INTRODUCTION Indian bean (Lablab purpureus L.) belonging to family Fabaceae, is one of the most ancient among the cultivated crops and is presently grown throughout the tropical regions in Asia, Africa and America. It is commonly known as Hyacinth bean, Dolichos bean, Avare (Kannada), Anumulu (Telugu), Avaria (Tamil), Indian bean, Lablab bean, Wal papdi (Gujarat) and Egyptian kidney bean. In India, the area under pulse crops is more than 176.17 lakh hectare and production is 93.14 lakh tonnes, with the productivity of 689 kg ha-1, as per 1st advanced estimates (DAC & FW) 2020-2021. Indian bean can be grown on a wide variety of soil types ranging from acidic to alkaline. Indian bean is primarily grown for green pods which are cooked as vegetables like others beans. The foliage of the crop provides nutritive hay, silage and green manure. It is also used in medicinal purpose (Smith, 1976). Plant diseases are one of the major constraints in crop production with drastic losses in the quality and quantity of the yield. Stem rot of Indian bean caused by Sclerotium rolfsii Sacc. has become a limiting factor to get production in South Gujarat. The species was first described in 1911 by Italian mycologist Pier Andrea Saccardo, based on specimens sent to him by Peter Henry Rolfs who considered the unnamed fungus to be the cause of tomato blight in Florida in 1892. S. rolfsii is a soil-borne pathogen that commonly occurs in the tropics, sub-tropics and other warm temperate regions of the world causing root rot, stem rot, wilt and foot rot on more than 500 plant species including almost all the agricultural and horticultural crops (Aycock, 1996). It is one of the most destructive soil inhabiting pathogen and causes heavy loss to the crops in both kharif and Rabi season. It is common where high temperatures exist during the rainy season. The present study was carried out for evaluating the cultural, morphological and pathogenic variability among the different isolates of S. rolfsii Sacc. causing stem rot of Indian bean collected from the different growing pockets viz., Surat, Navsari and Bharuch districts. MATERIALS AND METHODS A. Survey and symptomatology of Indian bean stem rot caused by S. rolfsii (i) Survey: An intensive survey was conducted to know the incidence of stem rot of Indian bean in Indian bean growing areas of South Gujarat viz., Surat, Navsari and Bharuch district. Random roving survey was followed for collecting the stem rot infected samples. The percent disease incidence (PDI) was calculated by following equation. Four Taluka of three Districts viz., Olpad of Surat, Jalalpore and Chikhli of Navsari and Amod of Bharuch district were selected under survey. The infected samples and data were collected from randomly four to five fields of two villages of each taluka. The information like variety, planting material used for isolation, location and isolates designation was also collected and tabulated. Symptomatology. The plants in the individual field were observed for the characteristic symptoms of stem rot caused by S. rolfsii. Indian bean plant showing the typical stem rot symptoms were collected and taken for further isolation. B. Collection and isolation of diseased samples, identification, mass multiplication and pathogenicity of the pathogen Collection and isolation of diseased samples. The isolation of fungus was done by following standard tissue isolation technique. The infected pieces were cut into small bits and then washing in running water. These bits were surface sterilized with 0.1 % Mercuric chloride (HgCl2) solution for one minute followed by washing in distilled sterile water, then aseptically transferred to PDA (Potato Dextrose Agar) plate and incubated at room temperature for seven days. From this fungal growth hyphal tip was used for further purification. Same procedure was followed to obtain the pure culture of S. rolfsii from all locations. The pure cultures that obtained were further stored in refrigerator for further investigation. Identification of the Pathogen. The pathogen was identified based on the characters mentioned below. — Types of mycellial growth on PDA. — In early stages of the growth, mycelium colour and luster. — Sclerotial colour and shape of different sclerotial stages till maturity. Maintenance of the pure culture. The fungus was sub-cultured on the PDA slants and allowed to grow at 27±1 ºC temperature for one week and later the culture was stored in refrigerator for further studies and was sub cultured periodically. Mass multiplication of S. rolfsii isolates. The study on mass multiplication of S. rolfsii was also conducted. A total eight numbers of isolates were multiplied on sorghum grains (200 