EVALUATION OF BIOLOGICAL CONTROL AGENTS FOR PHYTOPHTHORA CAPSICI DISEASE MANAGEMENT AND PLANT GROWTH PROMOTION IN PEPPER AND TOMATO

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1025991
• Grant No.: 2021-38821-34595
• Cumulative Award Amt.: $599,978.00
• Proposal No.: 2020-11016
• Project Start Date: May 1, 2021
• Project End Date: Apr 30, 2026
• Grant Year: 2021
• Program Code: [EQ]- Research Project

Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209

Performing Department
Agric. and Environmental Sc.

Non Technical Summary
Phytophthora capsici is one of the most destructive pathogens impacting pepper and tomatoes production. It infects all parts of the plant and causes severe losses in yield. After Phytophthora becomes established at a location is very difficult to eradicate because it affects many vegetable crops. Farmers rely on cultural methods to combat this disease, but these methods are inadequate. Although there are effective chemical fungicides, the development of fungicide resistance is a problem, and fungicides are toxic to farmworkers and contaminate the environment, soil, water, aquatic life, wildlife, and other organisms. There is clearly a need to find alternative tools to bolster cultural methods and reduce fungicide usage. This project will (1) advance the development of bacterial strains for Phytophthora disease management and plant growth promotion. (2) develop user-friendly formulations of the new bacterial products (3) generate information on how the bacteria work to control the Phytophthora diseases and promote plant growth; (4) show the cost and benefits of using this type of disease control; and (5) provide training to farmers and extension agents on environmentally friendly products that are available in the market and the new products from this project that can be combined with cultural practices and reduce fungicide usage. This project integrates research and outreach activities including cutting-edge research to better understand disease control by using the selected microorganisms. This project will impact vegetable farming and empower farmers to make informed choices on disease management and reducing toxic pesticide residues and environmental contamination.

Animal Health Component

85%

Research Effort Categories

Basic

5%

Applied

85%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1499	1160	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1499 - Vegetables, general/other;

Field Of Science
1160 - Pathology;

Keywords

biocontrol

endophytes

phytophthora

bacteria

biocides

disease management

pesticides

Goals / Objectives
1) Advance the development of three Bacillus species for plant protection against P. capsici and growth promotion of bell pepper and tomato(2) Develop user-friendly formulations of new products for easy adoption by growers(3) Analyze the disease management and plant growth promotion mechanisms and soil microbial diversity by using molecular and biochemical analysis.(4) Analyze economic cost-benefits of microbial-based disease management(5) Provide farmer training on microbial-based disease management systems.

