Characterization of morphological changes within stromata during sexual reproduction in Aspergillus flavus

Authors: LUIS, JANE - North Carolina State University; CARBONE, IGNAZIO - North Carolina State University; PAYNE, GARY - North Carolina State University; Bhatnagar, Deepak; Cary, Jeffrey; Moore, Geromy; Lebar, Matthew; Wei, Qijian - Mei Mei; Mack, Brian; OJIAMBO, PETER - North Carolina State University

Submitted to: Mycologia
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/21/2020
Publication Date: 8/21/2020
Citation: Luis, J.M., Carbone, I., Payne, G.A., Bhatnagar, D., Cary, J.W., Moore, G.G., Lebar, M.D., Wei, Q., Mack, B., Ojiambo, P.S. 2020. Characterization of morphological changes within stromata during sexual reproduction in Aspergillus flavus. Mycologia. 112(5):908-920. https://doi.org/10.1080/00275514.2020.1800361.
DOI: https://doi.org/10.1080/00275514.2020.1800361

Interpretive Summary: This work describes experiments that have been conducted in an effort to better understand the mechanisms that control sexual reproduction and development in Aspergillus flavus. Aflatoxins are toxic and carcinogenic compounds often produced by the fungus, Aspergillus flavus, during growth on crops such as corn, peanuts, cottonseed, and treenuts. Because of the potential health risks, aflatoxin contamination of food and feed crops is also of great economic importance to farmers who cannot sell their crops due to strict domestic and international regulatory guidelines with regards to aflatoxin contamination. In the field, these fungi are able to in essence have sex through a process called sexual recombination. This could lead to the expansion of more highly toxigenic and aggressive offspring that could lead to higher levels of aflatoxin contamination in crops. Using male and female parents of A. flavus with their own unique fluorescence signature, we were able to perform mating experiments. These studies suggested hyphae from the male parent may play a critical role in the earliest stages of sexual reproduction. These types of mating experiments will provide a better understanding of the biochemical and molecular mechanisms controlling sexual recombination in A. flavus and should aid in formulating strategies and fungal strains for control of aflatoxin contamination of food and feed crops.

Technical Abstract: Aspergillus flavus contaminates agricultural produce worldwide with carcinogenic aflatoxins that pose health risks to humans and animals. The fungus survives adverse environmental conditions through production of sclerotia. When fertilized by a compatible conidium of an opposite mating type, the sclerotia transform into stromata leading to the formation of cleistothecia, asci and ascospores. This transition from a sclerotium to a stroma, which is also influenced by female fertility (i.e., capability of a strain to produce cleistothecia in which meiosis occurs), is not well understood in A. flavus. Reciprocal crosses with different (i.e., low vs. high) levels of female fertility and unmated sclerotia, were plated in mixed cereal agar and incubated at 30 C in continuous dark. Samples of mated and unmated sclerotia were harvested at the time of crossing and every 2 weeks until 8 weeks of incubation and examined by microscopy to characterize morphological changes. Hyphal strands of germinated conidia of a green fluorescent protein (GFP)-labeled MAT1-1 strain were observed growing towards sclerotia of an mCherry (mCH)-labeled MAT1-2 strain after 24 hours of incubation. A network of interlocking hyphal strands of the GFP- and mCH-labeled strains was observed at the base of the sclerotia (i.e., region in contact with agar surface) after 72 hours of incubation. Intracellular green-fluorescent hyphal strands were observed within the stromatal matrix at 5 weeks of incubation. Morphological differences between unfertilized sclerotia and stromata became apparent at 4 weeks of incubation. Internal hyphae and croziers were detected inside multiple cleistothecia that developed within the stromatal matrix of the high fertility cross, but not in the matrix of the low fertility cross or unmated sclerotia. At 6 to 8 weeks of incubation, hyphal tips produced numerous asci, each ascus containing approximately eight ascospores that emerged out of the asci after breakdown of the ascus wall. These observations broaden our knowledge on early events during sexual reproduction and show that hyphae from the male parent may be involved in the early stages of sexual reproduction in A. flavus. When combined with omics data, these findings could be useful in further exploration of biochemical mechanisms underlying sexual reproduction in A. flavus and identify additional factors critical for sexual recombination in the ecology of this fungus.