EVALUATION OF TWO ENDOPHYTIC BACTERIA AS BIOLOGICAL CONTROL AGENTS FOR FUNGAL PATHOGENS AND AS HOST PLANT GROWTH PROMOTING AGENTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1012139
• Grant No.: 2017-38821-26418
• Cumulative Award Amt.: $297,476.00
• Proposal No.: 2016-06533
• Project Start Date: May 15, 2017
• Project End Date: May 14, 2022
• Grant Year: 2017
• Program Code: [EQ]- Research Project

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

Performing Department
Agriculture and Envir Science

Non Technical Summary
The overall goal of this project is to advance scientific knowledge on the role of microorganisms that inhabit plant tissue internally without causing harm to the host plants, to determine their role in protecting their hosts against diseases caused by fungi. The project will identify mechanisms involved in the plant defense against several pathogens of economic importance. Results from this project will confirm previous research results on the effect of the selected beneficial organisms on plant disease management. Results generated from our previous research on microorgansims that are found inside plant tissue have shown that some of the organisms help plant fight diseases naturally. The research looked at the effect of selected organisms on one disease (powdery mildew) that impact dogwood production. However the research will be expanded by testing the selected microorganisms on other plant diseases and crops of economic importance as follows: (1) Previous studies conducted in the laboratory will be tested on real plants in the greenhouse and field environments to assess the potential of selected microorganisms on diverse fungal pathogens and their potential utilization in different crops of economic importance. (2) Evaluation on how different soil conditions affect the colonization of plants by the selected beneficial microorganisms. ( 3) Evaluate mechanisms that underlie the beneficial interactions between selected organisms and their host plants at DNA level. (4) Identify compounds that are produced when the beneficial organisms interact with the host plant, interact with other beneficial organisms and with pathogens This project will result in utilization of a natural resource available in naturally existing beneficial microorganisms that may be developed into products marketable to the general public for controlling plant diseases. Therefore, the project fits well with sustainable agriculture on (1) Plant health and production and plant products, (2) Renewable energy, natural resources, and environment; and (3) Agriculture systems and technology. It is expected that the project results will generate information that would play a role in reducing the use of conventional fungicides; help in reducing health and environmental hazards that result from toxicities caused by chemical fungicides that are normally used to control plant disease. Other benefits from this project are including training of students especially minorities at BS, MS and PhD levels and help increase the number of underrepresented minorities in USDA and other agricultural employment sectors.

Animal Health Component

70%

Research Effort Categories

Basic

10%

Applied

70%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1419	1160	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1419 - Leguminous vegetables, general/other;

Field Of Science
1160 - Pathology;

Keywords

biochemical mechanisms

biological controls

fungicides

gene expression

diseases

pathogens

Goals / Objectives
The overall goal of this project is to advance scientific knowledge on the role of endophytes in protecting their hosts against fungal pathogens and identify biochemical and molecular mechanisms involved in plant defense. Results generated from our previous research on eco-friendly biological control agents (BCA) for powdery mildew will be advanced and new applications of those BCAs on other fungal pathogens and crops of economic importance will be developed with the following specific objectives1.Confirmation of in vitro studies on bioactivity of selected bacterial BCA on diverse fungal pathogens and assessment of their potential utilization in different crops.2. Identification ofthe point of ingress to plant hosts through foliage (spray applications), root (drenching), or seed (seed/germant coating)3. Evaluation ofsoil-ecological factors that may influence BCA colonization of selected plants and in the rhizosphere and non-rhizosphere environments.4. Evaluation ofmolecular mechanisms (Dual RNA sequencing) that underlie the BCA beneficial effect on their host5. Identification of potential novel biochemical products from BCA-Pathogen and BCA-BCA interactions in fungal disease control and plant growth improvement

