Location: National Peanut Research Laboratory2015 Annual Report
1. Determine the population dynamics of sexually reproducing Aspergillus flavus under field conditions. 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.
Sclerotia of A. flavus will be collected from corn grown in a randomized complete block design consisting of four overhead irrigation treatments to provide different degrees of drought stress. Experiments involving natural field populations of A. flavus will be conducted during years 1 and 2. The same procedure will be repeated for years 3 and 4, except that corn ears will be sprayed with a conidial suspension of a non-toxigenic biocontrol strain (NRRL 21882 [from Afla-Guard® ] or AF36). Sclerotia will incubated on the surface of nonsterile soil (100% relative humidity) for 5-7 months. Ascospores from fertile sclerotia will be germinated to obtain progeny strains. To detect genetic recombination, total genomic DNA will be isolated from progeny strains. Recombination events due to independent assortment of chromosomes and crossing over will be detected by multilocus sequence typing (MLST) and linkage disequilibrium/compatibility analyses. 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.
Aspergillus flavus and A. parasiticus, the two major species responsible for aflatoxin contamination of agricultural commodities, were crossed to obtain rare hybrid progeny. A. flavus typically produces B aflatoxins and cyclopiazonic acid (CPA) and A. parasiticus produces B and G aflatoxins but not CPA. Hybrid progeny showed extensive recombination throughout the genome as well as within the aflatoxin gene cluster. Some of the progeny exhibited enhanced aflatoxin production as well as unique mycotoxin profiles that included the production of B and G aflatoxins and CPA. Such hybridization events may account for the mycotoxin diversity observed among aflatoxin-producing species in nature and has broad implications for devising new strategies for controlling aflatoxins. This research was published in Molecular Ecology. In another study, sexual reproduction and genetic recombination were examined in Aspergillus tubingensis, which is used extensively in the industrial production of enzymes and organic acids. Crosses were performed between strains of the opposite mating type and 84% of the progeny showed genetic recombination among three loci. Recombination associated with sexual reproduction in A. tubingensis provides a new option for the genetic improvement of industrial strains for chemical production. This research was published in Mycologia. Large number of peanut breeding lines (148) went through the process of transformation with ribonucleic acid interference (RNAi), a biological process, in which RNA molecules inhibit gene expression. Such transformation was aimed at the potential silencing of fungal genes responsible for aflatoxin production. Presumably transformed peanut lines were grown in greenhouse for seed production, and the seeds obtained were screened by polymerase chain reaction (PCR). Mature and immature seeds of 12 RNAi-positive and RNAi-negative peanut lines, were tested by challenging them with Aspergillus flavus using the method that was recently developed at the NPRL. Samples were collected at 24, 48, 72 and 96 h in duplicate followed by their analyses for aflatoxin production by high performance liquid chromatography (HPLC), expression of the RNAi fragment by real-time PCR, and for complete stilbene phytoalexins profiles by HPLC–mass-spectrometry (MS). The manuscript that describes this research was accepted for publication. Three new phytoalexins were isolated from fungus-challenged peanut seeds. The structures of the new compounds were unambiguously elucidated using modern instrumentation. Investigation of antifungal properties of the new compounds is in progress. Twelve important Aspergillus species were fed with five major stilbene phytoalexins to determine their ability to detoxify the phytoalexins. At the same time, the toxigenic potential of the selected fungi was estimated. The content of stilbenoids modified/detoxificated by the fungi accounted for up to 50% of total stilbenoids. Aspergillus spp. from section Flavi were capable of degrading one of the major peanut phytoalexin, arachidin-3, into its hydroxylated homologue, arachidin-1 and a benzenoid, SB-1. However, A. niger from section Nigri as well as other fungal and bacterial species tested, were incapable of changing the structure of arachidin-3. None of the species tested, with the exception of a Cladosporium sp. and Rhizobium leguminosarum, were able to degrade SB-1. Elucidation of the phytoalexin detoxification system involved in infection of peanut by important Aspergillus spp. could provide strategies for preventing plant invasion by the fungi that produce aflatoxins. Two manuscripts have been written and are being submitted to a scientific journal. To quantify expression of 25 genes in the aflatoxin-biosynthetic pathway, primers were designed and tested; 14 of those passed the tests and are being used to determine the impact that individual phytoalexins, including newly discovered, have on aflatoxin-synthesis genes in Aspergillus flavus. Over five months of work were required to find a method for nucleic acid extractions suited for the experimental setup of feeding experiments.
1. New tool for improving industrial strains of Aspergillus. The fungus Aspergillus tubingensis (formerly A. niger) is used extensively in the industrial production of enzymes and organic acids. In the past, strains with enhanced chemical production have been created through laboratory mutations and various genetic transformations. ARS researchers at Dawson, Georgia, in collaboration with scientists at North Carolina State University have discovered sexual reproduction and associated genetic recombination in A. tubingensis. This sexual cycle provides a new option for the genetic improvement of industrial strains for enzyme and organic acid production.
2. New tool for evaluation of the ability of microorganisms that interact with peanuts to detoxify/degrade peanut defensive phytoalexins. Plants accumulate defensive phytoalexins in response to the presence of pathogens, which in turn produce phytoalexin-detoxification enzymes for successfully invading the plant host. ARS scientists at Dawson, Georgia demonstrated that Aspergillus spp. from section Flavi were capable of degrading the major peanut phytoalexins into its hydroxylated homologues and glycosides. However, fungal species from section Nigri as well as other fungal and bacterial species tested, were incapable of degrading the most abundant stilbenoids. This fact may indicate strong affiliation of aflatoxin-producing fungi with peanuts and their ability to overcome the peanut chemical defensive mechanism. Elucidation of the phytoalexin detoxification system involved in infection of peanut by important Aspergillus spp. could provide strategies for preventing plant invasion by the fungi that produce aflatoxins.
Olarte, R.A., Worthington, C.J., Horn, B.W., Moore, G.G., Singh, R., Monacell, J.T., Dorner, J.W., Stone, E.A., Xie, D., Carbone, I. 2015. Enhanced diversity and aflatoxigenicity in interspecific hybrids of Aspergillus flavus and Aspergillus parasiticus. Molecular Ecology 24(8):1889-1909.
Olarte, R.A., Horn, B.W., Singh, R., Carbone, I. 2015. Sexual recombination in Aspergillus tubingensis. Mycologia 107(2):307-312.
Moore, G.G., Beltz, S.B., Carbone, I., Ehrlich, K., Horn, B.W. 2011. The population dynamics of aflatoxigenic aspergilli. In: Guevara-Gonzalez, R.G., editor. Aflatoxins - Biochemistry and Molecular Biology. Rijeka, Croatia: Intech Open Access publishers. p. 347-366.