INTRODUCTION Peanut (Arachis hypogaea L.) is one of the most important oilseed and legume crop, which is highly used for edible purposes either as nuts or as oil extracted from nuts. It comes under the family Leguminaceae and originated from South Africa. Groundnut is of higher economic importance as it contributes to the Indian economy by exportation and exportation of groundnut is more than the domestic consumption. Although India is first in acreage, the yield/ ha is very low compared to china. The production of groundnut is declining over years and the main reason is considered as the yield loss caused by the economically important pathogens, Macrophomina phaseolina and Sclerotium rolfsii which infect the peanut crop and cause dry root rot and stem rot diseases respectively. These diseases are most destructive as it is soil borne and systemic, it infects the plants and cause complete destruction. Both the pathogens are soil borne and have a wide range of host infecting more than 500 crop species (Doley et al., 2012). These pathogens are considered to be detrimental due to the production of the fungal propagules, microsclerotium by M. phaseolina and sclerotium by S. rolfsii which survive in the soil for many years and it is difficult to destroy (Shifa et al., 2015). Also, the somatic structures of the pathogens are distributed in the soil randomly, it is very challenging to control by any means (Pardeep et al., 2015). The pathogens infect all the stages of peanut crop and their control has been difficult since. The incidence of dry root has been recorded as 33.33 per cent decayed seeds and post emergence mortality of 23.80 per cent (Moradiaand Khandar, 2011). And incidence of stem rot was recorded as 7.88 to 32.02% in various villages of Cuddalore district of Tamil Nadu (Sivakumar et al., 2016). Hence, the present study was aimed on survey of incidence of dry root rot and stem rot of peanut, isolation and characterization of M. phaseolina and S. rolfsii based on morphological and molecular characterization. MATERIALS AND METHODS Survey on the occurrence of root rot. The incidence of the root rot diseases were surveyed by roving method of survey (Archana et al., 2020) in major peanut growing districtsof Tamil Nadu viz., Coimbatore, Dharmapuri, Krishnagiri, Salem and Cuddaloreat different stages of the crop. Isolation of root rot pathogens. The symptomatic plants were collected for isolation from various districts of Tamil Nadu in which the incidence was surveyed. The symptoms were yellowing and wilting of plants, bark shredding in dry root rot infected plants and white mycelial propagules were grown on the infected parts in stem rot infected plants. Presence of sclerotia were observed on the infected parts of the plants and were visible to the naked eye. The pathogens were isolated by tissue segment method (Rangaswami, 1972). The infected roots were cut into small pieces of 0.2 to 0.5 cm in size. The root bits were washed in distilled water twice to remove the soil and debris and these bits were surface sterilized using 1% sodium hypochlorite once and then washed with sterile distilled water twice. Excessive moisture present on the root bits were dried by placing them on sterile tissue paper. After drying, root bits were placed in fresh sterilized PDA (Potato Dextrose Agar) medium which was poured half plate for isolation. Also, brown to black, rounded sclerotium present on the roots that were apparent to the naked eye were collected and placed on fresh sterilized potato dextrose agar medium (Shim et al., 1998). Morphological characterization. The pure cultures of the rot pathogens were observed in the naked eye for their colony characters. The colour and nature of the colony growth (Fluffy/Sparse/Compact) were observed in naked eye. The mycelial characters (hyaline/coloured, septate/coenocytic) and the sclerotial characters (size and shape of sclerotium) were observed under phase contrast microscope (LEICA DM30000, DST FIST lab, TNAU, Coimbatore) (Emayavarman et al., 2019). Molecular characterization. The DNA of the isolates of M. phaseolina and S. rolfsii were extracted separately from 14 days old PD broth inoculated with pathogens. DNA extraction was done by CTAB method (Almeida et al., 2003). The extracted DNA was amplified using the universal primers, ITS 1 and 4 with a reaction mixture, 5 µl of Master mix, 2µl of double sterile distilled water, 1µl of forward primer, 1µl of reverse primer. The reaction mixtures were prepared for all the isolates of M. phaseolina and