Project Methods
Objective 1: Confirmation of in-vitro studies on the effect of selected BCA on diverse fungal pathogens will be conducted in greenhouse using spore forming Bacillus isolate IMC8 and B17A to confirm results from in-vitro studies in which growth of Cercospora sp., Glomerella sp., Fusarium solani, F. oxysporum and Macrophomina phaseolina were significantly suppressed. Successful greenhouse experiments will be followed by field experiments. Since these pathogens impact broad host plants of agricultural importance in the U.S.A, studies will be conducted to concurrently show the potential utilization of these BCA on Cercospora sp, Glomerella sp. and F.oxysporum using flowering dogwood (C. florida) as a host and on charcoal/black rot (M. phaseolina) using Phaseolus vulgaris (snap and common beans) and bell pepper (Capsicum annuum) both of which are highly susceptible to M. phaseolina. The two food crops high susceptibility to the above fungal pathogens and their high aesthetic quality requirement often necessitate repeated fungicide applications. Host plant colonization of internal tissue of plants will be visualized using (a) re-isolation of the bacteria from inoculated plants (b) plant clearing and light microscopy and visualization of bacteria using fluorescence microscopyAssessment of growth-promoting ability of selected BCA that have shown potentials will be conducted on C. florida and two food plants, a legume (P. vulgaris) and a solanaceous plant (C.annuum). Plant growth responses to the selected bacteria, will be evaluated by visual growth vigor measured by plant height, leaf vigor (chlorophyll content), root morphology, and plant biomass measured by oven dry weight. Root and leaf tissues will be tested for increases in growth hormone production (e.g. auxins, cytokinins, gibberellins, and abscisic acid) using Liquid Chromatography Mass Spectrometry (LCMS) protocols for growth hormones and using high performance lipid chromatography (HPLC) protocols. These studies will be conducted in a greenhouse and repeated in field environment using a replication of four and a randomized complete block design. Data analysis will be done using SAS linear model for analysis of variance (SAS 2010).Objective 2. Evaluation of soil-ecological factors influencing BCA colonization of plants and soil colonization in rhizosphere and non-rhizosphere environments will be done using C.florida, P.vulgaris and/or C.annuum; edaphic factors that will be tested are: soil texture, soil temperature, moisture, N, pH levels and organic matter. Each edaphic factor will be evaluated separately using a randomized complete block design with four replicates and three levels of each edaphic factor. Soil created for different ecological factors will be inoculated with equal volumes and concentration of the BCA and planted with selected BCA. BCA tagged with green fluorescence protein (gfp) and, red fluorescence protein (rfp), will be used to facilitate bacteria visualization. Colonization of plants will be visualized and colonization efficiency will be determined by the abundance of bacteria in each treatment.Objective 3: Evaluation of molecular mechanisms underlying the beneficial interactions between the selected BCA and their host by using gene expression analysis. Dual RNA sequencing transcriptional analysis will be performed on 2 biological replicates of each treatment (inoculated and non-inoculated roots). Each replicate will consist of roots of ten C. florida seedlings and total RNA will be extracted using a commercial kit (Qiagen) and checked for purity and concentration using a Bioanalyzer platform (Agilent Technologies, Santa Clara, CA). To separate bacterial and C. florida libraries, the sequence reads will be first mapped to the reference sequences of Bacillus thuringiensis (IMC8) and Stenotrophomonas maltrophilia (B17A). The unmapped sequences will be assembled de novo also using the CLC Genomics Workbench platform. Differential expression in C. florida, IMC8 and B17A will be estimated using the DEseq package taking into account negative binomial distribution and shrinkage estimation (variance and size-factor in the distribution). Quantitative reverse transcription PCR (RT-qPCR) will be performed to confirm differential expression of 12 selected genes.Objective 4: Evaluation of biochemical mechanisms and identification of potential novel products from BCA-Pathogen and BCA-BCA interactions will include (i) Identification of volatile secondary metabolites by Gas Chromatrography Mass Spectrometry (GCMS) and (ii) Identification of non-volatile secondary metabolites by using Liquid Chromatography Mass Spectrometry (LCMS) and High performance lipid chromatography (HPLC) protocols. Individual bacterial isolates, and dual cultures of bacteria-pathogen, and bacteria-bacteria grown in 200 ml of nutrient agar broth and/or Potato dextrose agar broth will be analyzed and compounds identified to check for compounds of interest including compounds known to be precursors of compounds involved in host parasite interactions and those associated with induced systemic resistance (ISR) pathway such as Methyl Jasmonate involved in Jasmonic acid pathway (i.e. linolenic acid, 13(S)-hydroperoxylinolenic acid, and phytodienoic acid) and other precursors.Evaluation on whether the project objectives have been met will be by using the outcome metrics. Project outcomes include increased capacity by training students at M.S/ Ph.D. level and support of undergraduate experiential training as work study students. Students will have the opportunity to make presentations on their research and publish research results in abstracts at the University wide symposium available on-line at the TSU Research Sponsored Programs website and at Tennessee Academy of Science. In addition, students have the opportunity to present their research at other local and regional professional meetings like the 1890 Research Directors meeting, Southern Nursery Association Research conference and the American Phytopathological Society annual meetings. Being able to make presentations and publications in meeting abstracts or proceedings and full journal articles is a good measure that the project objectives are being met in terms of work done and student training. This project has broad objectives and can support 2 Masters student's research and 1 Ph.D. student research. Students will generate data that they will present in meetings, thus the number of publications including thesis, and student presentations in meetings each year of the project will be used to evaluate project success and progress made. Similarly, faculty involved in this project will present their work to scientific community in their respective professional meetings and also publish data in refereed journals and in meeting proceedings and abstracts. Having enough data to present and publish in journals will be a good measure that the project objectives are being met. It is anticipated that at least 2-3 articles will be published in refereed journals per year and 2-3 presentations in professional meetings every year as good indicators that good progress is being made and project objective are being met. At least 1-2 articles from M.S. degree thesis and 2-3 articles from Ph.D. dissertation are expected outcomes. By the end of this project, potential bioactive compounds or product(s) for disease management will be the ultimate measure of project success. Availability of such products for commercial potential evaluation is a bigmeasure of project success.