Project Methods
Objective 1. Evaluation of the effect of selected BCA onP. capsicidisease severity and growth promotion.Previously selected biological control agents (BCAs),Bacillus subtilis(Prt),B. amyloliquefaciens(PsL) andB. thuringiensis(IMC8) will be further evaluated on tomato and pepper cultivars susceptible toP. capsici.Greenhouse studies at TSU and field studies at three locations: (a) TSU research farm in Nashville, (b) Alabama A&M research farm or farmer's field in Huntsville, AL., and (c) farmer's field in Knoxville (Univ. of Tennessee) will be conducted. Treatments of plants with BCA suspensions by seed soaking have a total of 9 treatments (three BCAs, a mixture of the three BCAs, two chemical fungicides, two commercial biofungicides and water control). The three BCAs will be grown separately and mixed (1:1) equal concentrations before inoculation. Field planting will use raised beds, polyethylene mulch and irrigation by drip tape following standard production practices. Each raised mulched bed will represent a main plot in which treatments will be randomized as sub-plots with four replicates of single rows per plot and a one-meter separation between treatments. Farmers' fields identified to be Phytophthora infested and TSU research farm will have the presence of Phytophthora confirmed by isolations. Six to eight weeks-old seedlings colonized with BCA will be transplanted intoP. capsiciinfested soil in greenhouse and field studies. The BCA treatments will be reinforced by BCAs spray applications on plants at two weeks after planting, and equal volumes of BCA-colonized mulch applied on the base of each plant. These BCA treatments on the seed, the soil and the plants will be evaluated for efficacy against Phytophthoraq at all stages of plant growth. Plants will be monitored for disease development and disease severity on a scale of 0-5. Fruit yield in quantity and quality will be measured. Data analysis will use the SAS linear model for analysis of variance, and a series of t-tests according to SAS procedures in PROC. ANOVA (SAS/STAT. 2010).Objective 2. Development of user-friendly formulations of new products for easy adoption by growers.While the selected candidate BCAs, (Prt, PsL, and IMC8) have been evaluated for efficacy in plant disease control and growth promotion using aqueous inoculum suspensions for seed and for plant treatment, bacteria in the genusBacillusproduce spores within 1-2 days in culture and will be formulated as a dry powder using two techniques: (i) The slurry coating technique in which heat-sterilized peat will be inoculated and vacuum-dried and then ground into a fine powder and applied to seeds with a sticker and (ii) microencapsulation formulation that aims to produce a dry powder with a moisture content of 4-6%, as one of the most efficient methods for maintaining viability and stability of probiotics in the food industry and has been used for Rhizobium. Different materials that will be tested as carriers of the bacterial suspension include soluble starch, tapioca, dextran, various polysaccharides, skimmed milk, monosodium glutamate, vegetable oil, and glycerol which are commercially available. The formulated products will be stored at -20o, 4o, and room temperature and evaluated for viability every 2 weeks. The formulated products will be tested for efficacy in seed treatment for disease control and growth promotion in greenhouses studies.Objective 3.Analysis of the mechanisms of disease management and plant growth promotion and soil microbial diversity.This will be done by (i) Identification of Volatile Secondary Metabolites produced by bacterial endophytes will be by using GCMS analyses /or Electronic Nose (E-Nose) which uses the GC-MS technology. The volatile compounds will be identified by comparing with the National Institute of Standards and Technology (NIST) database on the mass spectrometer. ii) The non-volatile secondary metabolites will be analyzed by using HPLC-MS or Eletronic tongue for the identification, adaptation and quantification of polyphenols produced by the selected endophytes. (iiii) Plant defense mechanisms resulting from volatile compounds and non-volatile compounds produced by the selected bacterial strains will be based on BCA -pathogen interactions and/or BCA-plant interactions at 24h, 36 h, 48h, 72h, and 7d time points.Compounds induced by bacterial volatiles and/or enzymes such as peroxidase (PO), polyphenol oxidase (PPO), chitinases (PR3-family) and β-1, 3-glucanase (PR2) that are involved in induced systemic resistance (ISR) mediated plant defense will be determined and enzyme activity will be quantified using a spectrophotometer (iv) Protein related to pathogenesis (PR proteins) will also be extracted from the plant tissues and analyzed for induced proteins and/or ISR-related enzyme activity and compounds that are associated with the ISR pathways. Plant growth promoting traits such as IAA synthesis, phosphate solubilization, nitrogen fixation, ammonia and siderophores will also be analyzed. (v) The BCAs labeled with fluorescence tags (gfp and rfp) will be used to visualize the bacteria and track their plant colonization route inside plant tissues.vi) The effect of the biological control treatments on soil microbial diversity will be analyzed usingDNeasy Power Soil Kit (Qiagen, Venlo, the Netherlands) and Phyloseq package for microbial community composition and structure in 250 mg of bulk soil sample from different treatments. Bioinformatics analyses will be performed using standard procedures.Objective 4. Analysis of economic cost-benefits of microbial-based disease management.Data on costs will be recorded in each location with assistance from researchers and students. Yield and market price data and projections will be made based on a combination of primary experimental data and secondary data sources for different production systems in conventional organic regimes. Based on the information, we will compare and analyze costs, direct yield, and revenue potential from production systems. In addition, primary survey of consumers, contingent valuation, and data analysis will be done. A questionnaire and survey instrument will be designed to factors and information related to consumer perception and preference and estimate the willingness to pay for organic produce that was not treated with chemicals. Contingent valuation survey-based methodology for eliciting people's perceived value for goods, services and amenities with attributes such as 'good for health' and 'environmentally friendly' which are directly related to and require a response from human behavior.Objective 5. Provide farmer training on microbial-based disease management systems.This extension component of the project will provide information and education to farmers and extension agents from different parts of the State in Tennessee and Alabama and provide demonstrations in annual field days and workshops on microbial-based products as alternatives to chemical pesticide. University research farm and education centers as well as farmers' fields will be used to demonstrate research results on biological-based IPM forPhytophthoradisease management and plant growth promotion in vegetable production. Oral presentations on the IPM technology and success stories that are available from other states can be adopted in Tennessee and Alabama. In addition, publications in horticultural journals and popular magazines and extension journals as well as presentations in extension meetings and horticultural meetings will be used to disseminate information from this project. In addition, an "interactive project website" will be professionally developed for outreach and training to deliver data from this project on biological-based disease management as a tool to augment cultural practices in organic production, and for reducing pesticide usage in conventional farming.

