Location: Peanut Research
2013 Annual Report
2. Identify genes in Aspergillus flavus responsible for virulence during the infection process and elucidate the role of fungal gene products for overcoming peanut resistance mechanisms.
3. Determine the role of defensive peanut phytoalexins in mediating natural crop resistance against Aspergillus flavus.
Genes encoding putative phytoalexin-detoxification enzymes (PDEs) will be cloned from pathogenic A. flavus strains. PDE production by A. flavus will be induced in culture by the presence of purified peanut phytoalexins or peanut seeds. cDNA libraries will be generated and used as templates to amplify candidate genes by Polymerase Chain Reaction (PCR). Native in vitro-expressed proteins will be purified and their activity will be tested against a variety of purified peanut phytoalexins. Liquid chromatographic-tandem mass spectrometric (LC-MS) analysis of the phytoalexin samples after exposure to the various purified proteins will be used to detect potential enzymatic modifications of the phytoalexin compounds. Target PDEs will be analyzed from different genotypes of A. flavus and A. parasiticus to assess the genetic variability of these enzymes and thus predict the potential effectiveness of PDE inhibitors. A model system will be developed to screen PDE inhibitors. Pathogenicity tests will be conducted on single peanut seeds inoculated with A. flavus after the application of inhibitory compounds.
The bioactivity of phytoalexins will be assayed against economically important plant pathogenic fungi grown on micro-plates. The dynamics of phytoalexin formation will be studied by first determining the most fungal-resistant (high phytoalexin producers) and fungal-susceptible (low phytoalexin producers) peanut genotypes from a core collection of 108 genotypes. Peanut seeds from genotypes will be subjected to different biotic and abiotic elicitors to elucidate changes in the composition of phytoalexins and to detect possible degradation products due to detoxification. The embryos and cotyledons from seeds will be wounded and inoculated with fungi and bacteria, then extracted and analyzed with high performance liquid chromatography (HPLC)/MS. Data obtained from analyses of the core collection of peanut genotypes will be used to identify peanut germplasm with disease resistance. To further examine phytoalexin detoxification (degradation) products, feeding experiments will be conducted in which fungi and bacteria are fed peanut phytoalexins followed by HPLC/MS/Nuclear Magnetic Resonance analysis.
Three laccase genes from A. flavus were cloned into pET162 expression vector, and the three phytoalexin detoxification enzymes were purified using histidine tagging. Preliminary tests were performed for laccase activity, and production of the enzymes is currently being scaled up for further testing. Regulation of expression of the three laccases by A. flavus during invasion of living peanut kernels at different water activities was studied using Real-Time PCR. Phytoalexins produced by peanuts as a defense mechanism also were analyzed to determine the response of kernels to fungal invasion. The three fungal laccases were highly expressed in kernels under water stress (low water activity). Three major peanut phytoalexins were apparently degraded by A. flavus laccases and their degradation was correlated with high levels of laccase expression. Therefore, phytoalexin-detoxification enzymes produced by A. flavus may be important for plant invasion. The results were published in the journal Plant Pathology.
A high-performance liquid chromatographic/mass-spectrometric (HPLC/MS) method for simultaneous determination of 12 peanut phytoalexins was developed and used for the search of new phytoalexins in fungal-challenged peanut seeds. Two new putative phytoalexins were isolated. Investigation of their structures and bioactivity is in progress. The method was also used to study the dynamics of phytoalexin production in peanut seeds elicited by bacteria and fungi. Different biotic agents selectively elicited production of major peanut phytoalexins. Aspergillis species, compared to other biotic agents, were more potent elicitors of phytoalexins. Phytoalexins may be important for peanut resistance to microbial invasion. The results were published in the Journal of Agricultural and Food Chemistry.
Arias, R.S., Sobolev, V.S., Orner, V.A., Dang, P.M., Lamb, M.C. 2014. Potential involvement of Aspergillus flavus laccases in peanut invasion at low water potential. Plant Pathology. 63(2):354-364.
Horn, B.W., Olarte, R.A., Peterson, S.W., Carbone, I. 2013. Sexual reproduction in Aspergillus tubingensis from section Nigri. Mycologia 105(5): 1153-1163.
Shier, W., Abbas, H.K., Weaver, M.A., and Horn, B.W. 2012. Visualization of aflatoxigenic Aspergillus flavus contamination of coconut (Cocos nucifera) nutmeat (Copra) using ammonia treatment. Acta Horticulturae. 1: 177-182.
Sobolev, V.S. 2013. Production of phytoalexins in peanut (Arachis hypogaea) seed elicited by selected microorganisms. J. Agric. Food Chem. 61(8):1850-1858.
Sobolev, V.S., Gloer, J.B., Sy, A.A. 2010. The Peanut Plant and Light: Spermidines from Peanut Flowers and Studies of their Photoisomerization. Nova Science Publishers, Inc., Hauppauge, NY. 62 pp. (also published as a book)
Sobolev, V.S., Orner, V.A., Arias De Ares, R.S. 2013. Distribution of bacterial endophytes in peanut seeds obtained from axenic and control plant material under field conditions. Plant and Soil. 371(1-2):367-376.