Progress 05/15/17 to 05/14/22

Outputs
Target Audience: Nothing Reported Changes/Problems:The pandemic affected research progress and in addition, a tornadohit Nashville and destroyed our research facilities,including research greenhouses. What opportunities for training and professional development has the project provided?One undergraduate student received experiential learning and two PhD students completed their dissertation research, graduated and got hired in industry How have the results been disseminated to communities of interest?Presentation of research results in professional meetings and publications in peer-reviewed journals What do you plan to do during the next reporting period to accomplish the goals?The project has been completed

Impacts
What was accomplished under these goals? Studies on the point of ingress to plant hostand the identification of potential novel biochemical products from the two BCAs and BCA-Pathogen interactions in fungal disease control and plant growth improvement were completed as part of dissertation research andtwo PhD. students graduated on this project.

Publications

Progress 05/15/20 to 05/14/21

Outputs
Target Audience:Organic farmers, nursery growers, conventional farmers Changes/Problems:The project duration has changed from three to five years due to the following reasons: Student training was delayed for two years because the student could not start in the first year and then after starting, he needed additional time off due to unforeseen family problems. This put us back for 18 months. In summer 2019 TSU encountered electricity problems that knocked out electricity in the whole campus for over one month. This destroyed our research material that was in freezers awaiting analysis. We had to restart the project by growing new plants and apply treatment for analysis. Unfortunately, we could not easily start over in 2020, because Tennessee was hit by a destructive Tornado that destroyed our research greenhouses and research facilities and on-going research. Makeshift greenhouse spaces covered with plastic were used, but results were limited and could not be confirmed. The Covid 19 pandemic that closed our regular research activities and student training started in 13th March 2020. This impacted our research progress due to more time needed to prepare for safe lab work, ordering supplies, inadequate space for greenhouse work, and travel restrictions for dissemination of the results we had. What opportunities for training and professional development has the project provided?One graduate student was trained at MS level and one at PhD level and both gained experience in presenting their research results at professional meetings and in journal publications. One undergraduate student received experiential training in research. How have the results been disseminated to communities of interest?Presentations to other scientists and Extension agents in journal publications. What do you plan to do during the next reporting period to accomplish the goals?Complete the remaining activities that are in progress in different project goals: Dual RNA sequencing that underlies the BCA beneficial effect on their host. Use fluorescent endophytes to track the endophyte route of plant colonization and movement within plants when seed is the point of ingress. Complete the evaluation of plant colonization and effect of the selected endophytes on diverse crops to include Canola, mung bean, and cowpeas. Canola is a potential overwinter crop in vegetable production; its colonization would benefit perpetuation of the endophytes in the rhizosphere and may broaden the range of pathogens that can be controlled using the selected endophytes. Analysis and identification of volatile and non-volatile biochemical compounds associated with the endophyte-host plant interactions, BCA-Pathogen and BCA-BCA interactions in oomycete disease control and plant growth improvement.

Impacts
