Bacillus strains used for biological control of fusarium head blight: Identification, growth studies, and lipopeptide production

For the last several years, our laboratory has been working with four endospore-forming bacterial strains (designated as 1B-A, 1B-C, 1B-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 16S 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 1B-A and 1B-C had similar colony morphology, with a shiny and undulate appearance, whereas colonies of strain 1B-E were shiny but not wrinkled, and colonies of strain 1D-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 1B-A grew to over 10 times the optical density it achieved in the initial medium formulation. Plate count data also showed better growth of 1B-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 mug/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 1B-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. (Abstract shortened by UMI.)...