Journal of Pure and Applied MicrobiologyVol. 9 No. 2

Diversity Analysis and Comparison of ITS and SCAR Based Molecular Markers to Detect Rhizoctonia solani Using Conventional and Real-time PCR

Balendu K. Upadhyay1, Sunil C. Dubey2,Ravindra Singh3 and Aradhika Tripathi1

1Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi - 110 012, India. 2Division of Plant Quarantine, National Bureau of Plant Genetic Resources,?New Delhi - 110 012, India. 3Department of Botany, Mahatma Gandhi Chitrakoot Gramodaya Vishwavidyalaya, Satna, India.

Received on 08 April 2015 and accepted on 10 May 2015

 

ABSTRACT

The populations of Rhizoctonia solani (40 isolates) causing web blight/ wet root rot in mungbeanrepresenting 7 anastomosis groups (AGs) from 11 states of India were analysed for genetic diversity utilizing pathogenicity andinternal transcribed spacer(ITS) region. The isolates caused 23% to 100% disease incidence and the majority of the isolates (75%) proved to be highly virulent. The nucleotide sequences of ITS region of the pathogen varied from 571bp to 722bp.The phylogenetic analysis of 10 isolatesof R. solani from the present study and 9 isolates representing other parts of the world using neighbor joining and maximum parsimonyshowed high level of genetic similarity whereas, the northern and southern Indian populations were clustered separately. Northern Indian populations were clustered with the isolates of Asian origin. Thespecific markers viz., BKF1 and BKR1 were designed from ITS sequences. Sequence characterized amplified region (SCAR) markers viz., BKF2 and BKR2 were also developed from a specific RAPD fragment for detection of R. solani. ITS and SCAR based markers provided a specific and sensitive detection up to the DNA level of 100 pg through conventional PCRof R. solani. The markers BKF1 and BKR1 detected 1pg while BKF2 and BKR2 detected 5 pg of genomic DNA of R. solani using real-time PCR assay.

Keywords : Diversity, PCR marker, ITS,Real time QPCR, Rhizoctonia solani.