What was accomplished under these goals? In vivo studies on dogwood plants, snap beans, sweet pepper, tomatoes, soybeans, and sorghum confirmed results from the in vitro studies on the role of endophytes as biological control and growth promoting agents and revealed potential utilization of the selected endophytes in different crops. Their bioactivity on diverse fungal pathogens includes powderymildew, Phytophthora species and root rot pathogen Macrophomina phaseolina and revealed their potential use in diverse vegetables crops. Project activity on more crops and pathogens such as Canola, mung bean, and cowpeas have been initiated to be completed in 2021. Differences on endophyte efficacy were observed in different crops and pathogens, indicating some host-endophyte specificity that requires more studies on specific cases of endophyte-host interactions. The Identification of the point of ingress to plant hosts through foliage (spray applications), root (drenching), or seed (seed/germinant coating) have been confirmed and the movement of the endophytes within the host plant from seed to roots and lower stem/collar region was confirmed in sorghum, sweet pepper, and tomato. These observations indicate that the endophytes may be used in foliage spray applications, root (drenching), or seed treatment as needed in disease management. Studies on endophyte colonization of leaves and fruits when seed is the point of ingress have not been completed but use of fluorescent endophytes will facilitate the evaluation. Evaluation of soil-ecological factors that may influence BCA colonization of selected plants and in the rhizosphere and non- rhizosphere environments showed that high organic matter enhances efficacy, but autofluorescence interfered with the evaluation of colonization of sweet pepper in the rhizosphere and non-rhizosphere environments. The evaluation requires technique modifications to be completed in 2021. Evaluation of molecular mechanisms (Dual RNA sequencing) that underlie the BCA beneficial effect on their host. The evaluation is in progress and results are not available. Lock-down and working from home disrupted 2020 research and slowed down research progress. Identification of potential novel biochemical products from BCA-Pathogen and BCA-BCA interactions in fungal disease control and plant growth improvement identified volatile and non-volatile compounds associated with the endophye-host plant interactions and their analysis revealed compounds that are reported antimicrobials. Several volatile compounds were produced by the endophytes while different compounds were produced by the BCA-Pathogen interactions. Analysis of BCA- BCA interactions and pathogen emitted volatiles is in progress. Non-volatile compounds emitted by the BCA include diverse cell wall degrading enzymes including cellulase, amylase, chitinase, pectinase, lipase, and protease. The endophytes also produced plant growth promoting compounds such as phytohormone indole acetic acid (IAA), ammonia and siderophores and displayed the ability to solubilize phosphates and fix nitrogen. It was postulated that the biocontrol activity of the selected endophytes may be mediated by a combination of one or more enzymatic and/or plant growth promoting traits exhibited in this project.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Asha Maheshwari* and Margaret Mmbaga, Bandana Bhusal* and Christine A. Ondzighi-Assoume.2021. Effect of Volatile Compounds Produced by Selected Bacterial Endophytes in Promoting Plant Growth. Hortscience Journal (Accepted) Asha Maheshwari* and Margaret Mmbaga and Emily Rotich *2021.Antagonistic effects of selected bacterial endophytes against dogwood fungal pathogens to Frontiers in Plant Science (Submitted) Margaret Mmbaga Emily Rotich* and Jacqueline Joshua*. 2021. Studies on Stenotrophomonas Sp. And Serratia Marcescensas Biological Control Agents of Powdery Mildew of Dogwood. Biological control Journal. (In review) Asha Maheshwari* and Margaret Mmbaga and Emily Rotich* 2021.Antagonistic effects of selected bacterial endophytes against dogwood fungal pathogens to Frontiers in Plant Science (Submitted) Bhusal1*, and M.T. Mmbaga 2020. Biological control of Phytophthora Blight and growth promotion in sweet pepper by Bacillus species. Journal Biological Control Volume 150, https://doi.org/10.1016/j.biocontrol.2020.104373B. * Graduate student authors

