Mycotoxins: Biosecurity, Food Safety and Biofuels Byproducts (NC129, NC1025)

<P>NON-TECHNICAL SUMMARY: Research leading to a better understanding of the complex host-pathogen interaction will lead to novel mycotoxin treatment strategies and enhanced food safety for consumers and bioenergy security. </P>
<P>APPROACH: Maize hybrids with varying aflatoxin resistance have been inoculated with either toxigenic (NRRL 3357) and atoxigenic (NRRL 21882) strains of Aspergillus flavus or combinations of the two, in test plots in Mississippi. Tissue samples were collected from plants inoculated with both fungal strains, those inoculated with water, and from plants that were uninoculated. Using qRT-PCR and strain specific molecular marker we have begun to determine the A. flavus biomass. Additionally, we are developing a handleld FT-IR method for fungal detection in the field. The volatile metabolome and other small non-protein molecules of maize in response to A. flavus infection and aflatoxin accumulation are currently being profiled with SPME and liquid extraction coupled to GC/MS and GC-QTOF technologies. Identification of A. flavus induced phytoalexins has also been initiated. A detailed time-course analysis of acidic terpenoid phytoalexins, namely zealexin sesquiterpenoids and kauralexin diterpenoids, present in individually inoculated maize kernels was performed. The two primary maize varieties focused on included Va35 and Mp719, representing established lines susceptible and resistant to aflatoxin accumulation, respectively. Challenged at 18 days after pollination, Mp719 kernels displayed significantly greater zealexin accumulation 3 days post inoculation (dpi) compared to Va35 using the same toxigenic Aspergillus flavus isolate 3357. Similarly, kauralexin levels were significantly greater in Mp719 at 7 dpi compared to Va35. In contrast to the more rapid accumulation of zealexins and kauralexins in Mp719, levels of these defenses present in Va35 were significantly greater than those of Mp719 at 21 dpi. At this time point, total acidic terpenoid phytoalexins in the remaining Va35 kernel tissue were approximately 530 mg/g FW while those of Mp719 were 175 mg/g FW. Predictably, intact non-inoculated kernels of both lines contained relatively trace levels of phytoalexins at each time point. The high phytoalexin levels associated with Va35 display an intriguing correspondence with elevated aflatoxin B1 accumulation. Further studies confirming these patterns in additional susceptible and resistant lines of diverse origin will help establish the probable role of terpenoid phytoalexins in early and late term interactions with A. flavus and toxin production. Inoculated kernels, adjacent kernels, uninoculated kernels, and cob sections have been collected at various times after inoculation in field trials conducted at Mississippi State for several analyses: RNAseq technology is being used examine the gene response of different germplasm lines to both toxin producing and non-toxin producing A. flavus strains and to identify genes that are integral to the host/pathogen interaction. Tissues collected at Mississippi State have been provided to collaborators at other locations.</P> <P>Candidate genes are being evaluated by using qRT-PCR on kernels with or without inoculation of a toxin-producing A. flavus strain in different corn germplasm lines. Polymorphic DNA markers were designed and tested on the selected differentially expressed genes. Genes contributing to resistance with high statistical significance are being identified using Dr. Warburton's QTL and association study pipeline. Aflatoxin is being quantified by LC/MS-MS and cob sample are being analyzed via qRT-PCR for the expression of resistance related genes.Additionally, aflatoxin quantification (LC-MS/MS single kernal) procedures are being performed on control samples and those inoculated with both the toxigenic and atoxigenic strains of A. flavus. Analysis of toxin accumulation in a resistant line Mp719 and Va35 showed that both accumulated toxin but that the susceptible line had significantly more toxin after 21 days than the resistant line. Toxin accumulation remained relatively constant in Mp719 while it continued to increase over time in Va35.Furthermore, nucleic acid extractions are also being performed for use in fungal biomass quantification and gene expression experiments. Validation of the fungal biomass quantification technique that can differentiate between A. flavus strains NRRL 3357 and NRRL 21882 was presented at the Corn Utilization and Technology Conference in Indianapolis, IN. Data on the comparative structural analysis of maize homologous proteins associated with host plant resistance was also presented at this conference. Work pertaining to this funded project was also presented at four other conferences.Two-dimensional protein gel electrophoresis is a powerful tool for revealing differences in proteomic profiles under different treatments. Differentially expressed proteins across multiple samples can be identified and quantitatively evaluated by computational analysis of sets of 2D protein gel images. We have explored the use image processing methods (Matlab Image Processing Toolbox) for quantification of protein expression levels, and identified differentially expressed maize proteins associated with resistance to Aspergillus flavus infection and aflatoxin accumulation through the quantitative data analysis of 2D gel images. Resistant (Mp715, Mp719) and susceptible (Va35, Mp04:87) maize inbred lines were selected and developing kernels were collected from the primary ears of corn plants at 14 days after inoculation with A. flavus. Proteins were extracted using TCA/acetone precipitation in combination of a phenol extraction step. The 2-D protein gel electrophoresis was performed using PROTEAN IEF Cell (Bio-Rad) and PROTEAN II XL cell (Bio-Rad). Proteins were visualized with florescent dye Oriole (Bio-Rad). Gel images were obtained with an Alpha imager. We have established a working protocol for quantitative proteomic studies of maize proteins by computational and statistical analysis of 2D protein gel images. It is a novel analysis procedure for profiling and comparative investigations of differentially expressed proteins to facilitate the development of DNA markers for maize resistance breeding. </P>