Progress 05/01/24 to 04/30/25

Outputs
Target Audience:Farmer presentations and discussions on research progress addressing organic farming problems of farmers. Changes/Problems:1. Equipment breakdown was a big problem, including (1) Electronic Nose, (2) GCMS, and (3) the use of HPLC. However, we implemented the following solutions: a.The Electronic Nose repair was requested and approved, but it is still nonfunctional, and the results from the initial analysis cannot be retrieved. b. A new GCMS has been installed, and the training has been completed. Some research was conducted using desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) at Vanderbilt University. However, their equipment also broke down at some point, but it was fixed, which delayed work progress. The DESI-MSI technique is new for agricultural science and was used on only one bacterium. c. The biochemical analysis proposed using HPLC/LCMS will be conducted using nuclear magnetic resonance (NMR), and the material preparation is in progress. However, the analysis will be outsourced. 2. Farmer problems with labor were addressed by using a stipend, in which the farmers would benefit from labor provided by students, and students benefited from experiential learning. What opportunities for training and professional development has the project provided?Two students worked on the project for their MS research, with the opportunity for field work for one student and bacterial formulations for the second student, who acquired industry employment in biocontrol. Two other students continue on the project, working on their PhD dissertations, focusing on chemical and molecular research. The project provided other professional development opportunities for field research and working with farmers. How have the results been disseminated to communities of interest?Two journal articles have been submitted, and presentations at symposia and meetings have included ARD and University-wide workshops and an American Phytopathological Society annual meeting. Presentations to farmers were in TSU Farm EXPO and farmers' workshops in Huntsville and Nashville. What do you plan to do during the next reporting period to accomplish the goals?The bacteria formulations are being refined. Biochemical and molecular analyses are also in progress and will be completed this coming year. Farmer participatory research will also be completed this coming year.

Impacts
What was accomplished under these goals? (1) To advance the development of three Bacillus species for plant protection against P. capsici and growth promotion of bell pepper and tomato, the selected isolates were evaluated in a P. capsici-infested field. Disease severity was assessed on plants and fruits. Growth promotion was assessed by fruit yield. Results have been submitted for journal publication. (2) Developing user-friendly formulations of new products for easy adoption by growers was initiated. Protocols, ingredients, and parameters for media optimization were evaluated for optimum development and formulation of commercial prototypes. Spray drying and the production of primary powder will be refined for improved shelf life and production of liquid formulation (3) The selected bacteria are being evaluated for the mechanism of action using molecular and biochemical analysis. Genes associated with systemic resistance and enzymes associated with the degradation of fungal structures are being identified from whole-genome analysis. Volatile compounds are being analyzed using GCMS, and results have not been analyzed. Direct mass spectrometry imaging has provided spatial chemical information on non-volatile compounds as potential chemicals associated with Phytophthora capsici biocontrol. Additional biochemical Analysis using Nuclear Magnetic Resonance (NMR) spectroscopy will be used to analyze non-volatile compounds. (4) Farmer training on microbial-based disease management systems in collaboration with extension agents is continuous.