Progress 05/15/19 to 05/14/20

Outputs
Target Audience:Dogwood nursery production, Farmers in Vegetable production systems, Small farmers , rural farmers and minority farmers, organic farmers or farmers thinking of transitioning to organic farming without chemical pesticides and conventional farmers seeking to reduce pesticide usage. Changes/Problems:Equipment failure for biochemical analysis was a problem and the use of a food Science equipment "Electronic Nose" that uses GCMS technology was used for the remaining analyses. In Spring 2020, there was a severeTornado in Tennessee and TSU research farm,farm equipment,greenhouses and shade houses were completely destroyed. This severely affected our field research and greenhouse studies.Covid-19 also affected the country in 2020 resulting inunplannedteaching onlineand working from home in Starting Mid-March. This affected ordering of supplies, and delayed lab research thereby affecting our research progress. What opportunities for training and professional development has the project provided?FourMSgraduate students had training opportunity in thiis project. Three of the MS students graduated; twomoved to PhD programs One isat the Univ of Wisconsin, Madison, andone joined the TSU PhD program in Biological Sciences, while one took a job in extension services.Currently there isione MS student on biological control who will gradi=uate in the coming year with an interest to continue for PhD program either locally ot to another university. Two PhD students had dissertation research in this project partly supported by this project and theyboth graduated and got employed in their field os specialization.One of the PhDgraduates had focused on biomedical research and tested the selected endophytes on cancer treatment and as a sources of antibiotics;upon graduation, the young graduate got ajob in product development at a pharmaceutical company based inNashville, TN. The second PhD studenthad dissertation research on biological disease control andhad job placementas Senior research scientist at Bayer Crop Research. Two undergraduate students whoreceived experiential training on this project and graduated with job offersasa nursery plant Manager in Nashville, farmers market, TN and one in Railway company in Atlanta, GA. How have the results been disseminated to communities of interest?Presentations of project results at annual meetings of the TSU annual Farm Expo that brings invited farmers from all over Tennessee, at a symposium organized forsmall scale farmers and minority farmers and Southern Nursery Asociation, targeting ornamental nursery growers and vegetable growers. Presentations at the American Horticultural Society, American Phytopathplogical Scociety, 1890 Association of Research Directors and Tennessee Academy of science were made. Each student made presentations at TSU Unoiversity symposium targeting professional scientists in agricultural and biological sciences. Publications as abstracts and as journal articles also disseminated project results. What do you plan to do during the next reporting period to accomplish the goals?Refinement of seed treatment followed by spray or root drenching. Evaluation of soil-ecological factors that may influence BCA colonization in the rhizosphere and non-rhizosphere environments will be done using fluorescent tags to track and quantity the endophytes. Evaluation ofmolecular mechanisms (Dual RNA sequencing) that underlie the BCA beneficial effect on their host Continue the identification of potential novel biochemical products from BCA-Pathogen and BCA-BCA interactions in fungal disease control and plant growth improvement.

