction from Ft. Pierce. The standard prototype 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. So far we have not identified any new pathogens but we will continue to conduct this sampling effort Florida as well as expand it into Puerto Rico.
October 2013 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: All preliminary experiments have been completed for propiconazole, difenoconazole, and tetraconazole. Many concentrations were tested to find opitmal concentration ranges for each fungicide. We found an expected effect of light duration on the fungicide efficacy. We are exploring this further. Experiments with fenbuconazole are up-coming. Objective 2: The field trial was established in a commercial grove that was identified with ‘Valencia’ fruit with black spot. In spring 2013, the site was was scouted and rows of ‘Valencia’ were identified that had fairly even distribution of fruit with symptoms of black spot. Plots contained three trees with at least two trees serving as buffers between each plot. There were four replications per treatment arranged in a randomized complete block design. Sprays were initiated in May after fruit harvest. Applications were applied with an Air blast sprayer calibrated to deliver 126 gal per A operating at 200 psi and 3 mph with a 25 gallon mix. Applications continued until September. The grove was recently inspected for symptoms but none have developed yet. Objective 3: We are currently looking for suitable sites to conduct the spatial studies. This study will begin in earnest close to fruit maturity when symptoms become apparent. Objective 4: A contaminant was discovered in the G. citricarpa isolate used for the first set of experiments and steps taken to assure future work with clean cultures of the isolate. In addition, three additional Florida isolates were obtained for evaluation for possible use in the experiments. The government shutdown also disrupted work on the project. In an experiment repeating some of the earlier work and evaluating the effects of essential oils (Benzaldehyde, Cinnamaldehyde, Trans-cinnamaldehyde, Carvacrol, Eugenol, Citral, and Eucalypol) at two different concentrations on the organism growth in vitro, it was found that Citral (especially 100 mg/l) and Carvarol significantly inhibited mycelium growth often completely; Cinnamaldehyde, Trans-cinnamaldehyde, and Benzaldehyde inhibited mycelial growth moderately; and
Dr. Justin George assumed a postdoctoral position at USHRL in August and has begun work on this project, replacing Dr. Paul Robbins. Dr. Robbins has transferred to a position in the laboratory of Dr. Joe Patt where he will continue to work on ACP behavior and orientation to plant odors. Justin did his PhD at Pennsylvania State University in the laboratory of Tom Baker, an excellent chemical ecologist, where he worked on the behavior of mosquitoes. He brings expertise in electrophysiology and will contribute to our efforts to decipher the cues governing ACP behavior and orientation to host plants. There is no new progress to report at this time.
September 2013 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. We are in the process of hiring one technician for the project. Others have been hired and will be starting shortly. Small plots were set up an Immokalee area grove with 5% urea, CaCO3, Soil set, Compost Aid, Soil Set and Compost aid, and an untreated control. Leaves were collected and treated. The samples were collected and are still being processed. The DNA sequence of the mating type locus has been established for G. citricarpa. Forty-two isolates collected from Collier and Hendry counties have been screened to identify isolates of opposite mating types. This information will be used to establish compatible pairings of isolates to investigate the environmental conditions required for pseudothecium and ascospore development. To look at the effect of bagasse, the Senior Biological Scientist and the post doctoral associate worked on assembling the necessary supplies and components to carry out the controlled experiment portion of this project. Five treatments contained leaves which had been inoculated with conidia of Guignardia citricarpa and five treatments contained un-inoculated control leaves. Treatments were as follows: 1) Bagasse; 2) Bagasse + microbial consortium + molasses; 3) Microbial consortium + molasses; 4) Urea; and 5) Untreated. In addition to the above mentioned treatments, two additional treatments were included which did not contain citrus leaves. These were: 1) Bagasse + microbial consortium; and 2) Bagasse. The microbial consortiums were Ceriporiopsis subvermispora, Cellumonas flavigena, and Azospirillum brasilense. Soil weight, leaf weight, and then the total weight of each box was recorded. Leaves were collected at time zero and at fifteen day intervals. Leaf infection by G. citricarpa was verified. At each collection time, leaves were dried and weighed. Data collection and analysis is still in process. This experiment will be repeated during the next quarter. Based on observations and preliminary results, several modifications to the experiment will be implemented. The USHRL crosses have been cut back to continue the in vitro assays and we expect to receive samples within the next month for continued experimentation. The subcontracting process with the University of Queensland is complete. In QL leaf litter samples have been collected from 4 commercial blocks for the seasonal dynamics project. Fungal fructification is being enumerated. Because the number of structures is low, monthly sampling will continue until the onset of rains. An inventory of the sour orange crosses was taken and an prototype inoculation of procedure was tried. The results won’t be known for several months.
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