Publications

• Type: Other Journal Articles Status: Under Review Year Published: 2024 Citation: Robinson, J., Shepard, H., Demmissie, D., Eyegheleme, P., May, J., McLean, J., and Mmbaga. 2024. Spaciochemical Investigation of Potential Biocontrol Agents Against Phytophthora capsici Infection in Tomato. Phytopathology.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Robinson, J. and Mmbaga, M. 2024. Antifungal effects of VOCs produced by Bacillus Thuringiensis IMC-8 against oomycete pathogen Phytophthora capsici TSU University-wide Symposium, March 25-29, 2024. Nashville, TN.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Olawuni, M., Mmbaga, M., and Omondi, E. 2024. Screening of endophytes for Southern Blight disease management and plant growth improvements in Hemp production. 2024 Association of 1890 Research Directors (ARD) meeting, April 6-9, 2024. Nashville, TN.
• Type: Other Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: Ambachew, D., Mmbaga, M., Hall, R., Eyegheleme, P., Olawuni, M., Robinson, J., and Rotich, E. 2024. Three Bacterial endophytes enhanced plant growth and yield and reduced the severity of Phytophthora capsici in bell pepper and tomato plants in the field. MDPI Plants Journal (In Press)

Progress 05/01/23 to 04/30/24

Outputs
Target Audience: The target audience of the project includes: 1. Agricultural extension community including extension agents for vegetable production 2. Vegetable producers 3. Agricultural research and education community Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One PhD student and Two MS students were supported in this project. One undergraduate student was supported for one semester. A post-doctoral scientiest was also supported by this project. How have the results been disseminated to communities of interest?Field demonstration and oral presentations to organic farmers was done in Tennessee and Alabamaas communities of interest for biological control agents. Presentations to the sientific communityinclude Sientific symposia such as Tennessee Academy of Science,TSU University wide symposium,The 1890 Association of Research Directors and the American Society of Agronomy. Publication ofproject results will be done in scientific journals and in plant production-trade relate publications. Peter Egheleme 2024. Evaluation Of Growth Media and Cost-Effective Formulations forSelected Biological Control Agents. MS Thesis Submitted to The School of Graduate andProfessional Studies Tennessee State University What do you plan to do during the next reporting period to accomplish the goals?One PhD student will complete her studies on the project. The evaluation of biocontrol formulation'sshelf life will continue and refinement of formulations will also continue to improve productdevelopment. Field evaluation in farmers field will be repeated and oral presentations will be madein Farm Expo and other formats. Studies on microbial diversity and mechanism of actionwill be repeated for confirmation of results.

Impacts
What was accomplished under these goals? To advance the development of three Bacillus species for plant protection against P. capsici and growthpromotion of bell pepper and tomato, and the development of user-friendly formulations of microbial productsinvolved different studies including (a) the evaluation of different growth media, (b) the selection of the bestgrowth media for mass production of bacteria inoculum, (c) analysis of economic cost of media formulationmaterials for economic decision and budgeting of material for product development. Other studies includedevelopment of bacterial inoculum compositions, preparation of the probiotic solutions, cell cultivation conditionsfor optimum spore yields, conducting spray drying for powder formulation and evaluation of the formulatedproducts on pathogen using in vitro techniques. Determination of the shelf life of the formulated product wasinitiated for continuation over time. Analysis of disease management and plant growth promotion was conductedin field environment and plant yield was used a a parameter of growth improvement and economic gains. Onearticle has been submitted for journal publication. Analysis of changes in microbial diversity is in progress.

Publications

Progress 05/01/22 to 04/30/23

Outputs
Target Audience:The target audience of the project includes: 1. Agricultural extension community including extension agents for vegetable production 2. Vegetable producers 3. Agricultural research and education community Changes/Problems:There are no changes to the objective but rather the timing because of delays. The project is however progressing. What opportunities for training and professional development has the project provided?The project is providingan opportunity for training three graduate students. Some of these students have had the opportunity toattend both the regional and national conferences, including the Annual meeting of the American Phytopathological Society. At these meetings, the students networked and developedother professional skills. How have the results been disseminated to communities of interest?1. Presentations of research findings were made at the university wide workshop. 2. Presentations were made at the regional meeting of the American Phytopathological Society- Southern division. 3. The PDs incorporate the findings of the research into the materials they teach in class on Crop Protection and Plant-Microbe Interactions. What do you plan to do during the next reporting period to accomplish the goals?We are preparing for evaluation trials in farmers' fields. We are expecting to get the first result from these field trials in the next reporting system.

