INTRODUCTION Pearl millet (Pennisetum glaucum (L.) R. Br.) is one of the most widely grown arid and semi-arid crops in India and African sub-continents after rice, wheat and sorghum. It has a good energy source of carbohydrate, fats(5-7%), protein (9-13%), fibre (1.2 g/100 g)and contains antioxidant such as coumaric acids which helps in better digestion (Patni and Agrawal 2017). Pearl millet blast which was originally documented in 1942 from Kanpur, UP, India (Mehta et al., 1952) has become a serious threat in the last decade causing rampant in India's pearl millet-growing areas. In India, the incidence of Pearl millet blast (Magnaporthe grisea) disease, which was once considered a minor disease, but now it has been increased at an alarming rate, primarily on commercial hybrids, in numerous states (Thakur et al., 2009). Severe outbreaks of Pyricularia leaf spot known as blast disease was reported in major pearl millet producing states, including Gujarat, Rajasthan, Uttar Pradesh, Madhya Pradesh, Karnataka and Delhi were shown an upsurge in the occurrence of this disease both inpearl millet leaves and grains (Timper et al., 2002). The forage production of pearl millet (cumbu) causing chronic reductions in the last few years (Wilson and Gates 1993). The quality and productivity of the pearl millet crop are impacted by Magnaporthe blast, which has been found to be negatively connected with green-plot yields, dry matter production and digestion dry matter yield. The severity of the blast disease is exacerbated by humid weather conditions and dense plant stands. The fungus may infect plants at any stage of development, from seedling to adult, lowering grain and forage yields in varying degrees with occasionally dramatic negative consequences. The symptoms of Magnaporthe blast in pearl millet are most generally known as grey leaf spot. Initially, the symptoms appear as minute lesions or specks that expand and develop, necrotic elliptical greyish brown lesions, leading to extensive chlorosis and premature drying of leaves. The typical symptoms of the plants infected by Pyricularia pennisetigena showed small, brown, pinpoint, elliptical lesions with greying of the center in the later stages (Martinez et al., 2021). The M. grisea group is specific in its host range, but it is highly versatile in its ability to adapt to new environments. Molecular identification of the pathogen, which has shown to be a solid alternative to older approaches, is one of the rapid and easy ways for detecting the species diversity of fungal communities. To distinguish between the isolates, morphological characters and molecular by through internal transcribed spacer sequencing by using primers ITS 1 and ITS 4 were performed. The main intention of this research was to analyse different morphological characters of the fungi and confirming the pathogen using both morpho and molecular methods. Since, infectivity of the pathogen Pyricularia pennisetigena on pearl millet is not reported in Southern parts of India, this study paved a way for better understanding of the pathogen characters. MATERIALS AND METHODS Collection and Isolation of the blast pathogen: Leaves with typical spindle shaped lesions, associated with blast disease were collected from the pearl millet field from 8 districts of Tamil Nadu which were designated with the code as PMBT (Tirupur), CO-10 (Coimbatore), DP1 (Dharmapuri), KR2 (Krishnagiri), ERT (Erode), ALR (Ariyalur), SLM (Salem),and TVM (Thindivanam). The pathogen was isolated on potato dextrose agar medium. Infected leaves were washed using sterile water and the infected leaf portions were cut into small bits using sterile scalpel blade. Then the leaf bits were sterilized using 1% sodium hypochlorite solution for about 30 seconds followed by washing in sterile distilled water for three times and placed in Whatman no. 40 filter paper. Using sterile forceps the leaf bits were placed in petri plates containing sterilized PDA medium which was amended with streptomycin (50μg/ml) under aseptic conditions. The plates were incubated at room temperature (25oC). After initiation of growth is observed in the petri plates, single hyphal tip was transferred into new petri plate containing 15ml of sterilized PDA medium to obtain the pure culture of the fungi. For sporulating the fungus, the stem of the main host were cut into small bits (3-5 cm) and added to conical flasks, sterilized