INTRODUCTION Potato (Solanum tuberosum L.) is one of the most popular vegetables in the world, belonging to the Solanaceae family. There are nearly 200 different wild potato species, each with its own unique flavour and nutritional value. Potatoes are the fourth most commonly grown food crop in the world, after maize, wheat and rice. They are available throughout the year, and can help to meet nutritional needs in people who are deficient in these nutrients. It is also largely rich in antioxidants such as vitamin A, B1, B6, C and carotenoids (White et al., 2009). Phytophthora infestans (Anton de Bary) is the most destructive oomycete pathogen that causes late blight disease in potato and was responsible for the Irish famine in the 1840s (Montarry et al., 2008; Chowdappa et al., 2015). This pathogen is a heterothallic and hemibiotrophic in nature. The pathogen is sensitive and short-lived, and it destroys the plants it infects within two weeks under favourable conditions (Fry et al., 2008). The pathogenesis comparatively sensitive, short lived in infected potato leaf tissue, and the pathogen seems to possess a limited competitive saprophytic ability. In these aspects, the isolation and culturing of pathogen in artificial medium is relatively difficult. Several culture media have been used in various countries to isolate and maintain P. infestans isolates. It is very essential to supplement the necessary elements and compounds required for the growth and metabolism of the pathogen in the medium (Padmaja et al., 2015). Thus, different synthetic media were used in this study to see which was the best medium for the growth of P. infestans. Symptoms of the disease appear near the leaf tips as water-soaked irregular pale green lesions, which rapidly grow into large brown to black necrotic spots on the upper surface of leaves, and white hyphae growth can be seen on the lower surface of infected leaves (Mhatre et al., 2021). The infection on the stem appears as a brown lesion around the stem, eventually leading to collapse. In severe cases, the disease can affect the entire crop in a week and transform it into a dark blighted crop. The disease also affects tubers, making them hard, dry and rigid, resulting in rotting in the field and in storage (Agrios, 2005; Arora et al., 2014). In India prior to 2006, late blight was seen as a frequent threat to potato cultivation in the North Indian states (Chowdappa et al., 2011). Since 2008, severe outbreaks of the disease have been seen in South Indian potato crops, resulting in total crop loss (Jain et al., 2019). In addition, many diagnostic laboratories are not familiar with oomycete culture procedures, and the rapid growth of bacteria from leaf samples with secondary bacterial infection usually limits the isolation of P. infestans (George et al., 2017). Plant pathogenic oomycetes can be rapidly and efficiently isolated from infected plant tissues using antibiotic-enriched selective media following proper laboratory procedures. The selective media used for the isolation of oomycetes pathogen often contain antibacterial agents such as penicillin, ampicillin and rifampicin which prevent the development of bacterial growth and fungal growth. The molecular characterization of P. infestans by using the internal transcribed spacer regions (ITS) of rDNA may be helpful for easy detection and confirmation of species and the assessment of genetic variability among isolates. The main focus of this study is to i) isolate the pathogen from the infected leaf tissues, and ii) characterize the isolates of P. infestans with respect to their morphological and molecular features. MATERIALS AND METHODS Survey and sample collection. A roving field survey was conducted in major potato growing areas such as, Muthorai, Nanjanad, Wellington, Emerald and Palada in the Nilgiris district, Indiaduring Rabi 2021-22 to assess the severity and incidence of late blight disease in potato. Two fields were selected in each place. Totally, ten fields were surveyed at the time of survey, late blight infected leaves and stem samples were collected for pathogen isolation and characterization. The disease incidence in each field was determined by visually inspecting the plants and cutting two diagonal transects across the field (number of infected plants and healthy plants along each diagonal). Isolation of P. infestans and pure culture maintenance. The plants exhibiting typical symptoms of late blight were collected from the field during the survey and brought to the laboratory. Tissue segment method (Vasudeva et al., 1958) was employed for isolation of P. infestans on clarified V8 PARP medium supplemented with antibiotics such as pimaricin (400µl), penicillin (50µg/ml), ampicillin (250mg/ml), and rifampicin (100mg/ml) to suppress the growth of other microbes (Jadesha et al., 2020). Liquid soap was used to gently remove all soil-associated surface impurities from infected leaf samples. Small pieces (5×5 mm) of infected leaf tissues from the sporulating border of young lesions including a little bit of healthy leaf tissue was dissected using sterile scalpel. Leaf bits were placed in 