Rapid identification of endophytic fungi of sugarcane (Saccharum spp. hybrid) using PCR-RFLP of rDNA

Aim : Restriction fragment length polymorphism analysis of PCR-amplified internal transcribed spacer (ITS) regions of rDNA is a cheap and convenient molecular tool used in fungal taxonomy. This tool has been used for rapid identification of the endophytic fungi of sugarcane belonging to different genera for their further potential utilization. Methodology : Eight isolates belonging to three endophytic fungi viz. Coiletotrichum faicatum, Fusarium moniliforme and Trichoderma viride isolated from sugarcane plant were used for differentiation using PCR-RFLP pattern of rDNA. For this, the ITS1-5.8S-ITS2 region of rDNA was amplified with the help of universal primers and the PCR products were digested with various restriction enzymes. Results : The size variation of amplified products ranged from 637 bp (C. falcaturn isolates), 592-614 bp (F. moniliforme isolates) to 631-698 bp (T.viride isolates). Five restriction enzymes viz. Alu I, EcoR I, Barn H I, MSe land Smal used for PCR-RFLP of ITS fragments yielded a total of 54 bands that were arranged in three RFLP patterns. Interpretation : Banding pattern obtained with the combination of Alu I and Mse I enzymes presented the best profile to identify the endophytic fungi and allowed distinction at molecular level. This reflected the potential of PCR-ITS-RFLP using a combination of restriction endonucleases as a rapid and convenient differentiation tool to distinguish these endophytic fungi.