Use of molecular markers to compare Fusarium verticillioides pathogenic strains isolated from plants and humans

Abstract

ABSTRACT. Fusarium verticillioides is a pathogen of agriculturally important crops, especially maize. It is considered one of the most important pathogens responsible for fumonisin contamination of food products, which causes severe, chronic, and acute intoxication in humans and animals. Moreover, it is recognized as a cause of localized infections in immunocompetent patients and disseminated infections among severely immunosuppressed patients. Several molecular tools have been used to analyze the intraspecific variability of fungi. The objective of this study was to use molecular markers to compare pathogenic isolates of F. verticillioides and isolates of the same species obtained from clinical samples of patients with Fusarium mycoses. The molecular markers that we used were intersimple sequence repeat markers (primers GTG 5 and GACA 4 ), intron splice site primer (primer EI1), random amplified polymorphic DNA marker (primer OPW-6), and restriction fragment length polymorphism-internal transcribed spacer (ITS) from rDNA. From the data obtained, clusters were generated based on the UPGMA clustering method. The amplification products obtained using primers ITS4 and ITS5 and loci ITS1-5.8-ITS2 of the rDNA yielded fragments of approximately 600 bp for all the isolates. Digestion of the ITS region fragment using restriction enzymes such as EcoRI, DraI, BshI, AluI, HaeIII, HinfI, MspI, and PstI did not permit differentiation among pathogenic and clinical isolates. The inter-simple sequence repeat, intron splice site primer, and random amplified polymorphic DNA markers presented high genetic homogeneity among clinical isolates in contrast to the high variability found among the phytopathogenic isolates of F. verticillioides.

Analysis of the rDNA internal transcribed spacer region of the Fusarium species by polymerase chain reaction-restriction fragment length polymorphism

“…The ITS region was confirmed in the present study to be particularly suitable for the purpose of providing target genes for molecular identification of the Fusarium species. Variation in the nucleotide composition of the ITS region was successfully employed for recognition among the species (11)(12)(13). A variety of targets have been employed for DNA-based identification and dicrimination of pathogenic Fusarium species.…”

“…For these reasons, the molecular biological technique has been established in Fusarium systematics and the molecular variation at the DNA level has been investigated in numerous studies (8)(9)(10). The use of polymerase chain reaction (PCR) with primers targeted to the internal transcribed spacer (ITS) region of the ribosomal DNA (11,12) for identification of the Fusarium species has been reported. The ITS region sequences have shown to be highly variable in Fusarium genus (13).…”

Genetic variability and pathogenicity of Fusarium verticillioides isolates from the summer‐sown maize regions in China

“…ISSR is a preferred method because it is relatively simple, cost-effective and efficient. Importantly, ISSR has been successfully used in F. verticillioides (Chang et al, 2013).…”

Association of ectomycorrhizal fungi with Picea crassifolia (Pinaceae, Piceoidae) from high-altitude stands in Mount Helan Nature Reserve, China

“…The entire internal transcribed spacer (ITS) region was amplified with the fungalspecific primers ITS1F and ITS4 (Gardes and Bruns, 1993;Chang et al, 2013;Pei et al, 2014). The PCR mixture contained 2 mL undiluted DNA template, 2 mL each primer (10 mM concentrations) and 25 mL Master Mixture (containing Mg and 1U Expand TM High Fidelity Polymerase from Sangon Biotech, China).…”