gm) which was soaked overnight in water. About 200 gm of soaked sorghum grains were taken in 500 ml capacity conical flask tightly plugged. The conical flasks were then sterilized at 121°C for 20 min. After sterilization the sorghum seeds in conical flask were inoculated with the different eight isolates mycelial discs of five mm diameter from seven day old pure culture of S. rolfsii and flasks were incubated at 27°C ± 2°C for 15 days for mycelial growth. Pathogenicity of the pathogen. The potting mixture was prepared by thoroughly mixing soil, sand and farm yard manure at 1:1:1 ratio. The inoculum of S. rolfsii grown on sorghum grain based medium were separately mixed with the sterilized soil filled in 30 cm earthen pots at 10 days before sowing. Surface sterilized indian bean seeds were sown at the rate 5-10 seeds per pot and the pot without inoculum served as control. Soil moisture was maintained at moisture holding capacity of soil by adding sterilized water throughout the period. The typical wilting symptoms were observed after 15-20 day of inoculation. Reisolation was made from such infected plant tissue and compared with that of original isolates for conformity. C. Variability among the different isolates of S. rolfsii Studies on morphological variation of different isolates. The experiment was conducted in order to study the variation in the morphological characters of different isolates of S. rolfsii. For this purpose, 15 ml of potato dextrose agar was poured into sterilized petriplate. Mycelial disc of 7 day old culture of the respective isolates was placed at the centre of the PDA plate. Three replications of each isolate and allow to grown at room temperature (27±1°C) and colony characters, like diameter, radial growth and concentric rings were recorded. The radial growth of the each isolates (in mm) was measured four days after inoculation. The radial growth of each colony in two directions at right angles was measured. Visual observations on sclerotial formation were recorded. The total number of sclerotia produced per plate, sclerotial test weight, diameter of sclerotial bodies, size, colour and shape of sclerotia of individual isolates were also recorded and analyzed statistically. Virulence of different isolates of S. rolfsii. A pot experiment was conducted in the glass house of Department of Plant Pathology, N. M. College of Agriculture, Navsari to find out virulence index of different isolates on two different varieties namely GNIB 21 and GNIB 22. Each treatment was replicate thrice. Observations were noted on PDI and number of days taken for stem rot symptoms (latent period). Percent disease incidence (PDI) was calculated by following this equation: The numerical values of percent disease incidence and latent period were used to calculate the virulence index using the following equation (Thakur and Rao 1997). Virulence index = Percent disease incidence × Latent period-1. RESULTS AND DISCUSSION A. Survey and symptomatology of Indian bean stem rot caused by S. rolfsii Survey of stem rot of Indian bean: The random roving survey was carried out at monthly interval to know the incidence of stem rot in Indian bean during the kharif season 2019-20 in randomly selected villages of different taluka of South Gujarat. The data revealed that the incidence of stem rot disease in Indian bean is endemic in nature as it was observed in more or less severe form in all the selected fields of all the selected villages (Table 1). The percent disease incidence occurrence was ranged from 7 to 30 percent in different location (Table 1). Maximum disease incidence (30%) was recorded at NAU, farm, Eru (Jalalpore) while minimum disease incidence (7%) was recorded in Jamanpada (Chikhli). The variation observed in the disease incidence might be due to the prevalence of the strains of S. rolfsii with varied virulence in bean varieries and the environmental factors in the respective areas. The present studies are similar to results reported by Amrutha et al. (2019), which carried out random roving survey for studying groundnut stem rot disease incidences in Andhra Pradesh. Daunde et al. (2018) also studied prevalence of collar rot of chilli in Marathwada region of Maharashtra. Rani and Hegde (2017), Sivakumar et al. (2016), Divya rani et al. (2016) and Muthukumar and Venkatesh (2013) also carried out survey for studied prevalence of stem rot disease in different crops at different locations. Symptomatology of stem rot of Indian bean. First symptoms of stem rot indicated yellowing, browning and discoloration of leaf tissue followed by loss of vigour and premature death or wilting of lateral branches of Indian bean. If the main stem was attacked then the