Phylogenetic relationship among Bipolaris species based on morphological and molecular variability in internal transcribed spacer region of the nuclear ribosomal DNA

Phytopathogenic fungi belonging to Bipolaris species are responsible for severe economic losses to many cereal crops globally. Molecular variability among thirty eight isolates belonging to five Bipolaris species viz., B. sorokiniana (Bs), B. oryzae (Bo), B. maydis (Bm), B. spicifera (Bsp) and B. tetramera (Bt) was studied by sequence analysis of internal transcribed spacer (ITS) region of nuclear ribosomal DNA by PCR amplification, restriction analysis and direct sequencing. The PCR amplification with ITS1 and ITS 4 universal primers showed a single DNA band of 600bp (approx.) in all the isolates. The length of ITS 1 varied from 164 to 202bp and that of ITS 2 varied from 132 to 233bp. The variation in % G+C of ITS 1 among the isolates was lower than that of ITS 2. In silico analysis of full length ITS region sequences of all Bipolaris spp. resulted in formation of two clusters with more than 90% sequence homology. Cluster I consisted of isolates belonging to B. sorokiniana, B. maydis and B. oryzae while B. tetramera and B. spicifera formed cluster II. Clustering based on restriction enzyme site analysis also grouped the isolates into two major clusters with B. tetramera and B. spicifera isolates grouped together in a separate cluster. Restriction enzymes Hha-1, Hae-III and Rsa-1 produced distinct restriction pattern in different Bipolaris spp. On the basis of sequence analysis it was observed that Indian pathogenic isolates of Bipolaris spp. have evolved distinctly in tandem with their host preference and their variability can conveniently be elucidated by ITS region rDNA sequence. B. tetramera and B. spicifera were highly similar based on morphology and ITS sequences.