Recipient Organization
UNIVERSITY OF TENNESSEE
2621 MORGAN CIR
KNOXVILLE,TN 37996-4540
Performing Department
Entomology & Plant Pathology
Non Technical Summary
In the southeastern U.S., cultivation of hemp (Cannabis sativa), a crop grown for food, fiber, biofuel, and natural medicine, has increased significantly in the past 5 years due to the potential for agricultural profits. Hemp is a new crop in most locations, and research on plant diseases that cause significant crop losses and EPA-approved control measures have been developed at a slower pace. Development and spread of plant disease are highly influenced by environmental conditions, including weather, soil factors and availability of other plant hosts to maintain the pathogen. Pathogens that cause significant losses in one locale may cause minor or insignificant losses in another area. Identification of diseases that cause losses in hemp and development of cost effective and safe biopesticides is needed to minimize disease and economic losses of hemp producers. The overall goals of this project are to identify plant extracts that have significant biopesticidal activity against fungal, bacterial, and nematode pathogens of hemp that are suitable and safe for production of hemp food and natural medicines in the southeast.Our experimental methods are aimed at identification of hemp pathogens and assessing their ability to cause disease. Materials that aid Extension agents and hemp growers in identification of hemp diseases will be developed. Undergraduate and graduate students will be taught to recognize the causes of hemp disease through case studies presented in the classroom, and they will participate in the research studies and in development of educational materials for the public. Plant extracts will be tested for ability to suppress growth or disease development of fungi, bacteria, and plant-parasitic nematodes on hemp, and extracts that suppress plant disease will be promoted for commercial development. In addition, standard bioassay protocols will be developed that future scientists can use to evaluate plant extracts for plant disease control, and this information will be disseminated at national, regional, and local meetings for researchers and educators. Through the methods and outputs described above, the ultimate goals of this project are to identify biopesticides that are effective and safe for hemp production and minimize or eliminate plant disease losses. In addition, we aim to educate the current generation of producers and the next generation of agricultural scientists (students) in identification of hemp diseases. If our goals are met, disease losses of hemp from disease will be minimized and agricultural profitability will be increased for hemp growers in the southeastern U.S.
Animal Health Component
20%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Goals / Objectives
In the southeastern U.S., cultivation of hemp (Cannabis sativa), a crop grown for food, fiber, biofuel, and natural medicine, has increased significantly since research and pilot programs were legalized in 2014. Hemp is produced in both the field and in greenhouses, and several fungal, bacterial, and nematode pathogens have been identified. Disease development and spread are influenced by environmental conditions, including weather, soil factors and availability of other plant hosts. Pathogens that cause significant losses in one locale may cause minor losses in another area. Because this is a new crop in most parts of the U.S., significant new information on the losses caused by known and potential pathogens in different locations is needed by producers and the scientific community. Thus far, the U.S. EPA has approved ten pesticides for use on hemp. Of these, nine are biopesticides, and one is a conventional pesticide. Because most hemp grown in the southeastern U.S. is targeted for the cannabidiol (CBD) desired by the wellness industry, biopesticides, rather than conventional pesticides, are highly desired for both field and greenhouse production. Of the ten pesticides approved by the EPA, neem oil and its bioactive compound, azadirachtin, are the active ingredient(s) in five of the formulations, and little data regarding efficacy are available. The major goal of this project is to identify extracts from plants that have potential biopesticidal activity against fungal, bacterial, and nematode pathogens of hemp, and to determine their efficacy in greenhouse hemp production.The specific project objectives are the following.Identify and confirm pathogenicity of our laboratory collection of fungi and bacteria isolated from diseased hemp plants;Develop in vitro screening assays for rapid evaluation of the biopesticidal potential of plant extracts against fungal, bacterial, and nematode pathogens of hemp;Confirm biopesticidal potential and determine efficacy of selected plant extracts against fungal and bacterial pathogens of hemp with greenhouse plant disease assays; andEvaluate combinations of biopesticidal plant extracts and genetic resistance of hemp cultivars for management of pathogenic nematodes in greenhouse assays.
