Molecular identification and characterization of Fusariyum species causing leaf twister Disease of Onion

Abstract

Leaf Twister disease (LTD) is a major disease of onion (Allium cepa L.) cultivated in Sri Lanka. The pathogens causing the LTD of onion have been identified as Collectotrichum spp and Fusarium spp. However, species delineation of the two genera has not been done in detail. Morphological features are not a reliable criterion for species delineation due to diverse variations of colony characters of Fusarium spp. and minor variations of spore dimensions that could be detected morphometrically. Hence, the objective of the present study was to identify and characterize the Fusarium species associated with LTD of onion grown in Sri Lanka by DNA-based molecular techniques.

LTD-infected onion plants were collected from farmer fields at six locations of the Puttalam district, Sri Lanka. Possible causal organism/s of LTD were isolated from three different parts of the infected plants, namely leaves, pseudostem and bulb. Twelve morphologically-different isolates of Fusarium spp, were identified. Genomic DNA was extracted from each purified isolate using a modified CTAB method. Using genomic DNA, PCR amplification was done with ITS-Fu1f and ITS-Ful r primers to amplify the region of ITS2-rDNA subunit of the Fusarium isolates. All Fusarium isolates gave the expected PCR product of 466 bp. To determine the genomic variation among the isolates, PCR products were analyzed by PCR-RFLP using restriction endonucleases, Rsa I and Pvu II. Based on PCR-RFLP analysis, no molecular polymorphism was observed among the isolates of Fusarium spp.

DNA sequencing was done for a PCR product out of the 12 amplified products of the Fusarium isolates. DNA sequence data was compared with the available sequence information of the databases using Basic BLAST search option of NCBI. Sequence data of the query sample showed 99% similarity with Fusarium solani (Accession no. JX896998.1). Based on the results, all the isolates used in the present study were identified as Fusarium solani and they are not genomically-polymorphic with reference to ITS2-rDNA subunit. Therefore, Fusarium solani has been identified as one of the causal agents of LTD on onion (Allium cepa L.).