Semester of Graduation

Summer 2025

Degree

Master of Science (MS)

Department

Plant Pathology and Crop Physiology

Document Type

Thesis

Abstract

Aspergillus flavus is a major pathogen of cotton, maize, peanut, and other oilseed crops, capable of producing mycotoxins both in the field and during post-harvest storage. Among these mycotoxins, aflatoxins (AFs) are the most potent carcinogens and immunosuppressants, posing a significant threat to global food safety, with over 4.5 billion people estimated to be exposed to unmonitored levels of AFs. The effectiveness of using traditional methods to manage A. flavus infection and aflatoxin contamination is limited due to environmental toxicity of fungicides, inconsistent field efficacy of atoxigenic biocontrol strains, and difficulty in developing resistant cultivars. To address these challenges, this study explores the potential of RNA interference (RNAi), a mechanism of gene silencing, as an alternative strategy for suppressing aflatoxin production in A. flavus. In this study, eleven double-stranded RNA (dsRNA) constructs were designed to suppress the expression of aflN, aflR, aflV, aflO, 2,3- Dihydroxybenzoic Acid Decarboxylase (2,3 DHBD,) Translation Elongation Factor-1 (TEF-1), aflJ, aflE (norA), aflP (omtA), nucleoside hydrolase protein, and alk genes, which are involved key biological processes in A. flavus, such as protein and aflatoxin biosynthesis, to evaluate the potential of employing RNAi for reducing aflatoxin production by A. flavus. It was found that dsRNA treatments targeting aflN, aflR, aflV and TEF-1 genes led to a significant inhibition of germ tube elongation of A. flavus at 10 hrs post inoculation, and dsRNAs targeting aflN and aflR genes also significantly reduced spore germination compared to the control treated with dsRNA targeting a green fluorescent protein (GFP) gene. Further, treatment of A. flavus conidia with dsRNA targeting nucleoside hydrolase protein gene reduced not only spore germination, but also subsequent hyphal development up to 20 hours after inoculation in potato dextrose broth (PDB) medium. This study further showed that the application of aflR-targeting dsRNA at the time of A. flavus inoculation resulted in a significant reduction in aflatoxin B1 (AFB1) levels compared to the negative control (treated with dsRNA targeting GFP gene at day 0). Similarly, the addition of aflN- and aflV-targeting dsRNAs three days post-inoculation also significantly decreased AFB1 production when compared to the same control. Furthermore, aflatoxin levels were significantly reduced in liquid cultures treated with dsRNAs targeting aflJ, aflE (norA), and aflP (omtA) in vitro compared to the control containing only inoculum in A & M media with glucose. In addition, treating A. flavus cultures with all dsRNA led to a significant reduction in fungal biomass when compared to the control, which was treated with dsRNA targeting GFP. Overall, this study highlights the advantages of RNAi-based strategies over traditional approaches by offering a targeted, environmentally friendly, and effective means of reducing both aflatoxin production and A. flavus growth.

Date

7-30-2025

Committee Chair

Chen, Zhi Yuan

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Available for download on Wednesday, July 26, 2028

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