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United States Department of Agriculture

Agricultural Research Service

Research Project: Control of Toxic Endophytic Fungi with Bacterial Endophytes and Regulation of Bacterial Metabolites for Novel Uses in Food Safety

Location: Toxicology and Mycotoxin Research

2012 Annual Report


1a.Objectives (from AD-416):
1. Determine the potential and genetic mechanisms involved in horizontal gene transfer from Gram-negative pathogenic bacteria to a plant friendly Gram-positive bacterial endophyte Bacillus mojavensis affecting its use as a desirable biocontrol potential.

2. Determine the diversity, evolution, and function of bacterial endophytes in cornfield environments and their impact on the metabolic activity of Fusarium verticillioides, including the production of mycotoxins as well as the detoxification/inactivation of xenobiotic compounds.

3. Determine how the biocontrol organisms can be effectively utilized to prevent the accumulation of the fumonisins; specifically the enhancement of plant defense strategies and production of the antifungal agent surfactin by Bacillus mojavensis.


1b.Approach (from AD-416):
Objective 1: We will test to determine if pathogenic Gram-negative bacteria can transfer genetic information via conjugation to Gram-positive endophytic bacteria that could potentially alter an otherwise nontoxic plant friendly bacterium to a potential virulent human pathogen while maintaining its stable endophytic host relationship.

Objective 2: A multilevel approach using molecular genetics, bioinformatics, and in planta analyses will be used to provide data on variation among mycotoxin-producing Fusarium species. Emphasis will be on genes encoding diverse metabolic activities, including xenobiotic detoxification. The hypothesis that such genes were acquired via horizontal gene transfer will be addressed. The approach will evaluate the contribution of these metabolic activities to the general fitness and competitiveness of the fungi.

Objective 3: The same bank of strains of B. mojavensis used in the experimental procedures of Objective 1 will be used here as needed, as will the seedling bioassay developed for rating the in planta control of B. mojavensis strains under control conditions. Two cultivars of corn will be used, one rated resistant to Fusarium and the other susceptible. Most of the techniques, inoculations, measurements of disease expressions, endophytism, surface disinfections, microscopic and visual interpretation of diseases have been practiced or created in our laboratory over the years of studying this and other endophytic associations. All analytical analyses for surfactin and fumonisins will be determined as we have done over the past research accomplishments.


3.Progress Report:
Objective 1: To establish the appropriate conditions to approach this objective, transformation and effective selection of Bacillus mojavensis transformants were considered prerequisite for detection of potential DNA transfer events between bacteria. To this end, after trials with several methods, strain RRC101 was successfully transformed at low efficiency by natural competence. This result indicates the direction for further work on more solid footing, as among other benefits, we can test the function of shuttle plasmids via transformation. This is the better attempt at transformation for this bacterium, which now promises a better system to monitor all types of studies including in planta corn interactions.

Objective 2: Genomic DNA was sequenced along with cloned cDNA for all arylamine N-acetyltransferase (NAT) genes from Fusarium verticillioides, F. oxysporum, and F. graminearum, as well as from Aspergillus flavus and A. nidulans. We are evaluating gene expression on all of the NAT genes to quantify their respective expression when challenged with xenobiotic substrates. Many of the genes are constitutive, whereas the NAT1 homologs are inducible when the fungi are challenged with 2-aminophenol and/or 2-benzoxazolinone (BOA). This confirmed the unique inducible nature of the NAT1 homologs. We can now begin to assess the expression of these genes in planta during endophytic infection. We have also generated gene-deletion strains for NAT1, NAT2, and NAT3 in F. verticillioides. We are building a dataset of genes of interest that appear to be derived from horizontal gene transfer and could be critical for expanded metabolic capabilities.