Project Methods
Plant extracts will be prepared using hot water or solvent extraction. Extracts from chenopods and monarda with known chemistries and activities will be tested. Additional extracts, and active component mixes shown to be antifungal, antibacterial, insecticidal, or nematicidal, will be tested in subsequent studies. All extracts and mixes will be tested for phytotoxicity prior to greenhouse testing. Neem oil and azadirachtin will serve as industry standards in all tests.Project objective #1. Identify and confirm pathogenicity of our laboratory collection of fungi and bacteria isolated from diseased hemp plants. Working hypothesis: Disease assays will confirm that our collections of bacteria and fungi from diseased hemp will contain several plant pathogens of economic importance.The investigators have isolated several putative pathogens from diseased hemp plants that are currently being identified, including several putative Fusarium isolates collected from field plants and a collection of fungal and bacterial pathogens from seed and greenhouse-grown Katani hemp, an industrial grain cultivar. Identification of these putative pathogens will be confirmed based on morphological features and polymerase chain reaction and sequencing of the ITS (fungi) or 16S (bacteria) regions of the rRNA operon. Pathogenicity will be confirmed with Koch's postulates in greenhouse or growth chamber plant disease assays.Project objective #2. Develop in vitro screening assays for rapid evaluation of the biopesticidal potential of plant extracts against fungal and bacterial pathogens of hemp. Working hypothesis: Screening bioassays will predict plant protection in greenhouse production.In addition to the putative pathogens from our laboratory collections included in Objective 1, additional putative pathogens will be collected from diseased hemp in producers' fields and greenhouses and obtained from other scientists. These will include,but are not limited to Athelia rolfsii (asexual stage Sclerotium rolfsii), which causes southern blight of hemp and is a destructive soilborne pathogen. This fungus is extremely difficult to control because it produces long-lasting survival structures resistant to environmental stress, and it has a wide plant host range. Genetic resistance in hemp cultivars to southern blight has not been identified. Fusarium wilt and crown rot of hemp are of increasing importance and can cause significant crop loss. Species of the fungi Bipolaris, Cercospora, and Curvularia are also prevalent in hemp fields. Economic losses due to these pathogens can be ameliorated to some extent by use of moderately resistant hemp cultivars in field plantings, but control of these pathogens in greenhouses is more challenging.Significant bacterial pathogens that have been reported in the southeast include species of Pseudomonas and Xanthomonas. Unidentified bacterial isolates are in the Ownley lab collection. These isolates were collected from water-soaked lesions on leaf margins and veins, which are typical symptoms of these pathogens. Confirmed fungal pathogens will be included in the studies to develop screening assays. Impact of plant extracts on fungal pathogens will be determined by standard bioassay procedures such as poison food tests (culture media amended with extract) and spectrophotometric tests for spore germination and growth. Data collected will include spore or sclerotial germination, colony size, and dry weight. In tests by the Gwinn lab, sclerotia of another sclerotia-forming species, Sclerotinia sclerotiorum, were sensitive to combinations of essential oils found in monarda herbage. Methods used in those studies to fumigate sclerotia will be used to evaluate suppression of Athelia rolfsii. The Ownley lab routinely evaluates sclerotia of Athelia rolfsii and colonies of other fungi for sensitivity to volatile inorganic and organic chemical compounds and mycoparasites, such as Trichoderma. Viability of fumigated sclerotia will be determined by culture methods.Confirmed bacterial pathogens will be included to develop screening assays. Impact of plant extracts on bacterial pathogens will be determined by similar methods to those described for fungi. In previous studies on plant extracts against tomato pathogens, the investigators developed methods to evaluate impact of plant extracts on in vitro population growth of Xanthomonas, Pseudomonas, and other bacterial pathogens, as well as greenhouse challenge tests with pre- and post-treatment of tomato foliage coupled with inoculation of Xanthomonas and Pseudomonas. These methods will be modified for hemp assays.Project Objective #3. Confirm biopesticidal potential and determine efficacy of selected plant extracts against fungal and bacterial pathogens of hemp with greenhouse plant disease assays. Working hypothesis: Plant extracts will be identified that control hemp diseases in greenhouse assays.Extracts that are effective in vitro will be tested on greenhouse plants. Methods used for soilborne fungi will include mixing inoculum and biopesticide into growing mix prior to transplant or seeding. Leaf spot pathogens (bacterial and fungal) will be tested by application of the plant extract onto the leaf prior to and/or after inoculation with the pathogen. Cultivars with either no or moderate resistance to leaf spot pathogens will be tested.Project Objective #4. Evaluate combinations of biopesticidal plant extracts and genetic resistance of hemp cultivars for management of pathogenic nematodes in greenhouse assays. Working hypothesis: Plant extracts identified by in vitro screening, combined with resistant cultivars, will protect against plant parasitic nematodes.The Bernard lab has determined that some cultivars are severely impacted by root knot nematodes (RKN) whereas other cultivars may have moderate to high levels of resistance. Experiments on resistance of hemp cultivars to two RKN, Meloidogyne incognita (southern RKN) and M. hapla (northern RKN) have been underway for the last year. Southern RKN is ubiquitous in the southeastern US, while northern RKN is common in some southern localities. Cultures of both nematodes are available. The investigators have collaborated to investigate impact of chenopod saponins on nematode development. Methods used in these studies on Caenorhabditis elegans juveniles will be adapted to Meloidogyne studies. Briefly, impact of extracts on juveniles (J2) were evaluated in 96-well plates and percentages that had reduced mobility or were dead were determined at 24 and 48 hours. Impact of extracts on egg hatch will also be determined. Egg masses from source plants will be collected, and hatch in the presence and absence of plant extracts will be monitored.Extracts that have significant impact on nematode growth and development will be tested for efficacy in greenhouse tests. One sensitive and one moderately resistant cultivar will be tested. Greenhouse growing media augmented with sand to facilitate plant extraction will be treated with plant extracts. After transplanting into treated and untreated media, plants will be inoculated with J2s of M. incognita or M. hapla. Although outside the scope of this project, extracts inhibitory to nematodes may be evaluated for insecticidal and acaricidal activity when appropriate.Evaluation of experimental data. Experimental data will be analyzed in collaboration with The University of Tennessee Office of Information Technology - Research Computing Support. All data will be analyzed using mixed model analysis of variance with SAS (version 9.4, SAS Institute, Cary, NC, USA). Data will be evaluated for normality and homogeneity of variance and transformed as appropriate. Post hoc multiple comparisons will be performed with an F-protected Tukey's adjustment at P<0.05 significance level and untransformed means will be reported. Bioassays will be validated with greenhouse results or they will be rejected as screening tools.