2012 Annual Report
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.
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:.
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.