whole plant was affected and caused death of the plants. Moreover to this, there are formations of deep dark brown lesion on the stem region of the plant just near the ground. Advanced symptoms were developed when the lesion was covered by a radiating white mycelium with the rotting underneath it. In later stages of infection, mustard seed like dark brown or tan coloured, spherical or round sclerotial bodies were formed, which adhered around the infected stem region and such bodies were produced abundantly on stem. The infected plant showed poor root growth and rotting of the stem region. These types of symptoms are occurred at all the places from here those samples are collected; these symptoms are more or less similar. The similar symptoms and signs were observed by Sivakumar et al. (2016), they collected the infected groundnut plants showing the typical symptoms of stem rot due to infection with S. rolfsii along with rhizosphere soil. Nagamma and Nagaraja (2015) also observed disease symptoms of S. rolfsii on chickpea plant and notice that the pathogen infected first at collar region. B. Collection and isolation of diseased samples, identification and pathogenicity of the pathogen Collection and isolation of diseased samples. The stem rot infected samples of Indian bean were collected from the selected fields of different villages of different talukas from three districts viz., Surat, Navsari and Bharuch (Plate: 1). The collected samples were brought to the laboratory, which were then subjected to microscopic examination followed by standard tissue isolation technique using PDA medium. The pure culture thus obtained was further purified by single hyphal tip isolation technique and maintained on PDA medium. Identification of the Pathogen. The isolated pathogen was identified based on cultural and morphological characters as mentioned below. The isolated fungus was found to produce white, dense radiating mycelial growth on PDA. The mycelium of the pathogen was septate and hyaline with conspicuous branching at acute angles. The well developed mycelium had cord-like strands. In early stages of growth, the mycelium was silky whitish in colour; it gradually lost its lustre and became dull in appearance and form radial growth colony. The initiation of sclerotial was observed from 6th day onwards. At the initial stage, the sclerotial bodies were white in colour which was gradually turned into mustard seed like dark brown colour at maturity stage. Matured sclerotia were spherical to ellipsoidal (Plate: 2 and 3). These types of morphological and cultural characteristics were observed in all the isolates of S. rolfsii. They all named as SrKUD (Kudiyana), SrKUV (Kuvad), SrERU (Eru), SrKHR (Kharsad), SrPEL (Peladi bhervi), SrJAM (Jamanpada), SrBHM (Bhimpura) and SrGHM (Ghamnad) with more or less similar cultural and morphological features. Similar morphological characteristics of mycelial growth and sclerotial formation were observed by earlier workers, Kushwaha et al. (2019); Ghevariya and Patel (2019); Manu et al. (2018); Wavare et al. (2017); Sekhar et al. (2017). Pathogenicity of S. rolfsii. The pathogenicity test of S. rolfsii was proved by soil inoculation method with giant culture (sorghum grain based medium inoculated with isolate of S. rolfsii). It was carried out under greenhouse conditions as per the procedure described in methodology. Pathogen infect first at collar region. Leaves of such infected plants become yellow. During the advanced stage of infection the white mycelium grew around the collar region and completely covered it. The base of the stem was constricted and turned brown. The plant gradually dried and toppled. The sclerotial bodies were formed on infected parts. Severely infected plants showed death of plant and diseased plant could be easily pulled out from the soil. The fungus was reisolated from infected plant tissue and compared with the original culture, the reisolated culture and original culture showed similar cultural and micro morphological characters and thus identified as same. Hence, the pathogenicity of isolated fungal pathogen was proved as per Koch’s postulates showed in plate 4. B. Variability among the different isolates of S. rolfsii Morphological variation in different isolates of S. rolfsii on potato dextrose agar. Morphological characters of each of the isolates of S. rolfsii on potato dextrose agar were studied and observations were recorded. The characters like, colony growth diameter, color of sclerotia, shape of sclerotia, position of sclerotia in culture, numbers of sclerotia formation per plate, diameter of sclerotia and test weight of sclerotia were recorded and are presented in Table 2 and 3. (A) Growth