70% ethanol for 1 minute followed by 4% sodium hypochlorite solution for 60 seconds for surface sterilizing the tissue then rinsed thrice with sterile distilled water in laminar airflow chamber. The excess moisture was blotted with sterile Whatman filter papers. The sterile leaf bits were placed in half plate containing selective medium, clarified V8 PARP supplemented with combination of antibiotics. Inoculated plates were incubated at 20°C for 7-10 days under 12 hours of photoperiod in BOD (Martin et al., 2012). Phytophthora infestans was initially identified based on mycelial and morphological characteristics using standard mycological keys. The pure fungus culture was obtained using the single hyphal tip method (Tutte et al., 1969), which involved transferring the upper portion of actively growing mycelium from a 10-day-old culture into a 1.5ml microfuge tube containing 1 ml of sterile water. On the surface of V8 juice agar plates, three to five drops of suspension were added. The single hyphal bit was forced to ramify through the agar using this technique. Plates were examined under a dissecting microscope at 10x magnification after 5 days, and a single hyphal branch was transferred to V8 juice agar plates. On the surface of V8 juice agar plates, three to five drops of suspension were added. The single hyphal bit was forced to ramify through the agar using this technique. Plates were examined under a dissecting microscope at 10x magnification after 5 days, and a single hyphal branch was transferred to V8 juice agar plates. Pathogenicity assay under controlled conditions. Pathogenicity study of all four isolates (PPI1, PPI2, PPI3 and PPI4) was performed under controlled conditions on the highly susceptible cultivar Kufri Swarna. Inoculation of P. infestans four isolates were done by spraying zoospore suspensions (Becktell et al., 2005). Five 7mm mycelia discs of each isolate were placed in 90mm Petri dishes containing 15-20ml of diluted tomato juice broth. The cultures were allowed to grow for 24-48 hours at 20°C under continuous light conditions (Widmer et al., 2009). Zoospores were released from the sporangia after the cultures were refrigerated for 30 minutes and then incubated at room temperature. The released zoospores were diluted to the final concentrations. The motility of zoospores was verified under compound microscope. All the potted potato plants were inoculated by spraying a zoospore suspension (10,000-20,000 sporangia/ml) with handheld sprayer. Spraying of sterile water serves as control kept separately (Wang et al., 2020). The plants were watered daily for 3-4 times at 3hours interval. Each isolate containing 3 potted was considered as one replication and was replicated five times. Initial symptoms were observed after 48-72 hours after inoculation (Pande et al., 2011). Disease assessment. Late blight incidence was assessed using the formula (Ahmed et al., 2015) as mentioned below. "Percent disease incidence =" "Number of infected plants" /" Total number of plants observed" × 100 The pathogen was re-isolated from the artificially inoculated potato plant shown typical late blight symptoms and the culture was confirmed with the original culture for morphology and colony characters. To evaluate the virulence of four isolates, per cent disease incidence was calculated and virulent isolate was used throughout the research. Morphological characterization Sporangial induction and zoospores production. Six day old culture of P.infestans (PPI3) was taken for induction of sporangia and zoospores. Mycelial discs of 9mm diameter of P. infestans were cut using sterile cork borer from the edge of 5 days old culture grown on clarified V8 juice agar and inoculated into 90mm diameter to different Petri dishes flooded with 10-15 ml of 1% non sterile soil extract, tap water, distilled water, rain water, pond water (Malvi et al., 2022) and diluted tomato juice broth (Jadesha et al., 2020). The Petri dishes were kept for 48-72 hours at 19-22°C under light conditions. Consistent light condition and non-sterile liquids may promote the sporangial formation. Then, the plates were observed under stereo zoom microscope Induction of sporangia was initiated and abundant production was observed within 48-72 hours primarily towards the margin of the colony. The sporangial suspension was incubated at 4°C for 1 to 2 hours to release of zoospores from the sporangia (Widmer et al., 2009). Micro morphology. All the four isolates were isolated aseptically on the V8 juice agar plates and incubated at 21±2°C for 7 days and cultural characteristics such as colony pattern, growth rate and colony color were observed after 15 days of incubation (Vanegas et al., 2022). Mycelial characters and fungal morphology as described in were observed under LEICA DM2000 phase contrast microscope at 40X magnification of objective lens. Macro morphology Colony pattern. Twelve different culture media viz., Carrot agar, Clarified V8 juice agar, Corn meal agar, Czapek’s Dox agar Glucose asparagine agar, Potato carrot agar Oat meal agar, Potato dextrose agar, Potato leaf extract agar, Richard’s synthetic agar, Saboraud dextrose