DEVELOPMENT AND IMPLEMENTATION OF NEW CONTROL STRATEGIES FOR CITRUS HUANGLONGBING (GREENING)

This research is related to inhouse objective 1: Develop standardized protocols for screening and cleaning up the entire germplasm of the Florida citrus industry using our highly sensitive detection technology, and effective chemical compounds and 3. Verify if Murraya paniculata is, or is not, a preferred reservoir of HLB pathogens, whether it can be used as trap plants for the control of citrus HLB. Clean planting material (budwood and seeds) to eliminate recurring disease outbreaks: In addition to optimizing the Animal and Plant Health Inspection Service (APHIS) recommended standardized protocol for detection of Candidatus Liberibacter asiaticus (Las) in Florida, we have developed and standardized an additional supersensitive detection method that targets the nearly identical tandem repeats of two prophage genes in the Las bacteria. This method not only simplified the detection procedure, but also detected the huanglongbing (HLB) bacterium at very low titers, especially the seed-transmitted Las bacteria in seedlings. This new technology has been published in Molecular and Cellular Probes, and transferred to universities and regulatory institutions world-wide for extended applications in the detection and diagnostics of citrus HLB. Using information in the complete Las genome, finished in our lab, we have identified different population dynamics associated with different phenotypes of the HLB disease. We are currently optimizing strain-typing protocols for detection and differentiation of Florida�s Las populations with an emphasis on differentiating the seed-transmitted Las from HLB- causing Las populations. Most seeds from HLB-affected citrus plants carry a high titer of Las, but a high percentage (up to 70%) of the resulting seedlings carry an extremely low titer of Las. Most of these seedlings did not cause typical HLB. However, up to 30 % of the seedlings grew poorly compared to seedlings from healthy seeds. These growth-retarded seedlings can be rescued by rich nutrients after thermal therapy. For budwood therapy and preservation, we have developed a new heat treatment protocol and propagation system to clean up the pathogen. Determining if the ornamental Murraya species are HLB inoculum reservoirs: Murraya paniculata, or orange jasmine, is a common landscape plant that can be a host for both the psyllid and the bacteria. A systemic survey was conducted in which we monitored eight plantings of orange jasmine and adult psyllids that developed from nymphs on these plants for infection by Las. Extremely low rates of Las infection were found in both psyllids and orange jasmine. During the early spring (April) 0 to 7.4% of the Murraya samples from each site tested positive for Las. However, during the summer months (June and August), 0 to 28% of the Murraya samples tested positive. Despite the differences in Las prevalence in the plants, 0.8 to 1.8 % of the psyllids utilizing Murraya as a host were Las positive across all collections. It is possible that the low number of psyllids carrying Las is related to the extremely low titer of Las found in the plants since the titers found in Murraya are approximately 65,000 times lower than those in Citrus. Our results indicate that orange jasmine hedges may be a minor HLB inoculum source for nearby citrus groves. The manuscript entitle �Low Incidence of Ca. Liberibacter asiaticus in Murraya paniculata and associated Diaphorina citri� has been published in the journal Plant Disease. We have also conducted experiments testing the efficiency of psyllid transmission of Las between Citrus and Murraya plants. We successfully transmitted Las from infected Citrus to Murraya; however, the rate of infection was extremely low as was the titer of Las. We tested M. paniculata and Citrus sinensis grown in the same field for their Las titer. We also assayed D. citri from colonies on Las-infected M. paniculata and Citrus sp. We found the bacterium in both plant species, but 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 Murraya as a reservoir of HLB and indicate resistance to HLB in M. paniculata. Further work includes determining whether the D. citri that acquire a Las infection from M. paniculata are capable of transmitting Las that will cause typical HLB in Citrus. A manuscript entitled �Titers of �Candidatus Liberibacter asiaticus� in Murraya paniculata and Murraya-reared Diaphorina citri are much lower than in Citrus and Citrus-reared psyllids� has been submitted for publication. Application of new chemical therapies to eliminate �Ca. Liberibacter asiaticus�: The chemicals penicillin G and streptomycin (PS) have been evaluated as the most effective compounds for eliminating or suppressing the Las bacterium of HLB-affected citrus in greenhouse experiments. New optimized combinations of PS were injected into HLB-affected trees in field trials at the USHRL�s Pico�s farm. The Las bacterial titers in the PS-5 treated citrus were kept at less than 1000 cells per gram of plant tissue (Ct>36.0) for 14 months after the termination of PS treatments. Similar results were observed when PS was applied at PS-10, except that the Las bacterial titers were kept at less than 1000 cells per gram of plant tissues (Ct>36). In contrast, the Las bacterial titers from the water control treated plants remained at 2 x106 throughout the experiment. The results have been published in Phytopathology. We have developed a graft-based chemotherapy method to rapidly screen potential HLB-controlling chemical compounds. In addition, we improved transmission efficiency by using the most responsive HLB-affected scion- rootstock combination, and demonstrated the HLB bacterial titer was the critical factor in transmission. The HLB-affected lemon scions had a high titer of HLB bacterium, survival rate (83.3%), and pathogen transmission rate (59.9%). Trifoliate, a widely used commercial rootstock, had the highest survival rate (>70.0%) compared to grapefruit (52.6%) and sour orange (50.4%). Using this method, we confirmed a mixture of penicillin and streptomycin was the most effective compound in eliminating the HLB bacterium from the HLB-affected scions, and in successfully rescuing severely HLB-affected citrus germplasm. In addition, we screened more than 80 chemical compounds selected from the worldwide contest. A few of them are effective and may be used for field trials. These findings have been published in Phytopathology. Thermal therapies eliminate �Ca. Liberibacter asiaticus� from infected citrus trees under controlled conditions (additional accomplishment): We used controlled heat treatments to alleviate HLB caused by Las. In temperature-controlled growth chambers, we evaluated the time duration and temperature required to eliminate or suppress the Las bacterium in citrus, using various temperature treatments for time periods ranging from 2 days to 4 months. Results of quantitative polymerase chain reaction (qPCR) after treatment illustrated significant decreases in the Las bacterial titer, reaching an undetectable level, combined with healthy vigorous growth on all surviving trees. Repeated surveys confirm previously infected plants show no detectable levels of Las, while untreated control plants remain highly infected. The results indicate that continuous thermal exposure at 40-42�C for at least 48 hours was sufficient to significantly reduce Las titer levels in HLB-affected citrus seedlings. These results have been submitted for publication in Phytopathology. Striped mealybug (Ferrisia virgata) as a potential vector (additional accomplishment): Issues in our HLB isolation greenhouse with striped mealybug insects on infected periwinkle plants led us to investigate the hypothesis that the phloem-feeding mealybugs could (1) be infected with HLB and (2) transmit the disease to other plants in our greenhouse. We have confirmed these striped mealybugs carried the Las bacteria after feeding on HLB-infected periwinkles and citrus. However, transmission study using Las-positive stripe mealybugs gave no evidence of HLB transmission. Further studies will focus on the differences between mealybugs and psyllids in their vectoring mechanism for Las.