Rapid and sensitive diagnoses of dry root rot pathogen of chickpea (Rhizoctonia bataticola (Taub.) Butler) using loop-mediated isothermal amplification assay

Raju Ghosh,Avijit Tarafdar,Mamta Sharma

doi:10.1038/srep42737

Abstract

Dry root rot (DRR) caused by the fungus Rhizoctonia bataticola (Taub.) Butler, is an emerging disease in chickpea. The disease is often mistaken with other root rots like Fusarium wilt, collar rot and black root rot in chickpea. Therefore, its timely and specific detection is important. Current detection protocols are either based on mycological methods or on protocols involving DNA amplification by polymerase chain reaction (PCR). Here we report the rapid and specific detection of R. bataticola using loop-mediated isothermal amplification (LAMP) assay targeting fungal specific 5.8S rDNA sequence for visual detection of R. bataticola. The reaction was optimized at 63 °C for 75 min using minimum 10 fg of DNA. After adding SYBR Green I in LAMP products, the amplification was found to be highly specific in all the 94 isolates of R. bataticola collected from diverse geographical regions as well as DRR infected plants and sick soil. No reaction was found in other pathogenic fungi infecting chickpea (Fusarium oxysporum f. sp. ciceris, Rhizoctonia solani, Sclerotium rolfsii and Fusarium solani) and pigeonpea (Fusarium udum and Phytophthora cajani). The standardised LAMP assay with its simplicity, rapidity and specificity is very useful for the visual detection of this emerging disease in chickpea.

Highlights

In chickpea, Dry root rot (DRR) is often mistaken with Fusarium wilt and other root rot diseases, as the general symptoms of these diseases are nearly similar and visually undistinguishable in field conditions[1]
Since loop-mediated isothermal amplification (LAMP) assay has been reported to be very useful for quick detection and identification of a broad range of microorganisms, including viruses[21], bacteria[8], and fungi[10,11], the present study was proposed to develop highly specific and very sensitive LAMP assay for the detection of R. bataticola from infected plants and soil
For designing of LAMP assay primers for R. bataticola, the conserve region of the partial internal transcribed spacer (ITS) and 5.8S sequences was identified by multiple sequence alignment of corresponding nucleotide sequences of representative isolates in different crops (Table 2) and a set of six primers were designed (Table 3)

Summary

Introduction

DRR is often mistaken with Fusarium wilt and other root rot diseases (collar rot, black root rot etc.), as the general symptoms of these diseases are nearly similar and visually undistinguishable in field conditions[1]. PCR based methods like conventional PCR and real time PCR is being employed to detect fungal species and other microorganism[5,6,7], but it is not cost effective and need high-quality DNA due to the effects of inhibitors on PCR sensitivity[8,9]. Loop-mediated isothermal amplification (LAMP) has been developed as an alternative and reliable method for the detection of microbial pathogens and diagnosis of plant diseases[10,11,12,13,14]. Reliability of primer sets and DNA sequences of interest are the most important factors in development of molecular detection of targeted organisms. Since LAMP assay has been reported to be very useful for quick detection and identification of a broad range of microorganisms, including viruses[21], bacteria[8], and fungi[10,11], the present study was proposed to develop highly specific and very sensitive LAMP assay for the detection of R. bataticola from infected plants and soil

Methods

Results

Conclusion