Impacts
What was accomplished under these goals? The overall goal of this project is to advance scientific knowledge on the role of endophytes in protecting their hosts against fungal pathogens and to identify biochemical and molecular mechanisms involved in plant defense. Research activities in this goal are on-going.Confirmation of biological controlactivittiesfor powdery mildew disease management have been advanced using electron microscopy which confirmed collapse of spores and breakdown of hyphae cell walls that may be assiociated with parasitism, and/or biochemicalcompounds from the BCAs. Objective 1: Confirmation of in vitro studies on bioactivity of selected bacterial BCA on diverse fungal pathogens and on plant growth promotion included evaluation on pepper and tomatoes. Assessment of the BCA have displayed great potentialon Macrophomina phaseolina on beans and on cucumbers, on powdery mildew in cucumbers and squash and on Phythophthora capsici in pepper and tomato 2. Identification ofthe point of ingress to plant hosts through foliage showed potential effect using spray applications, root (drenching), or seed treatment (seed/germant coating) 3. Evaluation ofsoil-ecological factors that may influence BCA colonization of selected plants in the rhizosphere and non-rhizosphere environments is still in progressusing the effect of organic matter and other edaphic factors. 4. Evaluation ofmolecular mechanisms (Dual RNA sequencing) that underlie the BCA beneficial effect on their host is in progress. 5. Identification of potential novel biochemical products from BCA-Pathogen and BCA-BCA interactions in fungal disease control and plant growth improvement identified (i) some volatiles that are produced by the pathogen alone and new biochemical compounds produced when pathogen and BCA interact as well as biochemicals that are not produced when pathogen and BCA interact. (ii) BCA abilities in hydrolytic enzyme production including Cellulase, amylase, pectinase, chitinase, protease, andlipase associated with cell wall degradation. (iii) plant growth promoting traits in selected endophytic bacteria including IAA production, ammonia production, nitrogen fixation**Siderophore and Phosphate Solubilization

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Bhusal, B.*, and M.T. Mmbaga 2020. Biological control of Phytophthora Blight and growth promotion in sweet pepper by Bacillus species. Biological control Journal ( Accepted) Rotich* E. Mmbaga M.T and Joshua* JO 2019. Biological control of powdery mildew on Cornus florida using endophytic Bacillus thuringiensis isolate. Can J. of Plant Pathol. (https://doi.org/10.1080/07060661.2019.1641555) Joshua* J.O and Mmbaga M.T. 2019. Potential Biologicall Control Agents for Soil-borne Fungal Pathogens in Tennessee Snap 1 Bean. Hortscience J. (Hortscience 54: 1-7 https//doi.org/10.21273/HORSCI14081-19 * Graduate student training

Progress 05/15/18 to 05/14/19

Outputs
Target Audience:Growers including organic growers who need disease control measures that are environmentally friendly. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. Confirmation of in vitro studies on selected bacterial BCA was done in growth chambers and greenhouse . Confirmed bioactivity of the selected BCAs on diverse fungal pathogens included powdery mildews, Phytophthora species, Sclerotium rolfsii andMacrophomina phaseolina. Assessment of potential utilization of the BCA was on dogwood and diverse vegetable crops such as bell pepper, tomatoes, squash, cucumbers, andsnap beans. 2. Identification of the point of ingress to plant hosts was on foliage (spray applications), root (drenching), and seed soaking (seed coating). 4. Molecular mechanisms that underlie the BCA beneficial effect on their host is currently under evaluation. How have the results been disseminated to communities of interest?1. Presentationto organic vegetable growers. 2.Publications in professional meetings to disseminate results to other scientists and community at large. What do you plan to do during the next reporting period to accomplish the goals?1. Repeat greenhouse and field experiments to confirm bioactivity of selected bacterial BCA on disease control and plant growth in vegetable production. 2. Determine best application methodology for BCA by farmers. While all seed soaking, drenching and spray methodologies have been found effective depending on the disease being controlled, practical application that focus on shelf-life of the BCA and grower-friendly methodologiesin terms ofease to use and marketthe BCAs will be addressed, 3. Identification of soil-ecological factors that favor BCA colonization of selected plants and in the rhizosphere and non-rhizosphere environments will continue. 4. Evaluation ofmolecular and biochemical mechanisms that underlie the BCA beneficial effect on their host will focus on alternative methodologies due to problems on equipment breakdown(HPLC and GCMS) for biochemical analysis. Arrangements for sample paymentsat other institutions will be used, the number of samples will be limited and time will take a little longer.