S. rolfsii and were subjected to PCR in Thermocycler (Eppendorf Master Cycler Nexus gradient). The conditions for PCR fixed was, initial denaturation at 95°C for 1 min, denaturation at 95°C for 1 min (35 cycles), annealing at 58°C for 1 min, extension at 72°C for 1 min and final extension at 72°C for 10 mins. After the cycles were completed, the PCR product was checked for amplification in agarose gel electrophoresis by loading them in 1.2 % agarose gel amended with 2 µl of Ethidium Bromide. Then the gel was visualized under UV transilluminator and images were documented in gel documentation unit (BIO RAD, Gel DocTM EZ Imager, Bio-Rad Laboratories Inc.). To confirm the amplicon size at which the product were amplified, Molecular marker (DNA ladder, 100 bp) was used (Emayavarman et al., 2019). Statistical analysis. Design of experiment used was CRD and AGRES ANOVA package version 7.01 from Department of physical sciences, TNAU, India was used to analyse the statistical data (Senthilkumar et al., 2009) and the percent values are arcsine transformed. ANOVA was used to analyse the data at significant level 0.05 and means were compared using Duncan’s Multiple Range Test (DMRT). RESULTS AND DISCUSSION Survey on the incidence of root rot. The incidence of dry root rot was recorded from 18.60 per cent to 64.00 per cent and stem rot was recorded from 20.30 per cent to 66.20 per cent in various districts of Tamil Nadu. The highest incidence of dry root rot was recorded as 64 per cent in Vridhachalam, Cuddalore district and lowest incidence was 18.6 per cent in Pulikarai, Dharmapuri (Table 1). Moradiaand Khandar (2011) surveyed the incidence of dry root rot in Saurashtra region of Gujarat and reported 33.33 per cent of seed decay and post emergence mortality of 23.80 per cent in peanut crop due to dry root rot. The dry root rot incidence in the major peanut growing district in Tamil Nadu, Cuddalore, the incidence was recorded ranging from 21.73 per cent to 31.68 per cent (Raja Mohan et al., 2012). Similarly, Telugupalayam, Coimbatore district, recorded highest stem rot incidence of 66.2 per cent and lowest incidence of 20.3 per cent was recorded in Attur village of Salem district. Archana et al. (2020) stated that the incidence of wet root rot in Coimbatore, Tamil Nadu was recorded as 86.6 per cent during roving survey. The yield loss by stem rot was normally upto 25 per cent but in conducive conditions the yield loss maybe upto 80-90 per cent (Deepthi & Reddy 2013). Sivakumar et al. (2016) recorded stem rot incidence from 7.88 to 32.02 per cent in various villages of Cuddalore district of Tamil Nadu. A roving survey was conducted in major peanut growing areas in Kharif 2014, in which the incidence of stem rot was recorded as 10.11 to 59.33 per cent (Haveri, 2017). Isolation of rot pathogens. Dry root rot and stem rot infected specimens were collected and the pathogens were isolated separately under aseptic condition. The isolates were named as TNAU MP1, DPI MP2, KRI MP3, SLM MP4 and CUD MP5 for M. phaseolina isolates and TNAU SR1, DPI SR2, KRI SR3, SLM SR4 and CUD SR5 for S. rolfsii isolates. The isolates of M. phaseolina and S. rolfsii were periodically subcultured in fresh sterilized PDA plates for the maintenance of their axenic cultures. Similar isolation technique was used by Raja Mohan et al. (2012) for the isolation of M. phaseolina. The stem rot pathogen, Sclerotium was isolated by tissue segmentation method from the symptomatic stem parts by Archana et al. (2020). Colony and Morphological characteristics of M. phaseolina. The colonies of isolates of M. phaseolina were compact to fluffy in nature, the colour of the isolates varied from brown to black to olivaceous grey (Fig. 1). Similar observations made by Sarr et al. (2014) reported that the colony of M. phaseolina was fluffy, initially buff later turned to pale olivaceous grey and sclerotium were numerously produced. Dark brown to greyish coloured colonies were produced by M. phaseolina isolates on PDA medium (Pandey et al., 2020). The mycelium of all the isolates were observed under phase contrast microscope. The colour of the mycelium varies from light brown to dark brown and septations were present in all the isolates (Fig. 2). Emayavarman et al. (2019) reported the black coloured mycelium was produced by M. phaseolina and under microscope the mycelium was coloured and septate. The hyphae of M. phaseolina were found to be hyaline, with thin walls and the colour ranges from light brown to dark brown with more septa reported by