ENDOPHYTES AS SOURCES OF ANTIMICROBIALS TO CONTROL HUANGLONGBING (HLB)

Huanglongbing (HLB), also known as citrus greening is the most devastating citrus diseases in the U.S. HLB is caused by the bacterium Candidatus Liberibacter asiaticus (CLas) and is transmitted by a tiny insect, called the Asian citrus psyllid. Since HLB was detected in Florida in 2005, Florida's citrus production has fallen by 75%. The disease also threatens other citrus producing states. There is no cure for HLB, effective disease prevention and treatment methods have proven elusive. There is a high priority need for developing anti-microbial treatments that could reverse the disease impacts, specifically by reducing bacterial inoculum in infected citrus trees. The project proposes to first purify and characterize active anti-CLas compounds derived from bacterial endophytes isolated from survivor citrus trees. Next, uptake and anti-CLas activity within the citrus root system will be evaluated using CLas-citrus hairy root bio-assay. Finally, the protective and curative effects of purified antimicrobial compound treatment against CLas will be demonstrated under greenhouse conditions. Promising results from the project can readily be translated into products for the benefit of stakeholders through the agency of Marrone Bio Innovations, Inc. Initial information about the project will be shared with growers, promising results will be shared with growers through seminars, extension website, social media, newsletters, etc.

(Obj. 1) To identify compounds that are responsible for the anti-CLas activity found during the previous work (psyllid homogenate assay) we will use a bioassay-guided fractioning and purification scheme. we will analyze the crude extract using GCMS and UHLPC-MS/MS to identify the major groups of compounds found in the extracts. After, we will start initial fractioning of the crude extracts using C18 solid phase separation. This will yield four fractions based on the compound's polarity, the anti-CLas activity of these fractions will be tested once more. The most active fraction will be further separated via Automated Flash Chromatography paired with an ELSD detector. Each of the fractions obtained via flash chromatography will be teste once more for anti-CLas activity. If the chromatography yields more than 20 fractions, some of the fractions will be "pooled" together. Biologically active fractions will be analyzed via UHPLC-MS/MS to determine their composition. If multiple compounds are present, preparative HPLC will be performed. Pure compound fractions will be analyzed and identified via Fourier Transform Ion Cyclotron Resonance (FT-ICR) and Nuclear Magnetic Resonance (NMR). (Obj. 2) We will utilize the CLas-citrus hairy root bioassays to evaluate the efficacy of anti-CLas active fraction derived from endophytic bacteria and determine their inhibitory activity against CLas within the citrus root tissue. CLas-citrus hairy root cultures are periodically subjected to multiple quality control diagnostics to confirm and quantify CLas titers within the hairy root cultures. Next, the CLas-citrus hairy root cultures will be treated with the respective fraction/s, at multiple dosages (2%, 5%, 10% v/v), using the in vitro multi-well plate format, alongside untreated and DMSO/solvent alone controls. All the treatments are vacuum infiltrated to facilitate the penetration into the hairy root matrices and incubated for 72 hours. The hairy root tissues are subsequently sampled for molecular diagnostics. The effectiveness of the treatments will be determined by estimating relative CLas titers among the controls and treatments by qPCR amplification of ribonucleotide reductase β-subunit gene marker of CLas (RNR/nrdB, RNR-F/RNR-R). (Obj. 3) The most promising pure compound/s found to have activity against CLas in the CLas-ctirus hairy root assay will be tested in planta. We plan to test the compound/s on nursery trees that are approximately 1 year from budding. Scion and rootstock will depend on what is available from nurseries 3 to 6 months prior to initiation of the greenhouse studies but we will aim for sweet orange scions like 'Valencia'. Common rootstocks include 'Swingle' citrumelo, X-639, Cleopatra mandarin, and US-897. Two types of trials will be undertaken: 1) to determine the curative effect of compounds and 2) to determine the protective effect of compounds. Whether a compound is tested for a curative or protective effect will depend on the results from previous analysis and the likely mode of action. To investigate whether there is a curative effect on CLas in planta, trees will be inoculated with CLas by grafting 4 months prior to trial initiation with budwood that is known to contain active populations of CLas. The trees will be tested monthly after graft inoculation to confirm whether they have become CLas positive. Only trees that are confirmed CLas positive will be used in the trial. The compounds will be applied one of three ways, spray application, soil drenching, or possibly via injection. Soil uptake will be evaluated by seedling assay where 6-month-old seedlings in containers will be drenched with the compounds and the foliage tested to see if the compound can be detected. Similarly, the concentration range will also be determined prior to starting the greenhouse trial based on experiments with HLB-affected cuttings. These cuttings will contain one angular stem node with a leaf attached of approximately 10 cm in length. The cuttings will be placed in tubes containing a solution of the compound at multiple concentrations for 48 hours. The cuttings will then be transferred to water for a week. After a week they will be tested for the presence of live CLas cells with the RNA-based CLas viability assay. In the greenhouse experiment. there will be a minimum of five trees per treatment and preferably there should be 10. The leaf titer of CLas and viability will be taken at the time 0 just before the applications and 2 weeks, 4 weeks and 4 months post-application from two newly mature leaves per tree. At 6 months, the trees will be harvested. The total leaf area, trunk diameter, root mass, dry weight will be collected along with a disease rating. The titer will be measured in the leaves and roots. Two control treatments will be included in each trial, an untreated CLas inoculated and an untreated uninoculated control. The protective trial will be very similar with the exception of the inoculation timing to the compound application. In this case, the compound will be applied 2 to 3 weeks prior to inoculation with CLas. The trees will be monitored for CLas titer. The number of repetitions will need to be larger as there is a probability that some trees will not become CLas positive. The statistical analysis will be conducted with generalized linear mixed model methods and the distributions used will be dependent on the data. (Obj. 4) We understand outreach and extension of the project results to citrus growers is a key component and will be executed by all PIs. Members of the project team will communicate with stakeholders to identify needs and obstacles through surveys and grower forums. It is also essential to disseminate information rapidly as it arises from this proposed research. This will be accomplished efficiently via websites, presentations at industry and scientific meetings as well as through our stakeholder members. The information will be shared through regular dissemination of research reports, blogs, webinars (i.e., www.citrusresearch.ifas.ufl.edu), and participation in UF/IFAS hosted podcasts and newsletters focused on citrus. Findings and recommendations from this project will be conveyed to growers using these and other resources. Recommendations will be quickly delivered through multiple extension events through posters, displays, and handouts. Dissemination of information is also expected to take place via peer-reviewed scientific journals, extension publications, trade periodicals, oral presentations at field days held at the project sites, citrus industry events, professional society meetings, and citrus industry governing board meetings to be determined.