Impacts
What was accomplished under these goals? We have evaluated different growth media for growing bacterial isolated of our Biological Control Agents (BCA) for optimum yield. Through this, we have identified the best medium for growing bacteria for formulation studies. We are optimizing the formulation process to determine the best formulation of the BCAs for trials.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Asha Maheshwari and Margaret T. Mmbaga. Endophytic fungi residing within Cornus florida L in mid-Tennessee: Phylogenetic diversity, and enzymatic properties, and potential role on plant health. Fungal Ecology (2023).
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Mmbaga, Margaret, 2023. "Replication data for Endophytic fungi isolated from Cornus florida stem tissue.", Harvard Dataverse, VI. https://doi.org/10.7910/DVN/D5NP81,
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Mmbaga and Daniel Demmissie. 2022. Controlling Phytophthora capsici in tomatoes and Sclerotium rolfsii in hemp using endophytic bacteria and analysis of their biocontrol mechanisms. Presented at APS Annual Meeting, Pittsgurgh, PA
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Mustapha Olawuni*, Mmbaga and Daniel Demmissie. 2022. Field evaluation of three biological control agents for southern blight disease management and their effects on hemp plant growth. Presented at Tennessee Academy of Science Annual Meeting.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Peter Eyegheleme*, MT Mmbaga and Jamille Robinson. 2023. Effect of four bacterial endophytes on tomato biomass production. Presented at Tennessee State University Research Symposium.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Jamille Robinson* and Margaret Mmbaga. 2023. Biochemical analysis of bacillus thuringiensis isolate IMC8 in plant disease protection and growth promotion in tomato. Presented at Tennessee State University Research Symposium.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: M. T. MMBAGA, D. Demissie, J. Robinson, P. Eyegheleme. 2022. Field evaluation of bacterial endophytes for biocontrol of Phytophthora capsici and yield improvements in Capsicum annuum and Solanum lycopersicum Presented at Annual Meeting of American Phytopathological Society. Pittsgurgh, PA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Mustapha Olawuni & Margaret Mmbaga. Evaluation of Three Biological Control Agents for Southern Blight Disease Management. Presented at Tennessee State University Research Symposium.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Emily Rotich* and Margaret T. Mmbaga 2022. Data on Plant Defense Enzyme Activity Associated with Three Endophytes Against Cornus florida Erysiphe pulchra Powdery Mildew. Data In Brief Journal (2022)
• Type: Journal Articles Status: Published Year Published: 2022 Citation: E. Rotich and M.T. Mmbaga. Replication Data on bacterial endophytes providing plant defense (2022). Havard Dataverse V. https://doi.org/10.7910/DVN/YP61IT.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Margaret T. Mmbaga, Emily Rotich, and Jacqueline Joshua Naturally occurring bacterial endophytes of dogwood suppress diverse pathogens: the case study of Stenotrophomonas sp. and Serratia marcescens against powdery mildew pathogen (Erysiphe pulchra), Fusarium oxysporum, F, solani, and Macrophomina phaseolina. Forest Pathology Journal (2023).

Progress 05/01/21 to 04/30/22

Outputs
Target Audience:The target audience of the project includes: 1. Agricultural extension community including extension agents for vegetable production 2. Vegetable producers 3. Agricultural research and education community Changes/Problems:There has been a delay in starting the extension studies and establishing the subcontracts due to unforeseen circumstances. However, the project is progressing. What opportunities for training and professional development has the project provided?The project has provided an opportunity for training three graduate students. Some of these students were supported to attend both the regional and national conference of the American Phytopathological Society. The student got the opportunity in these conferences to network and develop other professional skills. How have the results been disseminated to communities of interest?1. Presentations of research findings were made at the university wide workshop. 2. Presentations were made at the regional meeting of the American Phytopathological Society- Southern division. 3. The PDs incorporate the findings of the research into the materials they teach in class on Crop Protection and Plant-Microbe Interactions. What do you plan to do during the next reporting period to accomplish the goals?1. We will work on growing bacteria in large quantities for studies on formulations. 2. Once we have an acceptable formation that works, we will evaluate it for shelf life and bacterial viability.

Impacts
What was accomplished under these goals? We have evaluated different growth media for growing bacterial isolated of our Biological Control Agents (BCA) foroptimum yield. Through this, we have identified the best medium for growing bacteria for formulation studies.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Robinson, J. 2022. Biochemical and molecular analysis of disease protection and growth promotion in tomato from selected biological control isolates. Presentation at 99th Southern Division Meeting of APS. March 7-10, 2022. Chattanooga, Tennessee.