Design and validation of a robust multiplex polymerase chain reaction assay for MAT idiomorph within the Fusarium fujikuroi species complex

Authors: MONTOYA-MARTINEZ, AMELIA - Universidad Michoacana De San Nicolas De Hidalgo; RODRIGUEZ-ALVARADO, GERARDO - Universidad Michoacana De San Nicolas De Hidalgo; FERNANDEZ-PAVIA, SYLVIA - Universidad Michoacana De San Nicolas De Hidalgo; Proctor, Robert; Kim, Hye-Seon; O Donnell, Kerry

Submitted to: Mycologia
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/26/2019
Publication Date: 9/5/2019
Citation: Montoya-Martinez, A.C., Rodriguez-Alvarado, G., Fernandez-Pavia, S.P., Proctor, R.H., Kim, H.-S., O'Donnell, K. 2019. Design and validation of a robust multiplex polymerase chain reaction assay for MAT idiomorph within the Fusarium fujikuroi species complex. Mycologia. 111(5):772-781. https://doi.org/10.1080/00275514.2019.1649956.
DOI: https://doi.org/10.1080/00275514.2019.1649956

Interpretive Summary: The genus Fusarium comprises over 20 species groups, many of which contain toxin producing plant pathogens that threaten agricultural biosecurity and food safety within the United States and among our trading partners worldwide. The species rich F. fujikuroi species complex (FFSC) ranks as one of the most important pathogen groups within Fusarium because it contains phytopathogens that are responsible for reduction in yields and species that contaminate economically important crops with toxins that pose a serious threat to human health. Food and feed heavily contaminated with FFSC toxins such as fumonisins, moniliformin, enniatins, among others, make it unsuitable for consumption by humans and other animals. Knowledge of a pathogens reproductive potential is important for developing robust disease control measures because ones that can reproduce sexually are much more likely to overcome crop resistance to disease. In this study we discovered that published molecular diagnostic tests for determining whether pathogens within the FFSC contain mating compatible strains failed to work on 50% or more of the species. Thus, the primary objective of this study was to develop a robust molecular diagnostic assay for determining strain mating type within the FFSC. To accomplish this objective, we mined mating type genes from the genomes of 60 FFSC isolates, representing 55 species, and from four species in a closely related group, the F. nisikadoi species complex (FNSC). Using the DNA sequence data, we designed a molecular diagnostic assay that worked on 100% of the FFSC strains tested. Our results suggest that if the FFSC and FNSC reproduce sexually, it requires the mating of two compatible strains. Through this research we developed the only robust molecular diagnostic for accurately determining whether FFSC species possess the ability to undergo sexual reproduction. This research will be of interest to plant pathologists focused on developing robust plant disease strategies and reducing toxin contamination by members of the FFSC in food and feed.

Technical Abstract: We discovered that published polymerase chain reaction (PCR) assays for determining mating type (MAT) idiomorph failed to genotype some of the Fusarium fujikuroi species complex (FFSC) isolates recovered from Mangifera indica (mango), Swietenia macrophylla (big-leafmahogany), Annona muricate (soursop), Bursera sp., and Tabebuia sp. inMexico. Thus, the primary objective of this studywas to design and validate a robust multiplex PCR–based diagnostic for typing MAT within the FFSC. To accomplish this objective, we mined the MAT1-1 or MAT1-2 locus from the genomes of 60 FFSC isolates, representing 56 phylospecies, and from four species in its sister group, the F. nisikadoi species complex (FNSC). Bioinformatic searches were facilitated by targeting DNA lyase (SLA2) and apurinic endonuclease (APN1), the genes that flank the MAT locus in Fusarium. As expected, three genes were identified within MAT1-1 (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and two in MAT1-2 (MAT1-2-1 and MAT1-2-9), using the ab initio prediction tool AUGUSTUS. Of the threemultiplex PCR assays we designed and tested, the one targeting MAT1-1-2 and MAT1-2-1 successfully genotyped the entire 71-isolate validation panel,which included 56 FFSC and 4 FNSC phylospecies. By contrast, the published PCR assays we tested produced positive genotypes for only 46.5–59% of the 71-isolate validation panel, but only when they were run as a uniplex assay. Although only one-fifth of the FFSC/FNSC are known to reproduce sexually, our results suggest that if they possess a sexual cycle, it is heterothallic (self-sterile).