2013 Annual Report
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.
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.
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.
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.
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. dx.doi.org/10.1016/j.fbr.2012.10.001
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