Document Type

Thesis - University Access Only

Award Date

2007

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

Abstract

For the last several years, our laboratory has been working with four endosporeforming bacterial strains (designated as lB-A, lB-C, lB-E, and 1D-3) isolated from South Dakota wheat foliage and residue that can antagonize Fusarium graminearum in laboratory plate assays and in greenhouse and field plot trials. We have attempted to identify these bacterial strains by different techniques, with different identification methods resulting in different genus affiliations for the strains. Sequence analysis showed that all four strains had identical sequences in the first 500 base pairs of their l 6S rDNA genes, and all were most closely related to Bacillus amyloliquefaciens with less but significant relatedness to Bacillus atrophaeus. In the work presented here, colony morphology, microscopic appearance, and phenotypic traits were evaluated and used to arrive at suggested identities for the strains. Strains lB-A and lB-C had similar colony morphology, with a shiny and undulate appearance, whereas colonies of strain lB-E were shiny but not wrinkled, and colonies of strain lD-3 were a dull color with dotted appearance instead of wrinkles. Results of phenotypic tests suggested that all four strains were most closely related to Bacillus firmus. These attempts to identify the four strains strongly suggest that they are tied to a phylogenetically and phenetically coherent B. subtilis group (group II). However, the four strains may all belong to a previously uncharacterized taxon with relatedness to B. amyloliquefaciens and B. atrophaeus, taxa that were split out of the old Bacillus subtilis taxon. There is a good amount known about the antibiotics produced by members of the B. subtilis group (group II). Among the many antibiotics produced by B. subtilis and its relatives are cyclic lipopeptides such as iturin and surfactin. We have cultured these bacterial strains in potato dextrose broth (PDB), a complex medium containing glucose, which may suppress iturin production to some degree. We have also cultured the Bacillus sp. in a defined broth medium lacking glucose, containing mannitol, glutamic acid and inorganic salts. Bacterial cell numbers in this original formulation were lower than desirable for application of cells to wheat plants, so a modification of the original medium was used increasing the mannitol by 2.3 times, and increasing the glutamic acid by 2.1 times. After 10 days of growth in the modified broth medium with increased carbon and nitrogen sources, bacterial strain lB-A grew to over 10 times the optical density it achieved in the initial medium formulation. Plate count data also showed better growth of lB-A in the modified defined medium having elevated carbon and nitrogen. Higher numbers of cells in the modified defined growth medium should allow better coverage of bacterial cells sprayed onto wheat surfaces when these bacteria are used in biocontrol trials. In addition, plate assays were done to see whether pure iturin would antagonize F. graminearum, and if the defined broth media in solidified form allowed the bacteria to antagonize the fungus. Purified iturin A was found to inhibit F. graminearum at a concentration of 40 μg/ml applied to a paper disk, challenging growth of the fungus on Potato Dextrose Agar. Analysis of extracts of broth cultures in defined media by absorption spectroscopy and HPLC indicated that iturin-like compounds were produced by all four Bacillus strains we have studied. Better understanding of the production of iturin, surfactin and other compounds that might act in concert with them would allow better understanding and use of these and related bacteria as biocontrol agents to control FHB. Bacillus sp. strain lB-A was cultured in a variety of defined (synthetic) and semi-defined broth media that lack glucose (which can suppress iturin production) to see if types and amounts of antibiotics produced differed in different media. The three broth media that were studied were: ( 1) a basal defined medium (BDM) containing mannitol, glutamic acid and inorganic salts; (2) a defined medium (DM) similar to BDM but containing increased amounts of mannitol and glutamic acid; and (3) a defined medium with the same composition as (2) but with increased concentrations of calcium and manganese, two elements which are known to be important in regulating different aspects of Bacillus metabolism. Broth cultures of Bacillus strain 1B-A were grown for different time periods in these three media. At selected time intervals culture samples were aseptically removed for measurement of optical density at 600 nm, and for iturin analysis using HPLC, to see if different phases of bacterial growth resulted in differences in iturin and surfactin production. Standard curves of iturin A (Sigma) were linear in a range from 50 ug/ml up to 250 ug/ml, with absorption maxima for iturin occurring at 214 nm and 275 nm for each iturin peak. In the BDM broth, maximum OD600 of 1. 7 was reached after 5 to 6 days of growth, and maximum iturin production occurred at this time (about 720 ug/ml). After this time, iturin levels declined greatly. In the DM broth having increased levels of mannitol and glutamic acid, maximum OD600 of 2.5 was not reached until 14 days incubation. However, maximum iturin production was reached around 5 days of growth (OD600 of 1.3; iturin production of 400 ug/ml). After 5 to 6 days of growth, iturin production sharply declined in this DM broth. In broth medium (3) containing the same components as (2) but with increased levels of Ca and Mn, maximum OD600 of 3.6 was reached after 8 days of growth, giving the highest cell yield of any broth medium. Iturin production in this medium had a much different pattern than the other two, with greatest iturin levels found within the first 24-48 hours of growth (132 ug/ml), and then declining sharply. Production of iturin in medium (3) was greatest during exponential growth, not stationary phase, and increased levels of Ca and Mn allowed iturin to be produced sooner, during early log to mid log phase of growth. Different growth media will result in different amounts of iturin; and the time of incubation will also affect iturin levels. This has implications for growing these bacteria for field application, in determining medium composition and incubation time.

Library of Congress Subject Headings

Wheat -- Diseases and pests -- Biological control

Fusarium diseases of plants

Fungal diseases of plants

Bacillus (Bacteria)

Format

application/pdf

Number of Pages

85

Publisher

South Dakota State University

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