Molecular diagnostics on the toxigenic potential of F usarium spp. plant pathogens

Abstract

AimsWe propose and test an efficient and rapid protocol for the detection of toxigenic Fusarium isolates producing three main types of Fusarium-associated mycotoxins (fumonisins, trichothecenes and zearelanone).Methods and ResultsThe novel approach utilizes partially multiplexed markers based on genes essential for mycotoxin biosynthesis (fumonisin—fum6, fum8; trichothecenes—tri5, tri6; zearalenone, zea2) in Fusarium spp. The protocol has been verified by screening a collection of 96 isolates representing diverse species of filamentous fungi. Each Fusarium isolate was taxonomically identified through both molecular and morphological techniques. The results demonstrate a reliable detection of toxigenic potential for trichothecenes (sensitivity 100%, specificity 95%), zearalenone (sensitivity 100%, specificity 100%) and fumonisins (sensitivity 94%, specificity 88%). Both presence and identity of toxin biosynthetic genes were further confirmed by direct sequencing of amplification products.ConclusionsThe cross-species-specific PCR markers for key biosynthetic genes provide a sensitive detection of toxigenic fungal isolates, contaminating biological material derived from agricultural fields.Significance and Impact of the StudyThe conducted study shows that a PCR-based assay of biosynthetic genes is a reliable, cost-effective, early warning system against Fusarium contamination. Its future use as a high-throughput detection strategy complementing chemical assays enables effective targeted application of crop protection products.

Multiple facets of response to fungicides – the influence of azole treatment on expression of key mycotoxin biosynthetic genes and candidate resistance factors in the control of resistant Fusarium strains

“…The chemotype and species determination as well as procedures used for their cultivation, identification and storage were described previously by Dawidziuk et al (2014). All investigated fungal isolates originated from culture collections of the Functional Evolution of Biological Systems Team (Institute of Plant Genetics, Polish Academy of Sciences, Poznan, Poland).…”

Molecular strategies for detection and quantification of mycotoxin‐producing Fusarium species: a review

“…But any PCR products were amplified by the primer sets, which taken into consider the toxigenic capacity of the pathogens in the maize samples. However, when the standard PCR was performed against the tissues of infected samples, the positive results only came from the infected wheat kernel, rather than the wheat chaff . Furthermore, Lezar and Barros reported an oligonucleotide microarray to identify potential mycotoxigenic fungi, in which the oligonucleotide probes were designed by EF‐1a or genes involved in the mycotoxin synthetic pathways.…”

“…The results clearly revealed a sensitive and specific detection of the toxigenic Fusarium isolates with the sensitivity (100%) and specificity (95%) for the trichothecenes-producing fungi, the sensitivity (100%) and specificity (100%) for the zearalenone-producing fungi, as well as the sensitivity (94%) and specificity (88%) for the fumonisins-producing fungi. 18 Nielsen et al reported a quantitative real-time PCR assay based on polymorphism of Tri12 gene in trichothecene biosynthesis cluster to identify and quantify the genotypes based on the trichothecene composition, i.e. 3-acetyl-deoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON) and nivalenol (NIV), in the Fusarium species in Danish cereals.…”

“…However, when the standard PCR was performed against the tissues of infected samples, the positive results only came from the infected wheat kernel, rather than the wheat chaff. 18 Furthermore, Lezar and Barros reported an oligonucleotide microarray to identify potential mycotoxigenic fungi, in which the oligonucleotide probes were designed by EF-1a or genes involved in the mycotoxin synthetic pathways. The method identified a total of 32 fungi with their potential to produce mycotoxins, such as trichothecenes and fumonisin.…”

Molecular identification and characterization of Fusarium spp. associated with wheat grains

“…Many Molecular techniques based on DNA analysis are being widely used to identify and discriminate among isolates within a species and to develop rapid, sensitive, and accurate detection methods (Mule et al, 2005;Nicolaisen et al, 2005). Furthermore specific PCR helps in accurate discrimination of fumonisin (FB) producing and nonproducing Fusarium isolates occurring on cereal grain (Dawidziuk et al, 2014). Researchers use genes from the FUM cluster as a good additional marker for phylogenetic and taxonomic studies of the fumonisin-producing Fusarium species (Baird et al 2008;Stepien et al 2011).…”