Anastomosis group typing of Rhizoctonia solani kuhn infecting solanaceous vegetable crops

Abstract

Rhizoctonia solani Kühn (teleomorph = Thanatephorus cucumeris (Frank) Donk) is an economically important pathogen of solanaceous vegetables, causing black scurf, damping-off, stem canker, and root rot in these crops. R. solani is a species complex of several anastomosis groups (AGs) that exhibit DNA base sequence homology and/or affinities. To date, thirteen AGs of R. solani have been internationally reported. The present study determines the occurrence of different AGs of R. solani on Potato, Tomato, and Chilli together with their morpho-molecular characterization. Survey of different locations of districts Rawalpindi, Jhelum, Attock, Chakwal, and Federal Capital Islamabad revealed maximum mean disease incidence on potato was recorded in Attock (37.4%) followed by Islamabad (35.8%), Jhelum (32.1%), Rawalpindi (30.5%) while minimum mean disease incidence was recorded in district Chakwal (20.2%). Maximum mean disease incidence on tomato was observed in Islamabad (38.7%) followed by Attock (36.3%), Rawalpindi (34.9%), and Jhelum (31.8%) while minimum in district Chakwal (29.6%). Mean disease incidence on chilli was maximum in Attock (30.9%), followed by Rawalpindi (30.1%), Islamabad and Jhelum (29.5%), while minimum in district Chakwal (27.0%). At least 63, 67, and 58 isolates of R. solani were recovered from potato, tomato, and chilli respectively. Fungal colonies isolated on malt extract agar (MEA) medium were light grey to brown in colour with plentiful mycelial growth and branched hyphae. A septum was always present in the branch of hyphae near the originating point with a slight constriction at the branch. The hyphal distance between two septa ranged between 66.6 to 150.3 μm and hyphal diameter from 4.8 to 8.3 μm. Majority of the isolates produced rough sclerotia and were superficially present on the hyphal mass. No conidia or conidiophores were observed from cultures on malt extract xxv agar (MEA) medium. All isolates were multinucleate when subjected to 4’-6 diamidino-2-phenylindole (DAPI) stain. Based on these morphological characteristics of fungal hyphae, isolates were identified as R. solani. Recovered isolates subjected to pathogenicity tests confirmed 47, 42, and 37 isolates from potato, tomato, and chilli respectively were highly virulent. Restriction analysis of PCR-amplified ribosomal DNA with four discriminant enzymes (MseI, AvaII, HincII, and MunI) revealed recovered isolates belong to; AG-2-1, AG-2-2, AG-3 PT, AG-4 HG I, AG-5, and AG-6. Isolates were further paired with tester strains of R. solani AGs which confirmed the results of AG composition revealed by RFLP analysis. Amplification of Internal transcribed spacer (ITS) region of rDNA with primers ITS1/ITS4 and sequence analysis exhibited 99-100% identity with already reported AGs. Isolates recovered from potato belong to AG-3 PT (76.5%), AG-5 (8.5%), AG-4 HG I (4.2%), AG-2-1 (6.3%), and AG-2-2 (4.2%). AG-3 PT was widely distributed to major potato growing areas while others were confined to distinct locations. Isolates recovered from tomato belong to AG-3 PT (64.2%), AG-2-1 (14.2%), AG-2-2 (9.5%), AG-5 (7.1%), and AG-4-HGI (4.7%). AG-3 PT was widely distributed to major tomato growing areas followed by AG-2-1 while other groups were confined to distinct locations. Similarly, AG-4 HGI (59.4%) was also widely distributed to chilli growing areas. Other AGs recovered from chilli belong to AG-2-1 (16.2%), AG-6 (10.8%), AG-3 PT (8.1%), and AG-5 (5.4%). This is the first study on AG composition, genetic variability, virulence, and molecular characterization of Pakistani isolates of R. solani. These findings will provide the basis for further understanding the infection of different AGs on differential hosts which will help in the development of control strategies for management of Rhizoctonia diseases on solanaceous vegetables and other economic crops being infected by this fungus.