Objective 3: Field studies on the biocontrol bacterium Bacillus mojavensis are underway. We have developed a Cooperative Research and Development Agreement (CRADA) designed to determine the development of commercial products for control of diseases caused by Fusarium spp. and for reduction of mycotoxin accumulation in grain crops, based on use of bacterial endophytic strains, and/or their exudates. Field studies on maize and wheat, planted at seven locations during the Northern Hemisphere (USA and Canada) growing season of 2012, are underway. These field trials represent the first plantings and have been monitored for plant performance parameters, including the fumonisin mycotoxin accumulation, seed germination, crop growth and development, disease symptoms, phytotoxicity, and components of yield. The season is still underway, but visual observations on wheat indicate that the wheat pathogen F. graminearum is not present or is greatly reduced as evidenced by reduced disease symptoms. Some preliminary data indicate that two insects are deterred by surfactins, although the concentrations are not yet established. These results provide further evidence that the biocontrol bacterium at the field level does not produce negative effects on wheat, and if visual markers are correct, the mycotoxin content will be reduced.


4.Accomplishments
1. The genomic sequence of Bacillus mojavensis strain RRC101 was determined. The sequence is 4.1 million base pairs in length and is most similar to sequenced strains of the type for the genus, B. subtilis. The impact of this work is expected to have large ramifications. Currently, the genomic data is assisting us in generating hypotheses related to mode of inhibition of Fusarium verticillioides by compounds produced by the bacterium. Combined with progress on transformations of this strain, we will explore direct methods to test these hypotheses over the coming year.


Review Publications
Allen, A., Islamovic, E., Kaur, J., Gold, S.E., Shah, D., Smith, T.J. 2011. Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut. Plant Biotechnology Journal. 9:857-864. DOI: 10.1111/j.1467-7652.2011.00590.x/abstract

Nadal, M., Gold, S.E. 2011. Assessment of autophagosome formation by transmission electron microscopy. In: Bolton, M.D., Thomma, B., editors. Methods in Molecular Biology--Plant Fungal Pathogens: Methods and Protocols. New York, NY: Humana Press. p. 481-489.

Glenn, A.E., Bodri, M.S. 2012. Fungal endophyte diversity in Sarracenia. PLoS One. 7(3):e32980.

Chacko, N., Gold, S.E. 2012. Deletion of the Ustilago maydis ortholog of the Aspergillus sporulation regulator medA affects mating and virulence through pheromone response. Fungal Genetics and Biology. 49:426-432. dx.doi.org/10.1016/j.fgb.2012.04.002

Klosterman, S.J., Subbarao, K.V., Kang, S., Veronese, P., Gold, S.E., Thomma, B.P.H.J, Chen, Z., Henrissat, B., Lee, Y., Park, J., Garcia-Pedrajas, M.D., Dez, B.J., Anchieta, A.G., De Jonge, R., Parthasarathy, S., Maruthachalam, K., Atallah, Z.K., Amyotte, S., Paz, Z., Inderbitizin, P., Hayes, R.J., Heiman, D.I., Young, S., Zeng, Q., Engels, R., Galagan, J., Cuomo, C., Dobinson, K.F., Ma, L-J. 2011. Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens. PLoS Pathogens. 7(7): e1002137.

Rodriguez-Kessler, M., Baeza-Montanez, L., Garcia-Pedrajas, M.D., Tapia-Morena, A., Gold, S.E., Jimenez-Bremont, J.F., Ruiz-Herrera, J. 2012. Isolation of UmRrm75, a gene involved in dimorphism and virulence of Ustilago maydis. Microbiological Research. 167:270-282.

Paz, Z., Garcia-Pedrajas, M.D., Andrews, D.L., Klosterman, S.J., Montanez, B., Gold, S.E. 2011. One step construction of agrobacterium recombination-ready plasmids (OSCAR), an efficient and robust tool for ATMT based gene deletion construction in fungi. Fungal Genetics and Biology. 48:677-684.

Bacon, C.W., Hinton, D.M., Mitchell, T.R., Snook, M.E., Olubajo, B. 2012. Characterization of endophytic strains of Bacillus mojavensis and their production of surfactin isomers. Biological Control. 62:1-9.

White, J.F., Bacon, C.W. 2012. The secret world of endophytes in perspective. Fungal Ecology. 5:287-288.

Last Modified: 8/1/2014
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