in an autoclave at 121oC for 20 minutes at 15 lbs pressure. The mycelial disc (9mm) of 7 days old culture was added to each flask. After 15 days the stem bits were taken out under sterile conditions and added to eppendorf tube containing distilled water, shaked vigorously to dislodge the spores and the conidial morphology was observed under the light microscope (Vanaraj et al., 2013). For additional research, the culture was maintained on PDA slants at 4 °C and stored at -20 °C for medium to long term preservation. Examination of cultural and morphological characteristics. Pearl millet blast infected isolates were grown at 25°C for 7 days on PDA medium, following which mycelium discs were transferred to the middle of a fresh PDA medium, within 10-14 days, fungal mycelium had covered the whole plate. All of the isolates on PDA media were evaluated daily for 5 to 10 days for their colony shape (morphology), texture and colour. The mycelial characters, colour and septations were observed. Pathogenicity assessment under in vitro and glasshouse conditions. Fresh leaves were collected and washed under running tap water. The leaves were then surface sterilized with 1% sodium hypochlorite and subsequently washed with sterilized distilled water for three times and placed on sterilized tissue paper for drying. The leaves were placed in petri plates which has been basally layered with cotton on which 4-5 drops of sterile water was added to maintain humidity, the leaves were placed on blotter paper placed above the cotton which was cut to fit the shape of the petri plate. A 9 mm actively growing mycelial disc was placed on the centre of the leaf. The plates were covered and incubated at a temperature of 22-24o C. After 7-10 days, the lesions were developed on the region around the disc. For conducting the pathogenicity assay under glass house conditions, Pearl millet seeds were sown in plastic pots filled with a combination of three parts soil to one part farmyard manure. The isolates grown on PDA plates with actively growing fungal mycelia were flooded with 10-15 ml of sterile water and fungal mass containing mycelia were scrapped with sterile scalpel blade. It was filtered through a muslin cloth and concentration of spores were adjusted using haemocytometer to 5 × 104 conidia/ml. Tween 20 (0.02%) was added as an adhesive agent to spore suspension before inoculation. Inoculation was performed on test seedlings at the three to four leaf stages (about 15 days old plants) by spraying the inoculum till run-off under glass house conditions. Polythene bags were placed over the infected plants for 24 hours. Following the incubation phase, the test plants were watered on a daily basis to maintain high humidity levels favourable for disease development (>90% RH). Symptoms occurred 5-7 days post-inoculation. Isolation and quantification of genomic DNA. Total DNA was extracted by using modified CTAB method. Isolates PMBT and CO-10 were cultured in PDA plates and 5 days after the cultures were transferred to potato dextrose broth (100 ml). The fungal mycelia (100-150 mg)were collected and ground to fine powder in a pestle and mortar by using liquid nitrogen. The ground and powdered mycelium was transferred to an Eppendorf tube (2 ml) which consists of a mixture of 1 ml of extraction buffer consisting of (2% CTAB buffer, 4M NaCl, 0.5M EDTA, 1M Tris-Cl, 0.02% β-Mercapto- ethanol). After 1 hour of incubation at 65°C, an equal amount of phenol, chloroform, and isoamyl alcohol (25:24:1) was added and the mixture was centrifuged at 12000 rpm for 10 minutes. After mixing well, the clear supernatant was put into an Eppendorf tube and an equal amount of chloroform and isoamyl alcohol (24:1) was added. This mixture was mixed and the tube was spun at 12000 rpm for 10 minutes. Then, ice cold isopropanol was added into the supernatant, mixed thoroughly and refrigerated at -20°C for overnight. After centrifugation the supernatant was discarded and the DNA pellet was washed with 100% ethanol, air dried after centrifugation again and resuspended in 100 μl sterile distilled water. The quality and amount of fungal DNA were determined using gel electrophoresis and the Nanodrop Spectrophotometer. For use in the PCR process, the DNA samples were diluted to a concentration of 30-50 ng/μl after nanodrop quantification. (Amplification via polymerase chain reaction and documentation) (Doyle et