Agronomy and Horticulture Department
Date of this Version
Root nodule is a unique environment formed in the legume roots through a highly specific symbiotic relationship between leguminous plants and nodule inducing bacteria. Over the years, Rhizobia have been thought as the only group of bacteria residing within the nodules. However, this consideration has been recently changed with the discovery of other groups of bacteria besides Rhizobia within the legume nodules. In an effort to identify beneficial bacteria for plant disease control and growth promotion, soybean nodules were studied as the source of nodule-associated bacteria. Metagenomics analysis of a single soybean nodule was conducted to determine the bacterial diversity of nodule microbiome. Surface sterilized single soybean nodule was used to isolate nodule-associated bacterial species. Fifty colonies were isolated from each nodule. A total of 500 colonies were tested individually against Clavibacter michiganensis subsp. michiganensis on solid media plates for inhibition of pathogen growth. From the initial screening, 54 colonies were selected based on significant growth inhibition of the pathogen. These colonies were further tested in vitro on two additional bacterial and two fungal pathogens on solid plates. In planta testing involved using 15 selected colonies from these 54 as inocula in tomato seedlings against Clavibacter michiganensis subsp. michiganensis. Tomato plants were infected using a soil inoculation method. In addition, one isolate from the selected 54 was tested in tomato seedlings against a Green Fluorescent Protein (GFP)-tagged Pseudomonas syringae pv tomato (Pst) by using stem inoculation method for the infection of tomato plants. Bacterial metabolites were extracted from 15 colonies with ethanol and tested against two bacterial pathogens on solid plates. These 15 colonies were identified by using MALDI-TOF mass spectrometry and 16S rRNA gene sequencing. Results reveal that many soybean nodule associated bacteria strongly inhibit the growth of plant pathogens in in vitro assays. In addition, a number of nodule-associated bacteria exhibit plant protection against the bacterial pathogen Clavibacter michiganensis subsp. michiganensis as well as the capability of plant growth promotion on tomato plants in in vivo assays. Pseudomonas spp. is the dominant bacterial group as nodule-associated bacteria within the soybean nodule. Isolation and identification of beneficial nodule-associated bacteria establish the foundation to study nodule-associated bacteria for their plant protection and growth promotion potential.
Advisor: Amitava Mitra
A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Agronomy, Under the Supervision of Professor Amitava Mitra. Lincoln, Nebraska: April, 2018.
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