IDENTIFICATION OF PLANT ENDOPHYTES FOR PLANT PROTECTION AGAINST FUNGAL DISEASES, PESTS AND ENVIRONMENTAL STRESSES.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: EVANS-ALLEN
• Reporting Frequency: Annual
• Accession No.: 1004771
• Project Start Date: Jan 9, 2015
• Project End Date: Sep 30, 2017
• Program Code: [(N/A)]- (N/A)

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

Performing Department
Agricultural and Environmental Sciences

Non Technical Summary
A wide range of endophytic bacteria and fungi that live inside plants without causing any symptoms are known to benefit their host plant in various ways including boosting plants defense system against pathogen, insect pests, environmental stresses and promoting plant growth. Fungal pathogens impose major constraints on agricultural production globally; despite the continued release of disease-resistant cultivars and chemical fungicides, fungal infections continue to cause crop yield losses and motivate the development of effective and durable disease management systems. These challenges are likely to worsen with climate change because of increased likelihood of drought and altered precipitation patterns. Novel, robust and naturally abundant microbial strains that colonize plants internally and enhance plant defense system against fungal pathogens are expected to be less vulnerable to external environmental fluctuations. Thus, endophytes can be an important source of biocompounds and provide valuable products for improving agricultural productivity with reduced agricultural chemicals, especially pesticides. There is need to identify such endophytes and utilize them to increase crop productivity. The main objective of this project is to identify endophytes that can be used to boost plant protection against diverse fungal pathogens. Specifically (1) identify endophytes that have utility as biological control agents against fungal pathogens and/or insect pests, and/or enhance plant tolerance to environmental stresses associated with climate change and (2) evaluate host range, plant colonization and plant growth-promoting ability of the selected endophytes in horticultural crops including diverse vegetables and ornamental plants

Animal Health Component

50%

Research Effort Categories

Basic

20%

Applied

50%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	2499	1160	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1160 - Pathology;

Keywords

biological control

climate change

horticultural crops, agricultural productivity

Goals / Objectives
This project will focus on culturable endophytes and their interaction with diverse host plants including food crops and horticultural crops.Objective: (1) Isolation and identification of endophytes for the studyObjective: (2) Evaluation of selected endophytes for host range, plant colonization and plant growth-promoting ability.Objective 3) Evaluation of endophyte-host plant interactions in protecting host plants against fungal pathogens.

Project Methods
Objective: (1) Isolation and identification of endophytes for the study. Endophytes will be isolated from internal tissue of stems and twigs of healthy C. kousa and C. florida plants in otherwise disease-prone areas. Between the two species, C. kousa, is generally less susceptible to diseases caused by foliage and soil-borne pathogens than C. florida. Endophytes will be isolated from inner tissues of stem and twigs using organ pieces surface disinfested with 70% ethanol. Fungal endophytes will be isolated on acidified potato dextrose agar (PDA) medium containing 0.1% lactic acid. Endophytic bacteria will be isolated on Nutrient agar (NA) medium using the same plant materials; pure cultures will be obtained by sub-culturing. Because many pathogenic organisms can colonize plants internally with an extensive period of asymptomatic growth and latent infection before symptoms develop, all isolates will be tested for pathogenicity using detached-leaf assays and pathogenic isolates will be eliminated from further study. Identification of the fungi and bacteria will use standard morphological features and DNA sequence-based analysis; the diversity of endophytes colonizing the disease-prone C. florida will be compared to that of endophytes isolated from the fairly disease-free C. kousa.Objective: (2) Evaluation of selected endophytes for host range, plant colonization and plant growth-promoting ability.Host range: Potential hosts including food crops, including legumes such as common bean (Phaseolus vulgaris), cowpea (Vigna unguiculata), lima bean (P. lunatus); horticultural crops including strawberry (Fragaria ananassa)], bell pepper (Capsicum annuum), tomatoes, eggplants and oil crops such as canola (Brassica napa) will be evaluated for colonization by the selected endophytes. Detection of plant colonization by the endophytes will be evaluated using standard light microscopy and fluorescence microscopy. The time needed by endophytes to colonize and become established and visualization of colonization will be conducted with fluorescence microscopy and scanning electron microscopy as needed. Visual growth vigor, stem diameter, plant height, leaf vigor (chlorophyll content), root morphology, and plant biomass (oven-dry weight) will be measured on selected plant host species to detect plant growth responses to endophyte -host interactions. Root and leaf tissues will be tested for increases in growth hormones (e.g., auxins, cytokinins, gibberellins, abscisic acid and other relevant phytohormones).Objective 3) Evaluation of endophyte-host plant interactions in protecting host plants against fungal pathogens To evaluate endophyte effect in reducing plant infections from soil-borne pathogens, two methods of inoculation will be used: seed/germinant treatment and root drenching. Plants will be challenged with the fungal pathogen inoculum either at the same time or post endophyte colonization/establishment; development of disease symptoms will be monitored throughout the study. Selected endophytes will be evaluated individually and as a population of two endophytes (for those that showed no inhibition when grown in vitro on dual culture plates). Control of diseases in presence of endophytes applied individually and in combinations will be quantified using standard disease rating scales. For foliage disease studies, plants will be spray inoculated with the endophyte at the two-leaf stage; plant challenge with the pathogen at pre-and post-plant colonization by endophyte will be by foliar spray. Disease development will be monitored and the level of disease control will be quantified and analyzed using SAS linear model (SAS 2010). Endophytes that provide protection against fungal pathogens will be selected.

