1a. Objectives (from AD-416):
1. Characterize the pathogen and elucidate its role in disease onset and development. 2. Investigate citrus plant response to pathogen infection. 3. Establish disease epidemiology and evaluate disease mitigation by cultural practices or competing microbes. 4. Define Asian Citrus Psyllid (ACP) biology and behavior important to HLB infection. 5. Develop and deliver ACP vector management. 6. Utilize emerging citrus genomics and transcriptomics to develop solutions to citrus diseases. 7. Develop transgenic and viral/bacterial-vector mediation of citrus resistance to disease. 8. Find solutions to citrus disease through use of model systems, including chemical screening and therapeutics development.
1b. Approach (from AD-416):
HLB is the most important exotic disease impacting Florida’s $9 billion industry and threatens other citrus-producing states (California, Arizona and Texas). Production costs have increased up to 100% per acre for just HLB and vector management, and some growers are considering abandonment of citrus. The Citrus Research and Development Foundation (CRDF), a Direct Support Organization (DSO) of the University of Florida and working on behalf of the Florida citrus industry, has utilized a strategic plan for research developed by the National Academies of Science (NAS)to establish an ongoing research agenda which currently involves projects with 22 institutions in 7 states, 3 USDA regional offices, 3 other countries, and 4 outside supporting institutions. The research proposed for support in this Specific Cooperative Agreement (SCA) comprises 8 components that focus on the pathogen, insect vectors, plant resistance, and delivery of short and long-term solutions directly to Florida’s 6,000 small and large growers. Funding to sustain this effort has largely come from grower research/marketing taxes, and will be complemented by this SCA support to help fund critical new research projects to develop and deliver solutions. Using the process recommended by the NAS strategic plan, CRDF request and evaluates proposals for new research addressing Huanglongbing (HLB). The CRDF national Scientific Advisory Board (SAB) provides peer review of all projects submitted for funding, This agreement proposes to use USDA funds to meet project costs during the agreement period for projects rated highly for their scientific merit and being evaluated for their impact on citrus production by the CRDF Research Management Committee. Solutions to diseases and pests affecting Florida and other US citrus producing areas will only emerge through aggressive research, product development and delivery. It is the mission of CRDF to partner with the strongest teams worldwide to develop the best science and application to the problems. Support from the SCA will advance our goals and sustain Florida’s longstanding premier citrus industry. Now in the third year addressing HLB research, CRDF manages a large portfolio of research being conducted by public and private institutions within Florida and beyond. Continuing costs of this portfolio or multi-year research projects addressing NAS priorities is in excess of $15 million, with new projects being solicited each year to build on advances and fill gaps in the NAS priorities. The goal is to identify and fund the best teams and proposals to mitigate citrus production loss from infectious disease in Florida.
3. Progress Report:
This research relates to inhouse project objective 1: Chemical Ecology: Develop semiochemical-based control methods for citrus pests, particularly Asian citrus psyllid (ACP). Objective 2: Biological Control: Develop new and improve existing biological control methods for ACP and sharpshooter species. Objective 3: Host Plant Resistance: Develop host plant resistance in citrus to key pests. Culturing Liberibacter asiaticus: 496 medium formulations have been tested for support of Candidatus Liberibacter asiaticus (CLas). None have supported continuous axenic culture of the bacterium. Various supplements, such as mucin, zinc sulfate, potassium nitrate taurine, betaine, folic acid, catalase, yeastolate, cyclic ampicillin (AMP), arabinose, sodium pyruvate, and lactoalbumin hydrolysate, have been tried. Some of the medium modifications appear to promote Las biofilm formation around the psyllid alimentary tracts that have been used as a source of inoculum, but planktonic growth has been minimal. Attempts to culture Las continue. Characterization of critical genes involved in spread of citrus canker pathogen Xanthomonas axonopodis pv. Citri: We have identified 12 EZ-Tn5 transposon mutants of Xac with reduced capacities of release from citrus canker lesions. The insertion sites of the 12 mutants have been identified with insertions in 11 different genes including xanA, btuB, gumC, gumB, gumK, gpsA, and several hypothetical genes. Currently, complementation analysis of the mutants is underway. The complementation constructs are being made for the selected mutants using pUFR053. Bacterial growth assays of the mutants and the wild type strain in grapefruit 'Duncan'and sweet orange 'Valencia' have been conducted. Evaluation of foliar Zinc and Manganese application for control of huanglongbing (HLB) or associated symptom development: The field study was set up with six treatments located in 4 plots of 150 trees per treatment (interior 10 trees in each block were identified for polymerase chain reaction (PCR), leaf nutrition sampling, tree health and yield evaluation). Four disease ratings have been taken so far and a slight decline in tree health has been observed, but no significant treatment effects have been observed. The 2011 harvest experienced a 27% yield increase in all treatments compared with the 2010 harvest, but no significant treatment effects were observed. Novel formulations and application methods for bactericides to control systemic HLB infection: In the greenhouse, 0.5-1.0 cm dia trunks of Hamlin orange trees (1 yr old) were painted with a Magna-Bon (MB) (Copper sulfate pentahydrate), Cop-R-Quik (copper nitrate), a copper phosphite (CP), an experimental copper (EXP) or oxytetracycline (OTC) mixed with 0.1% Pentra-Bark or left non-treated. The canker disease control effect was measured as the number of the canker lesions in a detached leaf in vitro assay. Preliminary trials conducted with potted nursery and young filed trees