Location: Subtropical Plant Pathology Research2012 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 is related to inhouse project objectives: 1. Characterize ecology, biology, epidemiology, molecular genetics, and vector and host (crop and weed) interactions of domestic, exotic, newly emerging, and re-emerging pathogens, and 3. Develop or improve comprehensive integrated disease management strategies. 1. Continue cleaning planting material (budwood and seeds) to eliminate recurring disease outbreak. a. Testing of planting materials to identify Candidatus Liberibacter asiaticus (Las)-free budwood and seed source: In addition to optimizing the Animal and Plant Health Inspection Services (APHIS) recommended standardized protocol for detection of ‘Candidatus Liberibacter asiaticus’ (Las), the causal agent of citrus Huanglongbing (HLB) in Florida, we have developed and standardized a supersensitive detection method that targets the nearly identical tandem repeats of two prophage genes of Las bacteria. A manuscript entitled ‘Improved real-time PCR detection of Candidatus Liberibacter asiaticus’ has been published in Molecular and Cell Probes: 26-90-98 (2012). b. Optimizing efficient methods of treatment to eliminate Las bacteria: In addition to the thermotherapy method used to cure HLB-affected plants, including clearing up budwoods and seeds, we have also developed a graft-based chemotherapy method for eliminating the HLB bacteria. Using this method, we confirmed a mixture of penicillin and streptomycin was the most effective compounds in eliminating the HLB bacterium from the HLB-affected scions, and in successfully rescuing severely HLB-affected citrus germplasms. 2. Continue to determine if the ornamental Murraya species are HLB inoculum reservoirs, and if thermotherapy can be applied to the Murraya industry. a. Determine the role of Las inoculum form in M. paniculata in HLB development: We have successfully transmitted Las from infected Citrus to Murraya; the extreme low rate of infection results in a low titer of ‘Ca. L. asiaticus’in the Murraya. The HLB bacterial titers of D. citri from colonies on ‘Ca. L. asiaticus’-infected M. paniculata and Citrus sp. were analyzed, and the results indicated that the titer was four orders of magnitude lower in M. paniculata than in Citrus. Psyllids reared on infected M. paniculata also carried bacterial titers four orders of magnitude lower (about 10,000 times fewer bacteria) than psyllids reared on infected Citrus sp. These observations question the importance of M. paniculata as a reservoir of huanglongbing and indicate resistance to huanglongbing in M. paniculata. Further work on M. paniculata is to determine whether ACP that acquires a Las infection from M. paniculata is capable of transmitting Las that will cause typical HLB disease in Citrus. b. Determine the effectiveness of thermotherapy in Murraya species and its possible value for clean Murraya production: Thermotherapy experiments have been planned and will commence shortly. 3. Continue application of new chemical and thermal therapies to eliminate Las. a. Optimization of chemical treatments: We have screened more chemicals for the control of HLB using the above-mentioned graft-based method, and several new chemicals, including antibiotics and non-antibiotics displayed effective results in suppressing or eliminating the HLB pathogen. Meanwhile, we are exploring different delivery system for the chemical control. b. Testing of application methodology: Thermotherapy treatment greenhouse trials. During this quarter, data from the controlled thermotherapy trials was summarized and a manuscript submitted to Phytopathology. The summary for this work is as follows: 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 quantitative polymerase chain reaction (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. c. Thermotherapy of citrus trees at USDA Pico’s Farm: Most of the trees at Pico farm that were previously heat treated between January and March 2011 were still positive for HLB in December 2011 (1 to 1.5 yrs post treatment) using qPCR. Based on temperature readings, we hypothesize that during the winter months the portable greenhouses do not reach high enough temperatures to effect the bacteria. In addition, tree roots may have not been exposed to as hot temperatures and may remain a source of inoculum. Now, that daily temperatures are in the 80°C-90°C additional thermotherapy trials at Pico’s Farm have commenced. Thermotherapy treatment commercial groves. In collaboration with two cooperators, trees at two commercial groves underwent thermotherapy and have been consistently monitored for HLB. Due to resets, more than 50% of the trees in one commercial grove have been removed from the study. We were unable to test the trees before they were removed. The remaining trees will continue to be monitored for HLB. It has been one year since heat treatment was applied to trees in the second grove. Nearly all trees remain at the second grove. Although most display HLB symptoms, many continue to flush, produce fruit, and appear healthier than before treatment. Samples have been processed for 1 year post treatment and statistics will be performed in the next quarter. 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 will continue to work with this cooperator and monitor the new set of trees. d. Thermotherapy of dooryard citrus: As a continuation of the thermal treatment project from August 16, 2011 to September 30, 2011, 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. During this period, temperatures reached over 40°C, surpassing the temperatures used with previous growth chamber and field experiments. Initial Ct values (qPCR using Las specific primers) were recorded from three different branches on each tree. Trees were sampled during the last weeks of March. The recent hardened spring flush was not sampled but rather the flush that appeared after the heat treatment was tested. All treated trees displayed more flush and from afar appear to be healthier than untreated citrus. Nutrient deficiency symptoms are present on the leaves. Despite the lack of typical HLB symptoms, all trees tested positive for Las in at least one of the branches sampled although with a lower titer than measured before treatment. It needs to be determined if qPCR results are from actively growing bacteria or from old dead bacteria. The trees will be fertilized and monitored throughout the summer. During this quarter, new methods will be developed in an attempt to combat the Las infection including defoliation and chemotherapy combined with thermotherapy. e. Combination of thermotherapy and chemotherapy on HLB positive citrus: In order to improve the control efficacy, we used a combination of thermotherapy and chemotherapy on HLB-affected citrus. In a study between September 22, 2011 and October 7, 2011, five Ortanique trees located at Pico’s Farm underwent 7 to 10 days of thermotherapy followed by 100 ml injections of a penicillin and streptomycin cocktail used in previous studies. One tree did not survive thermotherapy. The remaining trees displayed severe damage from the heat treatment but were able to recover and produce spring flush. Leaves continue to test positive for ‘Ca. Liberibacter asiaticus’. Statistics need to be performed to determine if there has been a decrease in bacterial titer. Due to the small sample size, additional studies using both chemotherapy and thermotherapy as control methods will be performed. Throughout the summer, trees will be treated with the antibiotic combination alone or with heat and also with aluminum chloride (related research has indicated that this chemical may reduce the Las titer) alone or with heat. 4. 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’ isolated from citrus, periwinkle plants and psyllids. Up to 1,000,000 cells/ml were obtained within 48 hrs based on qPCR estimation. 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.