Development of conventional and real time PCR assay for detection and quantification of Rhizoctonia solani infecting pulse crops
-
Sunil C. Dubey
,
Aradhika Tripathi
Abstract
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&R1, ARSF2&R2 and ARSF3&R3, ARSF4&R4 and ARSF5&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&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&R4 and ARSF5&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.
Acknowledgements
Authors are thankful to the Indian Council of Agricultural Research, New Delhi, India, for financial support through NFBSFARA project.
Abbreviations
- AG
anastomosis group
- dCTP
deoxycytidine triphosphate
- FAM
fluorescein amidite
- ITS
internal transcribed spacer
- qPCR
quantitative polymerase chain reaction
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© 2016 Institute of Molecular Biology, Slovak Academy of Sciences
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