Impacts
What was accomplished under these goals? 1. Confirmation of vitro studies was done in greenhouse and growth chambers. Bioactivity of the selected BCAs on diverse fungal pathogens has been confirmed on powdery mildews, Phytophthora species, Sclerotium rolfsii and Macrophomina phaseolina. Assessment of potential utilization of the selected BCA was conducted on dogwood, snap beansand diverse vegetable crops including bell pepper, tomatoes, squash,and cucumbers. 2. Identification of the point of ingress to plant hosts was evaluated on foliage (spray applications), root (drenching), and seed (seed soaking and coating). 4. Molecular mechanisms that underlie the BCA beneficial effect on their host is currently under evaluation.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Mmbaga, Margaret; Gurung Sunil and Maheshwari, Asha. 2018. Screening of plant endophytes for biological control agents against root rot pathogens of pepper (Capsicum annum). J. Plant Pathol. Microbiol. 9: 435. doi: 10.4172/2157-7471.1000435. Mmbaga, Margaret T., Mackasmiel Lucas A. and Mrema, Frank A. 2018. Evaluation of Biological Control Agents for Macrophomina Root Rot and Powdery Mildew in Flowering Dogwood (Cornus florida L.) Hortscience 53(10):16. 2018. https://doi.org/10.21273/HORTSCI13071-18.

Progress 05/15/17 to 05/14/18

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The experiential research opportunities provided by this project impacted one undergraduate and four graduate students who carried out independent research within this project. These students displayed professional development in conducting research including experimental design, data collection and analysis and research presentations in oral, poster and written reports from their projects. Presentations made at the University-wide symposium resulted in student winning a prize for the undergraduate oral presentations and one graduate student winning the first prize on oral presentations; one of the graduate students was selected as the outstanding graduate student in the college of agriculture. One graduate student had opportunity to present research results at the microbiology professional meeting that was held in Cookeville, TN. How have the results been disseminated to communities of interest?Publication of research results in journals and in meeting proceedings and abstracts was the main form of results dissemination. One presentation was made to farmers at the small farms workshop held at Tennessee State University, Nashville, TN. The presentation to farmers focused on chemical- free disease management strategies in vegetable production, and attendants included farmers from different counties in Tennessee and from neighboring states including Alabama and Kentucky What do you plan to do during the next reporting period to accomplish the goals?1. Colonization on treated seed and methods for improving seed treatments will be evaluated focusing on enhancement of BCA shelf life on treated seed. 2. Plant growth stimulation by different BCAs will be evaluated 3. Soil-ecological factors that may influence BCA colonization of selected plants and in the rhizosphere and non-rhizosphere environments will be evaluated. 4. Studies on biochemical and molecular mechanisms (Dual RNA sequencing) that underlie the BCA beneficial effect on their host will be initiated and biochemical products associated with BCA-Pathogen and BCA-BCA interactions will be evaluated.