Progress 01/09/15 to 10/30/14

Outputs
Target Audience:In this study, culturable endophytes colonizing flowering dogwood were isolated; the microorganisms were identified and their diversity was analyzed. Out of 197 endophytic isolates, 95% were fungi and only 5% were bacteria ; phylogenetic relationships between the endophytes was analysed. The endophytes were evaluated for pathogenicity and the nonpathogenic isolates were evaluated for potential application in biological control of known fungal pathogens including Fusarium solani, Fusarium oxysporum, Fusarium spp, Cercospora sp., Phytophthora species, and Macrophomina phaseolina using in-vitro dual cultures and detached leaf techniques. The majority of isolates evaluated did not cause disease symptoms on detached leaves indicating that they were not foliar pathogens on dogwood. However, a few isolates caused clearly defined symptoms within a few days indicating their pathogenic status as latent pathogens that may cause sporadic disease symptoms on the dogwood host when the environment for disease development becomes favorable . Several endophytic bacteria were found effective in controlling powdery mildew of flowering dogwood in greenhouse and field environments Five bacteria were found effective in controlling powdery mildew of flowering dogwood in greenhouse and field environments and some showed potential in controlling Phytophthora and macrophomina root rot in food crops under greenhouse conditions. One endophytic fungus suppressed mycelial growth of root rot pathogens, Fusarium solani, Fusarium oxysporum, Macrophomina phaseolina and Phytophthora capsici in dual cultures and reduced P. capsici root rot on pepper 'in growth chamber and greenhouse experiments Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One undergraduate, two MS and Two PhD students were trained in this project.. How have the results been disseminated to communities of interest?Yes, Dissemination to nursery growers through Research Conference at Nursery trade meetings, and through publications in nursery related meeting proceedings. Dissemination to the scientific community through meeting presentations and publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Out of 197 isolates of endophytes were isolated and identifiedduring thisstudy, some were latent pathogens while eight isolates seemed to suppressed growth of pathogenic fungi in culture InObjective: (2) Evaluation of selected endophytes for plant colonization and plant growth-promoting ability was conducted in ,Cornus florida, snap beans, soybens and bell pepper beans. Objective 3) Evaluation of endophyte-host plant interactions in protecting host plants against fungal pathogens.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Parikh, Lipi; Margaret T Mmbaga; Geoffrey Meru, Guorong Zhang, Lucas Mackasmiel, Phillip A Wadl, xinwang Wang, Robert N Trigiano. 2017. Quantitative trait loci associated with resistance to powdery mildew in Cornus florida. Scientia Horticulturae Journal. DOI: 10.1016/j.scienta.2017.08.044 Yonghao Li, Margaret T. Mmbaga, Boru Zhou, Jacqueline Joshua*,Emily Rotich, and Lipi Parikh*2016.Diseases of Hydrangea. Handbook of Florists' Crops Diseases. Part of the series Handbook of Plant Disease Management pp 1-19. DOI 10.1007/978-3-319-32374-9_36-1 (Print ISBN978-3-319-32374-9; Online ISBN978-3-319-32374-9) (Book chapter) Parikh*,L.P., Mmbaga, M.T. Kodati, S. Blair, M. Hui D. and Meru G. 2016 Broad-sense heritability and genetic gain for powdery mildew resistance in multiple pseudo-F2 populations of flowering dogwoods (Cornus florida L.). Scientia Horticulturae 213 (2016) 216221 Mmbaga, M.T.; Mrema, F.A. Mackasmiel L. and Rotich*, E. 2016. Effect of bacteria isolates in powdery mildew control in flowering dogwoods (Cornus florida L.). Crop Protection 89: 51-57 Jacqueline Joshua, Margaret T. Mmbaga and Lucas A. Mackasmiel 2016. Cherry Leaf Spot Disease Management in Ornamental Cherries in Mid-Tennessee. Canadian J of Plant Science. Can. J. Plant Sci. 97: 110118 (2017) dx.doi.org/10.1139/cjps-2015-0362 Parikh* L.P. Mmbaga, M.T., Kodati*, S., & Zhang G. (2015). Estimation of Narrow sense heritability of powdery mildew resistance in pseudo-F2 (F1) population of flowering dogwoods (Cornus florida L) European Journal of Plant Path. DOI 10.1007/s10658 015-0806-5 Joshua*, J.O. and M.T. Mmbaga 2014. Perpetuation of Cherry Leaf Spot Disease in ornamental Cherry. Phytopathology Journal Phytopathology Journal ; DOI: 10.1111/jph.12309 Maheshwari Asha , M. T. Mmbaga, J. Joshua, and S. Gurung. 2015. Diversity of Endophytes in Flowering Dogwoods and their Potential Applications. Proceed. Southern Nursery Association Research Conference .Vol 60: 158-164. Mackasmiel L. A and M. T. Mmbaga. A Survey for Soil-borne Pathogens of Woody Ornamentals in Mid-Tennessee Nurseries. Proceed. Southern Nursery Association Research Conference. Vol 60: 151-157. Rotich Emily. Margaret T. Mmbaga, Parama SikdarThe mode of action of bacterial biocontrol agents against powdery mildew in Cornus florida. Proceed. Southern Nursery Association Research Conference Vol 61:124-127 Maheshwari Asha M. T. Mmbaga and J. Watson. 2016. Screening of endophytic microorganisms from flowering dogwoods (Cornus florida L.) for their pathogenicity and potential biological control activity. Proceed. Southern Nursery Association Research Conference .Vol 61:120-123 Mmbaga, M. T. E. Rotich. 2016 (Abst). Microscopic analysis of parasitism mode of action by bacterial biological control agents on Cornus florida powdery mildew. American Phytopathological Society Annual conference. P 196