Title

Evaluation of Quantitative Polymerase Reaction and Microbial Volatile Organic Compounds to Determine Resistance to Aspergillus Flavus in Maize

Advisor

Baird, Richard E.

Committee Member

Henn, R. Alan

Committee Member

Mlsna, Todd E.

Committee Member

Windham, Gary L.

Date of Degree

1-1-2013

Document Type

Dissertation - Open Access

Abstract

Screening for resistance to Aspergillus flavus and aflatoxin contamination in maize is an ongoing effort by universities, state and federal agencies. We evaluated two techniques to screen for resistance; quantitative polymerase reaction (QPCR) and solid-phase microextraction (SPME). Methods were adapted to accurately detect and quantify the fungus in culture and in the vegetative stage of plant tissues. These assays can eliminate microbiological techniques. The primary objectives of the study were to utilize 1) QPCR to detect and quantify fungal biomass in maize stem tissues to evaluate resistance in maize genotypes to A. flavus colonization in situ and in vivo and 2) SPME to identify key MVOC’s to differentiate aflatoxigenic and non-aflatoxigenic strains of A. flavus in situ. A novel QPCR TaqMan probe (OMG3) was designed to detect a region in the aflP gene. The OMG3 probe detected 98.3% of the aflatoxigenic strains. The predominant MVOC’s extracted from both aflatoxigenic and non-aflatoxigenic strains were alcohols, ketones and hydrocarbons. The aflatoxigenic strain produced 39 compounds and the non-aflatoxigenic strain produced 41 compounds. Dimethylsulfide and 2-heptanol were key MVOC biomarkers produced only by the aflatoxigenic strain of A. flavus. Accuracy of the QPCR OMG3 probe, in vivo and in situ procedures were developed. A toothpick inoculation method was used to artificially inoculate maize stems in the vegetative stage five (V5). Plants were harvested at V7 and sampled at predetermined sites. This method was 91% consistent for infecting maize plants. The OMG3 probe was evaluated in in vivo and in situ studies conducted in the greenhouse, growth chamber, and field. Lesion length was greater in susceptible lines in 4 of 7 greenhouse trials. Based on inoculation data, subsequent research should focus on refining tissue-sampling methods and increasing length of plant growth time for tissue sampling post-inoculation.

URI

https://hdl.handle.net/11668/18580

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