Impacts
What was accomplished under these goals? The first objective was addressed while also providing experiential training to four graduate students and one undergraduate student. In vitro studies on bioactivity of selected bacterial biological control agents (BCAs) were expanded to include more diverse fungal pathogens. In addition to flowering dogwood, the BCA potential utilization in different crops was evaluated on bell pepper, cucumbers and snap beans root rot pathogens and powdery mildew in greenhouse environment. Effect of selected BCAs on Macrophomina phaseolina of snap beans was also confirmed in field conditions. Effective BCAs on Phytophthora capsici of bell pepper, and cucurbit powdery mildew will be tested in field environment The identification of the point of ingress to plant hosts was evaluated based on the target diseases. Spray application on the foliage was evaluated on powdery mildew of cucumber and seed coating and root drenching was evaluated on root rot pathogens including Phytophthora capsici and Macrophomina phaseolina both of which showed positive results similar to chemical fungicides. .

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Irabor A and Mmbaga MT 2017. Evaluation of Selected Bacterial Endophytes for Biocontrol Potentialagainst Phytophthora Blight of Bell Pepper (Capsicum annuum L.) J Plant Pathol Microbiol 2017 8:10. Journal of DOI: 10.4172/2157-7471.1000424 Mmbaga, Margaret T., Mackasmiel Lucas A. and Mrema, Frank A. 2018. Flowering Dogwood Infections with Macrophomina phaseolina Hortscience 53(3): 13. 2018. https://doi.org/10.21273/Hortsci12596-17. Asha Maheshwari1, Margaret Mmbaga & Quincy Quick, A. 2018. Nigrospora Sphaerica Products from the Flowering Dogwood Exhibit Antitumorigenic Effects Via the Translational Regulator, Ps6 Ribosomal Protein.CPQ Cancer, 1(1): 01-12. Mmbaga, Margaret; Gurung Sunil and Maheshwari, Asha 2018. Screening of plant endophytes for biological control agents against root rot pathogens of pepper (Capsicum annum). J Plant Pathol and Microbiol [Accepted] Mmbaga, Margaret T., Mackasmiel Lucas A. and Mrema, Frank A. 2018. Evaluation of Biological Control Agents for Macrophomina Root Rot and Powdery Mildew in Flowering Dogwood (Cornus florida L.) Hortscience [Accepted)
• Type: Book Chapters Status: Published Year Published: 2017 Citation: Yonghao Li, Margaret T. Mmbaga, Boru Zhou, Jacqueline Joshua*,Emily Rotich*, and Lipi Parikh*2017. Hydrangea diseases. In: Handbook of Florists' Crops Diseases. Part of the series Handbook of Plant Disease Management pp 1-19
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Asha Maheshwari and Margaret T. Mmbaga 2018. Phylogenetic Biodiversity of Endophytic Fungi in Cornus florida L. Proceed. Southern Nursery Association Research Conference. Vol 63: In Press
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Steve Osborne, Margaret Mmbaga, Asha Maheshwari 2018(abst). Biological Controls Agents against Botryosphaeria dothidea. TSU Research symposium Nashville, TN. April 2-6, 2018. Afona Irabor and Margaret Mmbaga. 2018(abst). Biological control of southern blight of tomato using selected bacterial endophytes. TSU Research symposium Nashville, TN. April 2-6, 2018. Asha Maheshwari and Margaret Mmbaga. 2018. (abst) Analysis of plant growth promoting traits of selected biological control agents. TSU Research symposium Nashville, TN. April 2-6, 2018. Nia Gordon, Wilson K. Ouma, Margaret T. Mmbaga, Arvazena Clardy, and Christine A. Ondzighi-Assoume 2018(abst). Controlling Powdery Mildew on Cucurbit Crops through Biological Control Agents. TSU Research symposium Nashville, TN. April 2-6, 2018. Jeremiah Sumpter and Margaret Mmbaga. 2018 (abst). Evaluation of Bacterial isolates for Powdery Mildew Control in Cucumbers (Cucumis sativus). TSU Research symposium Nashville, TN. April 2-6, 2018.