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Pak. J. Bot., 45(SI): 301-307, 2013.

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AMPLIFICATION AND SEQUENCING OF INTERNAL TRANSCRIBED REGIONS 1 & 2, AND 5.8S rDNA FROM LOCAL ISOLATES OF FUSARIUM SPECIES

S. KANWAL BATOOL NAQVI¹, SHEERAZ AHMED¹, CHAUDHARY ABDUL RAUF² AND S.M. SAQLAN NAQVI¹*

Abstract: Fusarium oxysporum is a phytopathogenic fungus. It is widely distributed around the globe. Conventional classification of F. oxysporum is based on phenotypic observations which not only vary highly, but are also sensitive to environment. In Fusarium taxonomy this problem is recently being addressed by sequence comparison at different loci. Internally transcribed spacer (ITS) region including 5.8S rRNA coding region in ribosomal DNA is one of the favorite targets for this purpose. The focus of present study was on the genetic diversity analysis of the ITS regions of rRNA gene complex of local isolates of Fusarium. The genomic DNA of these isolates was amplified using FoxF, FoxR and FoxIR primers designed at the end, and start of conserved 18S and 28S region and between ITS1 and ITS2 respectively. FoxF and FoxR primer set amplified ~500 bp product from all Fusarium strains. The amplified products were sequenced and sequence analyses have shown that F. oxysporum f.sp. ciceri strains possess a couple of SNPs. Similarly F. oxysporum f. sp. lentis has shown variations with two  strains of F. oxysporum f. sp. ciceri at two position. Comparison of F. moniliforme isolates with F. oxysporum  isolates have revealed that 5.8S region is identical in all isolates while significant sequence variation was observed in ITS regions of F. oxysporum  and F. moniliforme. Insertions and deletions of many nucleotides were observed at several positions which differentiate F. moniliforme from F. oxysporum. The phylogenetic analysis revealed no significant difference among local isolates and internationally reported sequences. From a clear grouping of F. moniliforme and F. oxysporum isolates into different clades it may be evident that ITS regions are useful for classifying F. oxysporum isolates at specie level.

¹Department of Biochemistry and ²Department of Plant Pathology, PMAS Arid Agriculture University, Rawalpindi, Pakistan

*Corresponding author e-mail: saqlan@uaar.edu.pk