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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Plant Pathology Research » Research » Research Project #422540


Location: Subtropical Plant Pathology Research

2013 Annual Report

1a. Objectives (from AD-416):
1. Continue screening and eliminating Huanglongbing (HLB) bacteria from nursery stocks using highly sensitive detection technology, coupled with effective Las-eradicant compounds and thermotherapy. 2. Continue determining if ornamental Murraya species are HLB inoculum reservoirs, and if thermotherapy can be applied to the Murraya industry. 3. Improve culturing of Las bacterium in vitro and conduct functional genomics to elucidate molecular mechanisms of HLB disease. 4. Continue to implement chemical control of citrus HLB by developing cost-effective chemical compounds and application technology.

1b. Approach (from AD-416):
1. Using the newly-developed super sensitive realtime polymerase chain reaction (PCR) to detect Las bacteria in the germplasms of citrus, and use the two effective chemicals coupled with thermal therapy to treat the budwoods and seeds to obtain real Las-free gerplasms. 2. Using realtime PCR and convential PCR to determine the type(s) of Las bacteria that can survive in Murraya plants and the dynamics of the bacteria in the plants, as well as their possibility to be transmitted to citrus by the asian citrus psyllids. 3. Optimizing the growth factors and conditions for improving the growth rate of Las bacterium in vitro, and knocking out and complementing some of the putative effector genes of Las. 4. Using the chemicals and thermotherapy device, we will apply and modify the application system for practical and cost effective control of citrus HLB.

3. Progress Report:
This research relates to inhouse project objective: 1. Characterize ecology, biology, epidemiology, molecular genetics, and vector and host (crop and weed) interactions of domestic, exotic, newly emerging, and re-emerging pathogens. Continue application of new chemical and thermal therapies to eliminate Las: A. Optimization of chemical treatments - We are exploring different delivery system for the chemical control, these trials are conducted both in greenhouse and fields. One penetration system has been developed for better delivery of the selected chemicals into citrus plants. B. Testing of application methodology - Thermotherapy treatment greenhouse trials. Using a temperature-controlled growth chamber, we evaluated the duration and temperature required to eliminate or suppress the Las bacterium in citrus, using three temperature treatments (40°C, 42°C and 45°C) for time periods ranging from 2 to 10 days. Results of qPCR after treatment illustrate significant decreases in the Las bacterial titer, reaching an undetectable level, combined with healthy vigorous growth on all surviving trees. Repeated qPCR testing confirmed that previously infected, heat-treated plants showed no detectable levels of Las, while untreated control plants remained highly infected. Continuous thermal exposure to 40-42°C for a minimum of 48 hours was sufficient to significantly reduce titer or eliminate Las bacteria entirely in HLB-affected citrus seedlings. Thermotherapy/chemotherapy of citrus trees at USDA Pico’s Farm: Previously, over 50 Valencia trees were treated with thermotherapy at Pico’s Farm using portable greenhouses and solarization. Treatment durations are weather dependent and trees are exposed to at least 25 hours of >40°C before tent is removed. Due to systemic infection of Las, trees are also being treated with various chemicals such as penicillin and streptomycin to reduce Las titer in the roots. Additional chemicals, compounds, and biocontrol agents are also being added to the treatment regime. Thermotherapy treatment commercial groves: We continue to extensively collaborate with one commercial grove in Fort Pierce, FL. A one year sampling was completed on the block of trees heated in the previous spring. Although most display HLB symptoms, many continue to flush, produce fruit, and appear healthier than before treatment. The grower believes the trees have significantly recovered and look similar to neighboring trees that have not shown any decline. Statistically, this block of trees that underwent thermotherapy for 3 days did show a significant decrease in Las titer after 22 months (two-tailed t-test of t31 = 2.77; two-sided p=0.0093). Due to the improved appearance of the treated trees, the grower built his own version of the portable greenhouse and began treating another group of trees with thermotherapy. We have added another block of trees into our study and will continue to work with this cooperator and monitor both set of trees. Thermotherapy of dooryard citrus: As a continuation of the thermal treatment project, 26 HLB infected dooryard citrus were placed under commercially available portable greenhouses for 7 to 10 days and 6 trees were left untreated for controls. The year and a half post treatment data is statistically significant (two-tailed t-test of t25 = 7.99; two-sided p<0.0001). In addition to the reduced Las titer in treated trees, most of the citrus trees are less symptomatic than before treatment and display a healthy, vigorous growth response. Thermotherapy has been discussed at many extension events including at the International HLB meeting in Orlando in February 2013, at multiple meetings with the Indian River Citrus League, Florida Citrus Production manager meeting, other USDA tours with politicians, CRDF members, growers, and local press. Improve Las culture in vitro, conduct functional genomics, and develop a high-throughput screening system for therapeutic molecules: A. Improve Las culture in vitro - We have developed a liquid media to culture ‘Ca. Liberibacter asiaticus’(Las) isolated from citrus, periwinkle plants and psyllids. The Las bacterial growth reached Stationary Phase and Death Phase in 48-72 hours in the liquid cultures. We are looking into factors affecting further growth. Although initial media formulations have reported an increase in the bacterial cell number of Candidatus Liberibacter asiaticus (Las), sustained growth over time has not been obtained. To try to achieve this goal studies involving specific components critical for the growth of Las, which may be found in both the phloem of plants and the gut of the psyllid host, are currently underway. The studies are designed to look at individual components to determine which ones may be playing a role. Thus far several of the proposed studies have been completed, which include the use of 94 different commercially available nutrient supplements and a comprehensive look into the proper pH for culture media using its psyllid host. It is believed that a culmination of the data from the individual studies will lead to media formulations that are better able to sustain growth. B. Functional genomics of the HLB bacterium - Since we do not have pure Las bacterial culture in vitro yet, we are using alternative host for gene expression and functional analysis. We have demonstrated Las encodes a functional flagellin that induce host defense response. We also have revealed Las encode two novel autotransporters that target to mitochondria. These two autotransporters were associated with pathogenicity and insect transmission. In addition, we have identified another effector of Las that target to host nuclei when this gene was transiently expressed in experimental host plants. C. Screening small molecules for the elimination of the bacterium - We have screened more than 80 chemicals/molecules for the control of HLB using the above-mentioned graft-based method, and several new chemicals/molecules, including antibiotics and non-antibiotic chemicals displayed effective results in suppressing or eliminating the HLB pathogen in greenhouse.

4. Accomplishments