al., 1990). Amplification via polymerase chain reaction and gel documentation. With the help of fungal universal primers, the ITS region of rDNA was successfully amplified using ITS 1 ((5'-TCCGTAGGTGAACCTGCGG-3') and ITS 4 (5'-TCCTCCGTTGATATGC-3') primers (White et al., 1990). Amplification of genomic DNA was carried out using a thermocycler (Master Eppendorf) using the following PCR conditions. The reaction mixture consisting of 10 μl of master mix, 4 μl of sterile water, each 2 μl of forward and reverse primers and genomic DNA 2 μl was used for the amplification process. The PCR was performed by initial denaturation at 94°C for 5 min which is followed by denaturation at 94°C for 60 sec, annealing at 58°C for 60 sec, extension at 72°C for 60 sec followed by a final extension at 72°C for 10 min for over 35 cycles (White et al., 1990). Agarose gel prepared at a concentration of 1.2 percent of agarose in TAE (tris acetic acid) buffer containing ethidium bromide at a concentration of 2μl/100 ml and allowed to solidified to form agel with place of comb to form a well. The amplified PCR products were loaded in the wells and run by electrophoresis at a voltage of 80 for 1 hour followed by documentation and assessment using a gel documentation Transilluminator system (GelDoc, BIO-RAD, Canada). The PCR product size was compared to the standard 1kb ladder. The DNA got amplified around the region of 560 bp. ITS region sequencing and phylogenetic tree construction. The PCR results were subsequently sequenced to authenticate the species. The resulting nucleotide sequences were analysed by using BLAST. Accession numbers were obtained after depositing the isolates in Gen Bank. Mega X Software was used to align the sequences of the isolates, and similar ITS1-ITS4 sequences were obtained from the NCBI gen bank in order to discriminate between the isolates. The neighbour-joining technique was used in the construction of a phylogenetic tree (Kumar et al., 2016). The resilience of clades was assessed using a 1000 bootstrap replication. RESULTS AND DISCUSSIONS Collection and Isolation of Magnaporthe grisea. Blast infected pearl millet leaf samples were collected from eight districts of Tamil Nadu among which the virulent isolates were pertaining to the samples collected from Tirupur and Coimbatore district (Table 1) (Fig. 1). The samples were collected based on the symptoms such as typical eye shaped lesion with dark brown borders (Yi et al., 2022). The leaf blast prevalent in hot western arid regions of Rajasthan were found to belong to the species Pyricularia pennisetigena which was validated from multiple sequence alignment verified from the deposited sequences in USA (Solanki et al., 2022). The host physical stimulus and hydration plays a key role in appressorium development of the pathogen (Sharma et al., 2019). The diseased leaf samples were isolated on PDA medium and incubated at 25oC (Fig. 2). The pure culture of the pathogen was obtained by single hyphal tip method. For sporulating the pathogen, stem bits of the host were autoclaved and the mycelial discs of the pathogen were inoculated. After 2 weeks the conidia were observed on the surface of the stem bits (Fig. 3). Magnoporthe grisea infects leaves with spores, the pathogen's most crucial stage for disease emergence, which tend to germinate on a hydrophobic leaf surface. After adhering firmly to the plant's surface, the fungus produces immense pressure within the melanized appressorium and enters the leaf's epidermal cell using a small penetration peg (Heath et al., 1992). The culture was kept on PDA slants and stored at 4 °C and -20 °C, respectively, for the course of the experiment. Examination of cultural and morphological characteristics. The mycelial characters of the fungi including the colour, colony characters and morphology (pattern) on PDA medium were observed. The radial growth and texture of the colony varied substantially for the isolates. The growth and colour of the mycelia ranged from greyish white to blackish grey in colour on PDA medium (Table 2). On potato dextrose agar, blast fungus had greyish colonies with smooth circular margins and concentric rings (Sonah et al., 2009). The pathogen was found to be Magnaporthe based on its conidial structures and colony characteristics (Ou, et al., 1985). The fungal pathogen identified as M. grisea in this investigation was based on the main morphological