Location: Peanut Research2013 Annual Report
1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
Corn was grown at Shellman, Georgia, in 2010 and 2011 under different levels of drought stress consisting of 0% irrigation (dry land cultivation) and 33%, 66% and 100% of the overhead irrigation recommended by Irrigation Pro for Corn software. A third year of corn cultivation has been added to the project and is in progress. Aspergillus flavus sclerotia, resistant structures produced by the fungus, were detected in approximately 1% of corn ears at harvest and showed no evidence of sexual reproduction. Sclerotia were then incubated under laboratory conditions on the surface of soil containing natural microbial populations. Sexual reproduction occurred in 0.1 to 6.1% of the sclerotia and resulted in the formation of viable sexual spores (ascospores). For sexual reproduction to occur in the field, A. flavus sclerotia likely require an additional incubation period on soil following dispersal at crop harvest. Sexual reproduction is likely responsible for the high genetic variation in A. flavus populations. 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.
1. Discovery of sexual reproduction in economically important Aspergillus tubingensis. Aspergillus tubingensis, A. niger and related ‘black Aspergilli’ are of great economic importance due to their invasion of crops, production of mycotoxins, and deterioration of stored foodstuffs. These fungi are also used extensively in industry for the production of enzymes and organic acids. Black Aspergilli were previously thought to be strictly nonsexual in their reproduction. ARS researchers at Dawson, Georgia, and Peoria, Illinois, and scientists at North Carolina State University collaborated in the discovery of sexual reproduction in A. tubingensis. Sexual reproduction may be useful for enhancing enzyme and organic acid production in industrial strains of A. tubingensis.
2. Microsatellite markers associated with salt tolerance in the warm-season turfgrass Paspalum vaginatum (seashore paspalum). Seashore paspalum is used for bioremediation and forage production, and the degradation and salinization of soils, in addition to increased use of secondary water sources for irrigation, have increased the demand for salt-tolerant accessions/cultivars. ARS researchers from Dawson and Tifton, Georgia, generated microsatellite-enriched libraries, then used high-throughput DNA sequencing and bioinformatics to identify markers linked to salt tolerance in seashore paspalum. The selected markers distinguished between salt tolerant and salt susceptible cultivars and accessions. These markers will help identify salt tolerant cultivars. A total of 2,576 DNA sequences of microsatellite markers of P. vaginatum were submitted to GenBank at the National Center for Biotechnology Information (NCBI) and they are now available to breeders and the general public.
3. Distribution of bacterial endophytes in peanut seeds. Knowledge of peanut bacterial endophytes and their interactions with the host plant and fungal pathogens is of great importance due to significant economic losses caused by aflatoxin-producing fungi. ARS researchers at Dawson, Georgia, demonstrated that mature peanut seeds contain several species of nonpathogenic endophytic bacteria, among which Bacillus thuringiensis is dominant. Furthermore, all Bacillus amyloliquefaciens isolates, the second most abundant bacterial species, demonstrated activity against aflatoxin-producing Aspergillus flavus. Endophytic bacteria originated from local soil and not from the seed source, and the peanut plant accommodated only select species of bacteria from diverse soil populations. Endophytic bacteria may play a role in the defense mechanism of peanut against fungal invasion.
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.