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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 & Mycotoxin Research

2013 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 (see Appendix, Table 1), as will the seedling bioassay (Appendix, Figure 6) 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: A strain of a Gram negative bacterium has been developed (by another RRC scientist) to consist of several markers that can be used in conjugation recovery experiments. However, a major component of the current objective was to demonstrate the degree of infection that the newly constructed bacterium has on plant infection and internal colonization. These experiments have not been conducted due to timing constraints directed at work dealing with B. mojavensis. A major issue was the low efficiency of transformation by natural competence. We report that in addition to the above we have obtained vectors commonly used for conjugation experiments based on gene deletions methods. These vectors have the additional potential as shuttle vectors for testing horizontal gene transfer between Gram negative and B. mojavensis. Objective 2: This objective extends the ongoing collaborative project with the Democritus University of Thrace in Alexandropoulos, Greece. All genes encoding an arylamine N-acetyltransferase (NAT) from Fusarium verticillioides, F. oxysporum, and F. graminearum, as well as from Aspergillus flavus and A. nidulans were cloned and recombinantly expressed in E. coli, allowing for purification of these important proteins that degrade and detoxify agriculturally important compounds and environmental pollutants. We have also employed enzymatic assays to evaluate functionally the activity of the enzymes in the fungi. These in vivo assays allow us to assess directly the activity of the native enzyme. Additional genes involved in detoxifying chemical compounds that the fungi may encounter were also studied. In particular are genes encoding beta-lactamase enzymes, which are well known for their role in antibiotic resistance in bacteria. For that reason, these genes are also of interest since the fungi may have acquired some of them from bacteria through horizontal gene transfer. We have investigated the expression profiles and possible functions of some of these genes and are in the process of deleting genes for further evaluation. Objective 3: The milestone for FY 2013 was to establish other locations that can be used to reduce fumonisin and to reduce the accumulation/entry of other mycotoxic species such as A. flavus. Field studies conducted by our CRADA partner on the use of three strains of Bacillus mojavensis on protection of maize and wheat planted at seven locations. Data indicated some reduction in yield by one strain, while the other increased the yield; mycotoxin analysis of fumonisin and aflatoxins showed some reduction over controls. The other mycotoxic species used was Fusarium graminearum and its mycotoxin DON. Work conducted in Canada indicated the infection of F. graminearum in wheat was reduced, as was DON. This data was rated good from the Canadian field trials. The effect on F. graminearum was unexpected and will have to be repeated over the winter, which calls for an extension of the CRADA period, and this has been requested.

4. Accomplishments
1. Field characterization of bacterial strains for biocontrol of toxic fungi. Two species of fungi, Fusarium graminearum and F. verticillioides, are pathogens of corn and wheat and they also produce mycotoxins on these grains resulting in toxicities to human, livestock, and poultry. Scientists in the South Atlantic Area, Russell Research Center, Athens, Georgia have identified a group of biocontrol bacteria, Bacillus mojavensis, which under greenhouse conditions colonize the interior of corn as endophytes preventing the infection of the toxic fungi. It is unknown how this bacterium will control the fungi under field condition. Tests conducted under field conditions on corn and wheat at several U.S. and Canadian locations suggest success. These tests have determined that concentrations of inoculum required for a positive field effect and have identified several strains of Bacillus mojavensis that are candidates for patents due to their inhibitory effects on the target fungi.

2. Additional fungicidal compounds identified in the endophytic plant friendly bacterium Bacillus mojavensis. A collection of biocontrol bacteria, B. mojavensis, is being used to control pathogenic and toxigenic fungi associated with corn and wheat. Not all strains produce the surfactins, a class of fungicidal compounds. These non-surfactin producers have now been analyzed and found to produce two additional fungicides that explain the antagonism of such strains to fungi reported earlier. These substances are less toxic than the surfactins, but the production of the new class of fungicides is beneficial to this cadre of strains that are equally plant friendly and highly antagonistic to fungal pathogens as the patented surfactin producing strain.

3. A fungus identified that has the unique capability to detoxify antimicrobial compounds native to corn. The fungus Fusarium verticillioides produces the fumonisin mycotoxins, which are of great concern due to their contamination of corn. ARS Researchers in Athens, GA, have discovered that the fungus has evolved an effective way of protecting itself from the protective chemical compounds produced by the corn plant, thus giving the fungus a unique competitive advantage in the cornfield. At least two unique enzymes detoxify the photochemical, with both encoding genes being turned on to high levels when the fungus is exposed to the compounds. The results demonstrate how fungi may evolve unique metabolic functions depending on their environmental challenges.

Review Publications
Garcia-Pedrajas, M.D., Paz, Z., Andrews, D.L., Baeza-Montanez, L., Gold, S.E. 2012. Rapid deletion plasmid construction methods for protoplast and Agrobacterium based fungal transformation systems. In: Gupta, V., Tuohy, M., Ayyachamy, M., Turner, K., O'Donovan, A., editors. Laboratory Protocols in Fungal Biology. New York, NY: Springer-Verlag New York Inc. p. 375-394.

Schardl, C.L., Young, C.A., Hesse, U., Amyotte, S.G., Andreeva, K., et al. 2013. Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the Clavicipitaceae reveals dynamics of alkaloid loci. PLoS Genetics. 9(2):e1003323. DOI:10.1371/journal.pgen.1003323.

Leyte-Lugo, M., Gonzalez-Andrade, M., Gonzalez, Maria, D., Glenn, A.E., Cerda-Garcia-Rojas, C.M., Mata, R. 2012. (+)-Ascosalitoxin and vermelhotin, a calmodulin inhibitor, from an endophytic fungus isolated from Hintonia latiflora. Journal of Natural Products. 75:1571-1577. DOI: 10.1021/np300327y

Allen, A., Islamovic, E., Kaur, J., Gold, S.E., Shah, D., Smith, T.J. 2013. The virally encoded killer proteins from Ustilago maydis. Fungal Biology Reviews. 26(4):166-173.

Last Modified: 05/23/2017
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