First Report of Collar and Stem Rot Caused by Rhizoctonia solani AG-1-IA on Sesbania sesban in India

Abstract

Sesbania sesban (L.) Merr., (family Fabaceae) commonly called as “dhaincha” in India, is a multi-purpose crop used as a cover crop, as green manure, in the paper industry as well as animal fodder. The leaves of Sesbania contain high amounts of pinitol, which acts as an anti-diabetic agent (Misra and Siddiqi 2004). During July/August from 2017 to 2019, Sesbania plants exhibiting typical Rhizoctonia-like symptoms, including collar rot, wilting, and necrotic lesions on stems were regularly observed at ICAR-Central Potato Research Institute Regional Station, Meerut, Uttar Pradesh. The disease incidence ranged between 5 and 10% in a Sesbania crop being grown on 25 ha in Sesbania-potato rotation. Ten diseased plants were collected from different fields and brought to the laboratory for diagnosis. Affected stem pieces approximately 5 mm in size were surface sterilized with 2% sodium hypochlorite, washed twice in sterilized water and air dried. Four diseased pieces per plate were inoculated on 2% water agar amended with 2% streptomycin sulfate and incubated at 28±1℃ in the dark. All four affected pieces began to produce Rhizoctonia-like colonies after 48 h of incubation and in total eight isolates were purified and stored at 4℃ for further use. The colonies of eight isolates were evaluated and all were whitish during early growth and became light brown after 72 h. Dark-brown sclerotia appeared in the random pattern on PDA after 120 h. Microscopic observations showed that all isolates had hyphal branching at right angles with slight constriction at the base of the branch, presence of dolipore septum near the branching and multinucleate individual hyphae compartments (Sneh 1991). Based on these morphological characteristics, the fungus was identified as Rhizoctonia solani. All isolates were further characterized to determine anastomosis group (AG) by pairing with a known AG tester of R. solani AG-1-IA (ITCC 7650), AG-1-IB (ITCC 5650), and AG-3 (RS-20) procured from Indian Type Culture Collection, Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi and ICAR-Central Potato Research Institute, Shimla, Himachal Pradesh, India, respectively. All eight isolates showed positive anastomosis with a known AG-1-IA tester isolate while no anastomosis was observed with other known tester isolates (Carling 1996). Furthermore, a single ~265 bp amplicon was amplified with AG-specific primer, which was specific to R. solani AG1-IA group; confirms the AG-specific identity of the isolates (Matsumoto 2002). Amplification was not observed with AG1-IB, AG3 and AG2 specific primers (Khodayari et al. 2009). The selected four isolates were molecularly characterized by amplifying the internal transcribed spacer (ITS) and ribosomal DNA (rDNA) 5.8s regions by polymerase chain reaction using ITS1 and ITS4 primer pairs (White et al. 1990). The nucleotide BLAST (BLASTn) analysis of the resulting four sequences i.e. GenBank acc. no. MT105386, MT105387, MT105388, and MT105389 supported the identification of the isolates as AG-1-IA sub-group and showed 95.12%, 98.93%, 96.79%, and 98.04%, respectively, sequence homology with known cultures of R. solani AG1-IA isolated from rice in China (KC285893), and India (MK481078). To confirm pathogenicity, Sesbania plants were grown in pots and maintained in the greenhouse at 25℃ with a 12-h-light/dark photoperiod. After 35 to 40 days of growth, the stems of ten Sesbania plant were artificially inoculated with PDA plugs containing R. solani mycelia (Jia et al. 2007) and covered with aluminium foil. Plants inoculated with noncolonized agar plugs served as control. After 96 h of incubation, all the plants inoculated presented the typical collar and stem rot symptoms. No symptoms were observed in the control plants. R. solani was re-isolated cent percent from these ten infected plants fulfilling Koch’s postulates. R. solani AG1-IA has been reported to cause sheath blight and banded leaf and sheath blight diseases of rice and maize, respectively (Ogoshi 1987). To our knowledge, this is the first report of Sesbania sesban infected by R. solani AG1-IA, and serve as a host for the pathogen. The result from our findings will be helpful for planning of crop rotations in an agro-ecosystem.