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A specific and sensitive conventional and real-time PCR assays were developed for the detection and quantification of  Rhizoctonia solani  populations infecting pulses. Five different sets of  R. solani  specific molecular primers ARSF1&amp;R1, ARSF2&amp;R2 and ARSF3&amp;R3, ARSF4&amp;R4 and ARSF5&amp;R5 were designed from the internal transcribed spacer sequences of  R. solani  isolates associated with pulse crops representing 7 anastomosis groups (AG1, AG2-2, AG2-2LP, AG2-3, AG3, AG4 and AG5). The designed primers yielded variable amplicons ranging from 174 to 563 bp specific to  R. solani  populations. The markers proved to be highly sensitive to  R. solani  and were able to detect the pathogen from both mycelium and infected mung bean plants at various concentrations of genomic DNA ranging from 0.025 to 1.0 ng using conventional PCR. The markers were also found suitable for real-time PCR assay with enhanced sensitivity up to 1.24 pg genomic DNA. The efficacy of markers for early detection of  R. solani  infection in mung bean plants was also tested and the marker ARSF4&amp;R4 was the most sensitive in detecting the pathogen at 24 h after artificial inoculation under field and at 6 h after artificial inoculation under  in vitro  conditions. The radioactive and fluorescein amidite labelled probes for markers ARSF4&amp;R4 and ARSF5&amp;R5 were designed and tested for cross-reactivity and sensitivity by Southern blotting and real-time PCR analyses, respectively. Both probes proved to be highly sensitive and specific only to  R. solani . Both quantitative PCR and probe-based diagnostics have been developed for  R. solani  detection in pulses.

Development of conventional and real time PCR assay for detection and quantification of Rhizoctonia solani infecting pulse crops