agar, Tomato juice agar were prepared in accordance with the manufacturer's (HiMedia, India) specifications and compositions (Dhingra and Sinclair, 1985). Mycelial discs of 7mm diameter were taken from ten days old pre-cultured Petri plates by using sterile cork borer and placed in all the Petri dishes containing twelve different media. Three replications in each media were maintained and the mean mycelial growth was recorded. The plates were incubated at 19±2°C in 12 hours dark and 12 hours light conditions for 7-10 days. This study was conducted for evaluating the best culture media for the radial growth of P. infestans using the twelve different solid media (Rangaswami et al., 1958). The average colony diameter of the fungal growth was measured in millimeters (Jadesha et al., 2020). Molecular characterization DNA extraction. Isolates of P. infestans were cultured on oat meal agar for 7-10 days. Mycelia were scrapped from the plates and ground at liquid nitrogen. Extraction of DNA is done by cetyl trimethyl ammonium bromide (CTAB) procedure (Wangsomboondee et al., 2002). The frozen mycelia were added with 750µl CTAB buffer and transferred the extract into 2ml centrifuge tubes. Then the tubes were vortexed and incubated in water bath at 65°C for 30 minutes. After incubation, 750µl of phenol: chloroform: isoamyl alcohol (25:24:1) was added to each tube and centrifuged at 13000 rpm for 15 minutes at room temperature. The uppermost aqueous phase was removed to a new tube and ice cold isopropan-2-ol (600µl) was added and tubes were incubated at -20°C for overnight incubation. After incubation, tubes were centrifuged at 13000 rpm for 15 minutes. The supernatant was discarded and pellets were washed with 70% ethanol, then air dried. DNA was suspended in 30µl TE buffer and stored in -20°C. DNA concentration was checked with 0.8% agarose gel for one hour. PCR amplification. The identification of Phytophthora was confirmed later by performing ITS-PCR assay. Employing the universal primer pairs ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’), amplified the ITS regions between small nuclear 18S rDNA and the larger nuclear 28S rDNA, including 5.8S rDNA (Hussain et al., 2014). PCR was performed in a volume of 25µl, containing 10mM of oligonucleotide primer (1 µl), 20mM of each of the four deoxynucleotide triphosphates (0.75 µl) 25mM MgCl2 (1.0 µl), 0.35 µl of Taq DNA polymerase, 10 × Assay buffer (2.5 µl) and 1.0 µl template DNA (60 ng/µl) (Nath et al., 2015). All PCR reactions were carried out using Mastercycler® Nexus gradient X2 PCR cycler (MA, USA) with the following PCR conditions, initial denaturation at 94°C for 3 min, 35 cycles of amplification with denaturation 94°C for 30 sec, annealing at 56°C for 1 min and 72°C for 2 min for extension with final extension 72°C for 10 min (George et al., 2017) and the PCR products were checked for amplification by 1.0 percent agarose gel electrophoresis. The UVITEC Gel doc EZ Imaging system was used to view the size of amplicons and sanger dideoxy sequencing was done to analyze the nucleotide sequence of DNA. The resulted high quality sequences were analyzed with NCBI BLASTn to confirm the authenticity of isolates. Statistical analysis. The obtained data were statistically analyzed by IBM SPSS Statistics 22. Duncan’s multiple range test and post hoc tests were performed at 5% level of significance (Gomez et al., 1984). RESULTS AND DISCUSSION Survey and assessment of potato late blight incidence The survey results have shown that the disease was prevalent in all the surveyed areas with different magnitude of infection. The small circular to irregular-shaped water-soaked spots were observed on leaf surface with whitish mildew-appearing area is visible at the edge of the lesions or along petioles. The maximum disease incidence was recorded in Muthorai (95.63%) followed by Palada (87.29%) and Emerald (82.56%). The minimum disease incidence was recorded in Nanjanad (59.37%) followed by Wellington (69.01%) have shown in Fig 1. Isolation of P. infestans Mostly Phytophthora spp. was hard to isolate, especially from infected potato leaf tissues due to the hasty growth rate of other saprophytic fungi according to Henricot et al. (2014). Successful isolation of Phytophthora spp. can be accomplished by the use of selective media that either impedes the development of other fungus and bacteria. Five different media viz., potato dextrose agar, corn meal agar, carrot agar, lima bean agar, and potato leaf extract agar were screened for isolation of P. infestans from the infected leaf bits (Fatima et al., 2015). In the present study, among all the media tested, V8 PARP (V8 juice amended with pimaricin, ampicillin, rifampicin, penicillin) medium was found effective in supporting the growth of P. infestans which minimized the bacterial contamination by the presence of antibiotics supplemented in the medium. The results of the present study was in compliance with findings of (Jadesha et al., 2020). Totally, four isolates (PPI1, PPI2, PPI3 and PPI4) were identified form this study (Table 1). Morphological characterization Evaluation of different media on the growth