Preliminary Identification and Typing of Pathogenic and Toxigenic Fusarium Species Using Restriction Digestion of ITS1-5.8S rDNA-ITS2 Region

Abstract

Background: Fusarium species are capable of causing a wide range of crop plants infections as well as uncommon hu­man infections. Many species of the genus produce mycotoxins, which are responsible for acute or chronic diseases in ani­mals and humans. Identification of Fusaria to the species level is necessary for biological, epidemiological, pathologi­cal, and toxicological purposes. In this study, we undertook a computer-based analysis of ITS1-5.8SrDNA-ITS2 in 192 GenBank sequences from 36 Fusarium species to achieve data for establishing a molecular method for spe­cie-specific identification.
Methods: Sequence data and 610 restriction enzymes were analyzed for choosing RFLP profiles, and subsequently de­signed and validated a PCR-restriction enzyme system for identification and typing of species.  DNA extracted from 32 refer­ence strains of 16 species were amplified using ITS1 and ITS4 universal primers followed by sequencing and restric­tion enzyme digestion of PCR products.
Results: The following 3 restriction enzymes TasI, ItaI and CfoI provide the best discriminatory power. Using ITS1 and ITS4 primers a product of approximately 550bp was observed for all Fusarium strains, as expected regarding the se­quence analyses. After RFLP of the PCR products, some species were definitely identified by the method and some strains had different patterns in same species.
Conclusion: Our profile has potential not only for identification of species, but also for genotyping of strains. On the other hand, some Fusarium species were 100% identical in their ITS-5.8SrDNA-ITS2 sequences, therefore differentia­tion of these species is impossible regarding this target alone. ITS-PCR-RFLP method might be useful for preliminary differ­entiation and typing of most common Fusarium species.