and cultural features described by (Bourett et al., 1990). The morphology of pathogenic cultures varied from one culture to others and based on medium also. The quantity of aerial mycelium varies greatly, ranging from a thin to a dense cottony mass. The cultural and morphological differences revealed among the pearl millet blast pathogen isolates matched prior results on P. grisea made by other researchers investigating on the same topic (Srivastava et al., 2014). The spores were examined under the microscope. The shape, presence of septation, hilum etc. were observed. The isolate's conidiophores were discovered to be thin and straight, containing clusters of conidia that were generally pyriform or obclavate and 2-3 septate (Getachew et al., 2014). The pyriform shape of the conidia indicates that Pyricularia grisea is an asexual anamorph of the Magnaporthe isolate. Pathogenicity assessment under in vitro and glasshouse conditions. The virulent isolate among the two was finalized as virulent based on the assay conducted under in vitro in which fresh untreated leaves of the host were taken. The leaves were surface sterilized using 1% sodium hypochlorite solution followed by washing with sterile distilled water 3 times. The 9mm mycelial disc of 7 days old culture was placed in the leaf surface. The lesions were formed around the mycelial disc after a week (Fig. 4). The pathogen becomes necrotrophic at 48 hours after inoculation by producing thin-invasive hyphae which is followed by the development of peg which differentiates into lobed- bulbous infectious hyphae that will expand intracellularly and intercellularly causing blast lesions (Howard et al., 1996). For conducting the pathogenicity test under glass house conditions, seeds were sown in pots containing mixture of FYM and soil. The spore suspension was sprayed at a rate of 5 × 104 conidia/ml with tween 20 (0.02%) which was used as an adhesive (Fig. 5). Based on the symptom development the isolates PMBT and CO-10 showed virulent nature compare to other isolates. In accordance with Koch's postulate, pathogens were determined to be related with disease at all stages of crop development and were able to produce disease when re-inoculated into the same host. Hence study was conducted by testing the pathogenicity of the detected field strain on the same pearl millet variety from which the organism was obtained. The re-isolation of the pathogen was done and it was found to be the same kind as the previously isolated one. Thus, Koch's postulate was demonstrated. Molecular characterization of the pathogen: Amplification via polymerase chain reaction and gel documentation. DNA extraction, PCR amplification using ITS PCR primers, and Sanger sequencing were used to identify and validate the molecular identity of the field sample. ITS rDNA sequencing and molecular analysis were done. Two isolates viz., PMBT and CO-10 which were virulent among others were opted for performing molecular approaches. Molecular detection of Pyricularia grisea and Pyricularia pennisetigena using the primers ITS 1 and ITS 4 for amplification of isolates were done (Fig. 6). The amplification values were found to be approximately 560bp which was similar to our experiments (Chuwa et al., 2013). The amplicon of size 520 bp was observed in course of amplifying, Magnaporthe oryzae isolates from Eastern India using universal primers ITS 1 and ITS 4 (Priyanka et al., 2021). To confirm the pathogen at species level, the PCR products were purified up and sequenced. ITS region sequencing and phylogenetic tree construction. Phylogenetic relationship of the isolates with other nucleotides available in NCBI are done to know the category in which the isolates are genetically related with (Fig. 7) Isolates of Pyricularia infecting different hosts were taken for phylogenetic analysis (Klaubaf et al., 2014). The Pyricularia grisea isolate showed 98.90% and Pyricularia pennisetigena isolate showed 100% identity when compared with sequences available in NCBI and these isolates sequenced were deposited NCBI genbank (Accession number- Pyricularia grisea -OM883863 and Pyricularia pennisetigena ON116174) (Table 3). Phylogenetic tree constructed by using MEGA X software revealed that the isolate Pyricularia grisea showed 26% similarity and Pyricularia pennisetigena showed 91% similarity with the isolates in various clades taken for phylogenetic tree construction.