of P.infestans. The suitable culture media and physiological conditions for mycelial growth was evaluated by placing of 7 mm mycelial discs into twelve different culture media (Table 2). Among twelve different media evaluated against P. infestans, the maximum mycelial growth of pathogen was observed in clarified V8 juice agar (90 mm) followed by carrot agar (89 mm), potato dextrose agar (88 mm), potato carrot agar (79.30 mm), and potato leaf extract agar (70.30 mm). The results are in agreement with George et al, (2017) who found the suitability ofV8 juice agar for culturing the P. infestans. The minimum mycelial growth was noticed in glucose asparagine agar of 22.70 mm followed by Richard’s synthetic agar of 31 mm was recorded. The obtained results were similar to findings of (Henricot et al., 2014) that recorded maximum growth of P. pachyplura was noticed in V8 juice agar of 90mm and in potato dextrose agar (84 mm). The least mycelial growth was recorded in glucose asparagine (41.80 mm) was observed in P. parasitica (Prasad et al., 2017) which is similar to that of our results. Colony pattern. In the present study, four isolates were obtained from the infected potato leaf and stem samples collected from the Nilgiris district. The colony colour of P. infestans was observed cottony white to dull white with rosette pattern. Mycelium was long, aseptate, hyaline, sympodial branching pattern, uniform in diameter. The mycelium was too sensitive and had a watery nature. The pathogen growth was achieved in transparent media. So, thin film of media is enough for establishment of the pathogen under artificial conditions. The sporulation was not observed in culture media. The sporangia were observed in 1% soil suspension with mycelial discs which were incubated for 48-72 hours (Mehmood et al., 2022). The lemoni form sporangium with semi papilla was seen under stereo zoom microscope. The limitations in availability of nutrients and temperature might be the reason for lack of sporulation in Phytophthora spp. On the basis of the study of cultural and morphological characteristics, it was concluded that the species closely resembled to P. infestans (Prasad et al., 2017). However sporulation was not observed in any of the medium tested. Sporulation of P. infestans. The study showed that different methods given by research workers were tried for induction of sporangia of P. infestans and results were given in Table 3. The result revealed that out of six methods tried, diluted tomato juice broth produced more amounts of lemon shaped sporangia with semi papilla and oospores also observed under stereo zoom microscope. The obtained results were similar to that of Jadesha et al. (2020). The sporangial production was formed in a lesser amount in 15 ml of rain water and river water. Non-sterilized liquids and non-sterile soil extracts may induce sporangial development in P. infestans (Fig. 2). Molecular characterization of P. infestans. The internal transcribed spacer (ITS) regions of 18S rDNA were amplified by employing primer pair ITS1 and ITS4 (Cespedes et al., 2013). The initial identification was confirmed by the amplicon size of 850 base pairs in all the four isolates (Fig. 3). Similarly, George et al. (2017) obtained the amplicon size of 850 base pairs in Phytophthora isolates. BLASTn analysis revealed that the sequence of ITS regions of all the four isolates were shown 100% sequence homology with sequences of P. infestans in GenBank, NCBI and the nucleotide sequences were submitted and accession numbers were assigned by NCBI GenBank. Pathogenicity test. Twenty five days old potato plants of highly susceptible variety (Kufri Swarna) was artificially inoculated with P. infestans by spraying zoospore suspension method. The first symptom of late blight disease was recorded during the pathogenicity test was almost identical to the symptoms at open field conditions. Initially, a small water soaked lesion of late blight disease was appeared on upper leaf surface during third day after inoculations. The infection progressed and lesion became enlarged with whitish downy growth on its lower surface after fifteen days from inoculation. The fungus was re-isolated from the infected leaves were compared with original culture of P. infestans. Jadesha et al. (2020) observed the symptom development in redgram after 48 hours of inoculation with P. infestans pathogen. Virulence of P. infestans isolates Artificial inoculation was employed for assessing the virulence of four isolates viz., PPI1, PPI2, PPI3, PPI4 in potato plants (Kufri Swarna) and the results were presented in Table 4. It was found that PPI3 was most virulent with disease incidence of 95.30 percent on 20th day after inoculation followed by PPI1 (81.05 percent). The least virulent with disease incidence was found in PPI4 and PPI3 with percent disease incidence of 73.14 and 66.30 percent, respectively. Hence, the isolate PPI3 was used throughout the study. The infective potential of zoospores and concentrations were observed more in PPI3 when compared to other isolates. The results obtained from this study were similar to that of Widmer et al. (2009).