Lakhran et al. (2018). The microsclerotium were present in abundant numbers in culture plates of M. phaseolina and the colour of microsclerotium varies from dark brown to black and oval, spherical and irregular shapes of microsclerotium were also observed in different isolates (Fig. 3). Similarly, Marquez et al. (2021) described that themicrosclerotia were spherical, oval, or oblong masses of hardened fungal mycelium that are initially light brown in colour later turns to dark brown to black. Similarly, Sarr et al. (2014) reported that black coloured sclerotia were produced by M. phaseolina. TNAU MP1 isolate produced larger sized microsclerotium of an average size of 84.896 µm and the growth of TNAU MP1 was also rapid at a growth rate of 36.68 mm/day compared to other isolates (Table 2). Raja Mohan et al., (2012) recorded that size of microsclerotium produced by M. phaseolina MP5 was 85.70 µm. The size of the microsclerotium for 64 isolates of M. phaseolina was recorded as 72.8 to 127.8µm by Manici et al. (1992). Lakharan et al. (2018) have reported that the size of microsclerotium varied from 82.5 to 105 µm. Colony and Morphological characteristics of S. rolfsii. The colonies of S. rolfsii were sparse to fluffy in nature and spreading radially in vein like manner. The mycelium were initially resembled silk white threads later turned to cotton white colour with sparse to fluffy growth (Fig. 4). Mahadevakumar et al. (2018) reported that the colonies of S. rolfsii were white in colour with dense, fluffy growth. Sivakumar et al. (2016) investigated the colony characters of S. rolfsii and reported that the colonies were initially silk white which later changed to dull white colour and profuse mycelium which grow radially to give fan like appearance. The mycelium of different isolates were hyaline and highly septate when observed under phase contrast microscope (Fig. 5). The sclerotium of all the isolates were observed in naked eye and were dark brown, reddish brown, golden brown and black colours (Fig. 6). The size of the sclerotium for different isolates was varied from 480.95 µm to 778.42 µm. The isolate TNAU SR1 was found to produce sclerotium on 5 days after inoculation (DAI). The isolates DPI SR2, SLM SR4 and CUD SR5 produced sclerotium on 7 DAI and KRI SR3 isolate produced sclerotium on 8 DAI. The number of sclerotium produced by different isolates also varies from 77.68 to 434.22 on 9 DAI in which TNAU SR1 isolate produced the higher number of sclerotium and the mycelial growth is also rapid at a growth rate of 33.70 mm/day (Table 3). Pandi et al. (2017) reported that light brown, dark brown and reddish brown coloured sclerotia were produced by 8 isolates of S. rolfsii and the size of the sclerotium varied from 1002 to 1224 µm. The number of sclerotium produced by eight isolates ranged from 274 to 360/plate and the isolates grown with a growth rate of 21.62 to 31.45 mm/day. Sarma et al. (2002), reported that the colonies produced by 18 isolates of S. rolfsii were fluffy, 8 isolates were compact with the growth rate of 23-31 mm/day and the no. of sclerotium produced were 80 to 500 sclerotia/plate. The average diameter of the sclerotium was recorded as 1 – 1.2 mm and were dark to reddish brown in colour. Molecular characterization. The DNA of all the isolates of M. phaseolina and S. rolfsii were extracted separately and they were subjected to PCR and agarose gel electrophoresis was also performed for each isolate to find the amplicon size and were visualized under UV illuminator. All the M. phaseolina isolates were amplified and the amplicon were visible at app. 550 – 560 bpand for all the S. rolfsii isolates the amplicon were visible at app. 650 – 700 bp. The positive control with fungal DNA shown amplicon and in the negative control with bacterial DNA amplicon was not obtained. This confirms that the pathogens were fungi. The DNA of four isolates of M. phaseolina were extracted and 560 bp amplicon size were obtained in agarose gel electrophoresis in ITS 1&4 primers (Emayavarman et al., 2019). Similarly, Chakraborty et al. (2011) also obtained an amplicon size of app. 550 bp for the M. phaseolina isolates in ITS 1&4 primers. In case of S. rolfsii, Jebaraj et al. (2017) observed the amplicon size of 650 – 700 bp in ITS 1&4 universal primers. Similar results were also reported by Durgaprasad et al. (2008). Yu et al. (2019) extracted DNA of S. rolfsii on PCR and agarose gel electrophoresis reported that it was amplified at an amplicon size of 683bp in ITS 1&4 universal primers.