Date of Graduation

7-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Biological Sciences

Advisor/Mentor

Burton H. Bluhm

Committee Member

John C. Rupe

Second Committee Member

Griffiths G. Atungulu

Third Committee Member

Jackson Lay, Jr.

Keywords

Aflatoxins, Biological control, Fumonisins, Maize grains, Mycotoxigenic fungi, Pathogenic fungi

Abstract

Mycotoxin contamination of cereal crops, such as maize and sorghum, is a global concern because of the potential health effects on humans and animals. Although substantial research has been conducting regarding mycotoxin prevention and mitigation, little information is available about the association of mycotoxin-producing fungi with corn and sorghum grain in Iraq. Identifying and refining indigenous atoxigenic strains to reduce mycotoxin contamination of maize and sorghum has the potential to enhance the nutritional value of these grains while reducing economic losses. However, to our knowledge, this tactic has not yet been adopted by agricultural authorities and farmers in Iraq. To survey mycotoxigenic fungi associated with corn and sorghum grain in Iraq and identify potential biological control agents ‘customized’ for Iraqi production conditions, a collection of corn and sorghum grain samples were imported from Iraq. DNA-based diagnostic analyses were integrated with morphology-dependent methods to identify seedborne mycoflora in corn and sorghum samples collected from different regions in Iraq (north, central, and south). The most common fungal genera, i.e., Fusarium spp., Alternaria spp., Chaetomium spp., Penicillium spp., and Aspergillus spp. were found frequently in both kinds of grains. However, Exserohilum, Anthracosystis, Bipolaris, Sporisorium, Curvularia, Sarocladium, Humicola, Byssochlayms, and Stenocarpella were found associated with some samples, which is the first such reporting of these genera associated with corn in Iraq. Moreover, species borders of Aspergillus spp. and Fusarium spp. were delimited successfully through multi-locus sequence typing analysis of four conserved genes within each genera (ITS, B-tub, CaM, and RPB-2 for Aspergillus spp. and EF-1ɑ, CaM, and H3 for Fusarium spp.).

Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of Iraqi corn and sorghum samples revealed that most samples were contaminated with relatively low levels of fumonisin-B1, ranging from 0.01-8.6 ppm. However, neither corn nor sorghum samples were contaminated with aflatoxin-B1. Also, analyses of mycotoxigenic potential of Aspergillus flavus (27 isolates) identified three non-toxigenic A. flavus isolates (A-1-9, A-4-54, and A-4-75), whereas the rest varied in aflatoxin-B1 production. However, all Fusarium species (86 isolates) produced fumonisin-B1 at different levels, ranging from high to low concentrations. This is potentially the first report of F. incarnatum and F. acuminatum (well-known as deoxynivalenol –DON- and T2 producers, respectively) producing fumonisin mycotoxins.

Co-inoculation experiments between Iraqi native nontoxigenic isolates (A-1-9, A-4-54, and A-4-75) and native Iraqi toxigenic isolate (A-15-49) or the reference A. flavus NRRL 3357 substantiated the competitive ability of the Iraqi native nontoxigenic isolates to suppress aflatoxin biosynthesis in toxigenic strains. Aflatoxin-B1 was suppressed by approximately 99.2% compared to control treatments in which toxigenic isolates were inoculated individually. This is the first report of indigenous non-toxigenic A. flavus isolates from Iraq with documented biological control activity against toxigenic strains. Further investigations are required to evaluate the performance of these isolates in corn fields in Iraq.

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