Identification and differentiation of Fusarium species using selected molecular methods

Abstract

The taxonomy of Fusarium at the species level is based on morphological characteristics which include hyphae, conidia and microconidia. These features require expertise in taxonomy for an accurate and reliable diagnosis which is crucial as it aids in disease management and genetic diversity studies. This study aimed to develop alternative and/ or complementary taxonomic tools through the use of molecular based techniques. Initially, 113 morphologically identified Fusarium isolates were selected for polymerase chain reaction (PCR) amplification with a set of six inter-simple sequence repeat (ISSR) primers and four universally-primed (UP-PCR) primers to establish identifiable differences and similarities. Genetic variation was also assessed by amplification and sequencing of internally transcribed spacer (ITS) regions of rDNA from 13 isolates using Fusarium specific primers. ISSR amplifications employing four primers and UP-PCR analysis using one primer pair revealed scorable polymorphisms among an average of 86 isolates per primer. Some of the isolates did not yield amplifiable DNA with the selected primers used. The four ISSR primers yielded a total of 500 polymorphic bands and the UP-PCR primer pair revealed 126 polymorphic bands. Genetic similarities among the isolates were calculated using Jaccard’s coefficient while cluster analysis was used to generate dendrograms showing genetic relationships. The isolates were grouped according to similarity levels.

Results obtained indicated a high degree of genetic variability in the genus Fusarium. High intraspecies diversity was observed in Fusarium oxysporum and Fusarium solani isolates. Some unexpected genetic similarities were observed among the isolates indicating non-agreement between morphological and molecular identification of the isolates. This suggests the need to use species-specific primers in further analyzing the revealed genetic relationships. Sequencing of the amplified ITS regions among the 13 Fusarium species revealed two groups with 85% genetic similarity. The ITS regions in the two groups showed a relative similarity ranging from 87 to 100%. A 100% genetic similarity was noted between two F. oxysporum isolates which indicated an agreement between morphological and molecular identification. Another 100% genetic similarity was noted among three species, F. moniliforme, F. pallidoroseum and F. lateritium suggesting that species designation can be unreliable if based on morphological data alone. Based on the overall results, the use of molecular methods constitutes an important complement of the morphological criteria needed to allow fungi to be more easily identified