indicate that soil drench is a more effective and consistent application method for increasing copper status of leaves than trunk painting. The bactericides chosen for further evaluation include EXP, Cop-R-Quik, MB, and a novel copper phosphite (CP) and zinc phosphite (ZP). Terminal leaves will be collected periodically to measure copper or zinc concentration to test for systemic movement of the metals. Establishment of Citrus Health Management Areas (CHMAs): A program assistant was recently hired to serve as the CHMA program assistant within IFAS. The CHMA program assistant has been contacting CHMA grower leaders to help facilitate their communication of ongoing psyllid control efforts through the CHMA website and has also been working to summarize data from previous and current psyllid scouting reports in a grower friendly format. Transmission of the emerging citrus pathogen cytoplasmic citrus leprosis virus by endemic mites: Cytoplasmic citrus leprosis infected samples previously sent from our cooperator in Colombia continued to be negative in PCR and antibody tests for cytoplasmic citrus leprosis virus. Viral particles similar to those previously published for cytoplasmic citrus leprosis were discovered in all the samples. Sequencing was done and a new virus was identified by our lab. With our new primers we have found that he has transmitted this new virus with Brevipalpus mites from Colombia. Symptoms of the new virus are identical to citrus leprosis. How the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development: The objective is to examine initiation of HLB infection after psyllid inoculation to investigate how introduction of the pathogenic bacterium into different types of flushes of a tree affects establishment of infection. For this purpose, we have been setting a number of trials using sweet orange plants that have young growing flushes and plants that have only matured flushes. These plants have been exposed to HLB-infected psyllids. Leaves on which psyllids fed were analyzed by PCR to see if the HLB bacterium could be detected soon after the exposure of leaves to infected psyllids. We were able to detect presence of the bacterium fairly early after the initial exposure. Plants exposed to infected psyllids have been monitored for the development of infection. How does infection of Asian citrus psylid (ACP) with Candidatus Liberibacter asiaticus (CLas) affect the behavioral response of the vector to healthy versus diseased citrus trees? Our objective was to determine how psyllid behavior is affected by Las-infection of citrus. In the last quarter, we have been successful in making nutrient deficient uninfected plants to use in these experiments. We are in the process of assessing the volatile profile of three plant types: Healthy, Nutrient Deficient, and HLB infected citrus. Upon analysis of Healthy, Nutrient Deficient and HLB infected plants, we will determine the characteristic components of HLB infected plant odor. These odors will then be applied to the plants. Mass rearing and release of parasitic wasps to augment biological control of the Asian citrus psyllid (ACP): This year T. radiata were produced in colonies from South China, Pakistan, North Vietnam and Florida strain previously established from Taiwan and South Vietnam and maintained at DPI, Gainesville. Wasps from these colonies were used for research, to maintain the colonies and to release in conventional citrus mostly in southwest Florida and organic citrus, mostly in North Florida. We have observed parasitism rates of up to 60% or more at release sites in conventional citrus in southwest Florida during spring and summer compared to < 20% at sites with no releases, showing that augmentative release can potentially increase incidence of parasitism by T. radiata in the field. Testing of existing botanical insecticides for activity against Asian citrus psyllid to identify potential new tools for psyllid management: Our objective for this project was to evaluate botanical compounds as repellents of Asian Citrus Psyllid (ACP). Botanical oils and their constituent compounds are promising as repellents of ACP because many plant chemicals have shown repellency in other insect systems. In the previous quarter, a majority of the odors we screened for repellency were individual compounds. Unfortunately, our results suggest that these compounds are not likely to be useful for control of ACP in situ because the required dosages would be too high and cost prohibitive. Therefore, we selected additional botanical oils based on their availability and cost (less than $100/kg oil) to use in olfactometer and toxicity assays potential repellency. Improving psyllid management by optimizing: 1) adjuvants for low volume sprays, 2) targeted border-row treatments, and 3) location of spray applications Low volume (2-10 gallons per acre) are currently routinely used by many Florida citrus growers. The objective of this investigation was to study potential alternative methods for implementing low volume technology, as well as, to understand the potential limitations of the technology. We investigated longevity of activity of low volume sprays, using leaves from field trials with fenopropathrin (16 oz/acre), phosmet (1.5 lb/acre) and imidacloprid (15 oz/acre) and compared with high volume applications. A bioassay was then performed with citrus leaf discs in a Petri dishes. The mortality of adult psyllids after 4 days was between 60 and 100% after high volume applications, but <10% after low volume applications. These results indicate very limited residual activity of low volume treatments. Insecticidal and antimicrobial peptides for management of Asian citrus psyllid: There is an urgent need to develop and evaluate alternative management programs for ACP and greening disease. Previously, we have reported the success of two putative insecticidal peptides (A and B) in imparting deleterious effects on the growth and development of ACP. Plants containing peptides A and B have elicited reduced feeding, host selection, fecundity, and longevity of eggs and nymphs of ACP when compared to control plants. ACP surviving on plants containing peptides A and B also exhibited increased developmental time for egg and nymph when compared to control plants. Currently, we are evaluating effects of plants containing peptide C on the growth and development of ACP.