rate of colony Wide range of variation was noticed among the eight isolates of S. rolfsii with respect to various attributes studied. Here highest colony growth diameter found in SrERU (89.00 mm) isolate. Whereas, isolate SrBHM (87.00 mm) was statistically at par with the SrERU isolate, which followed by SrKUV (86.00 mm), SrKHR (83.00 mm), SrPEL (80.00 mm), SrGHM (79.00 mm), SrKUD (78.00 mm) and SrJAM (74.00 mm) isolates. The growth rate was ranged from 18.50 to 22.25 mm per day. (B) Time taken for sclerotial initiation The isolates varied with respect to time taken for sclerotial formation. Isolates were grouped into three groups. Group I consisted of three isolates (SrERU, SrKHR and SrPEL) which took just four days for sclerotial initiation. Group II consisted of four isolates (SrKUV, SrJAM, SrBHM and SrGHM) in which sclerotial initiation was noticed on 5th day. Group III consisted of one isolate (SrKUD) took maximum number of days (06) for sclerotial initiation. In all isolates sclerotial bodies were initially white, with time turned dark brown. (C) Colour, shape and position of sclerotia With regards to sclerotial colour, 3 types of colour were observed visually among the isolates. Isolates SrKUV, SrJAM and SrBHM had chocolate brown sclerotia, isolates SrKUD and SrGHM showed light brown coloured sclerotia and remaining isolates SrERU, SrKHR and SrPEL had dark brown coloured sclerotia. Isolates SrKUD, SrERU, SrKHR, SrJAM and SrGHM showed round shape sclerotia whereas, others viz., SrKUV and SrPEL and SrBHM showed oval shape sclerotia. With respect to position of sclerotia, maximum isolates (SrKUD, SrERU, SrKHR and SrBHM) of sclerotia were produced irregularly scattered all over the petriplates. SrJAM produced sclerotia at centre in concentrated form. Whereas isolates like (SrKUV, SrPEL and SrGHM) produced sclerotia at the edges of petriplates (Plate: 5). (D) Number of sclerotia formation in plate Here the higher numbers of sclerotia recorded per plate in SrERU isolate (300) which followed by SrKUD (283.33), SrKHR (259), SrJAM (227.66), SrPEL (189.33), SrGHM (185.67), SrKUV (176.99) and SrBHM (176.66) isolate. (E) Size of sclerotia The variation in size of sclerotia of isolates under study was found significant. SrJAM isolate produced largest sclerotia with mean diameter of 1.22 mm, whereas SrGHM isolate (1.21 mm) was found statistically at par with SrJAM isolate, which followed by SrBHM isolate (1.12 mm), SrKHR (1.093 mm), SrERU (0.99 mm), SrPEL (0.96 mm), SrKUD (0.91 mm) and SrKUV (0.83 mm) isolates. (F) Test weight of 100 sclerotia The test weight of sclerotia revealed significant variation among all the isolates. Maximum test weight was recorded in SrJAM isolate (301.66 mg) followed by SrGHM (285.60 mg), SrBHM (263 mg), SrERU (226.33 mg), SrKHR (207.33 mg), SrKUD (182 mg), SrPEL (166.66 mg) and SrKUV (165.33 mg). Pandi et al. (2017), Naresh et al. (2017), Mahato and Biswas (2017), Kakade et al. (2017), Ekka et al. (2016), Kuldhar et al. (2014), Rakholiya and Jadeja (2011) and Bagwan (2011) also carried out an experiment to study cultural and morphological variability in different isolates of S. rolfsii causing stem rot disease. Virulence index. All the eight isolates were inoculated on two different varieties GNIB-21 and GNIB-22 of Indian bean to study the variation in pathogenicity. Observations on percent disease incidence and number of days taken for stem rot symptoms (latent period) were recorded and presented in the Table 4 and 5. For GNIB-21 variety of Indian bean, maximum degree of virulence was recorded in SrERU (10.00) which took less number of days (10) for disease infection to plant followed by SrKUD (7.49), SrKUV (6.23), SrPEL (4.93), SrKHR (4.00), SrBHM (3.41), SrJAM (2.84) and SrGHM (2.18) which required 12, 13, 15, 16, 17, 19 and 22 days for stem rot infection respectively. SrGHM was least virulent with minimum virulence index of 2.18 and required maximum period for completely rotting of plant. Isolates with maximum virulence index required less period of days for causing stem rot of plants presented in Table 4 and plate 6. For GNIB-22 variety of Indian bean, maximum degree of virulence was recorded in SrERU (7.58) which took less number of days (12) for disease infection of plant followed by SrKUD (4.87), SrKUV (3.78), SrPEL (3.36), SrKHR (2.66), SrBHM (2.27), SrJAM (2.0) and SrGHM (1.53) required 16, 19, 19, 21, 22, 23 and 26 days for stem rot infection respectively. SrGHM was least virulent with minimum virulence index of 1.53 and required maximum period for completely rotting of plant. Isolates with maximum virulence index required less period of days for causing stem rot of plants presented in Table 5 and plate 7.