INTRODUCTION Okra (Abelmoschus esculentus (L.) Moench), generally known as lady's finger or bhendi, is a popular vegetable all over the world. It is a member of the Malvaceae family. It is a major vegetable crop in India, and its notable varieties are commercially farmed in many areas of the world for its delicious taste and high nutritional content (Benchasri, 2012). In India, a variety of fungal, bacterial, and viral diseases have been recorded causing damage to the bhendi crop. Diseases are a key limitation to lower the bhendi yields (Sastry and Singh, 1974). Among them, powdery mildew is one of the most destructive disease and cause 65% yield loss. The genera of different powdery mildew fungi, obligate biotrophic pathogens are causing infection more than 10,000 host plant species. To protect the crops from powdery mildew infection, fungicides are indiscriminately applied frequently with inadequate dosage which cause fungicide resistance against powdery mildew fungi. In addition to that the fungicide are causing harmful effect on biodiversity, natural ecosystem and possess the residual problem (Maurya et al., 2013). Physical and biological approaches have been proposed to support and replace chemical management of powdery mildews. Mycoparasites (fungi that parasitize other fungi) are abundant in the majority of terrestrial environments, with the most well-known species attacking fungal plant diseases (Kiss 2003). Numerous mycoparasites have been investigated extensively and economically used as crop disease biocontrol agents (BCAs) (Viterbo and Horwitz 2010). A. quisqualis is a unique mycoparasite of Erysiphales (Angeli et al., 2013), and it is classified as an endoparasitic fungus because its conidia (pycnidiospores or spores, as some authors refer to them) penetrate into Erysiphe cichoracearum and generate pycnidia inside powdery mildew structures (Romero et al., 2003). Molecular studies have shown that the mycoparasities are genetically diverse (Szentivanyi et al., 2005). All of these studies were analysed on the basis of the nuclear ribosomal DNA gene internal transcribed spacer (ITS) region (rDNA). Ampelomyces spp. have a phylogenetic position in the order Dothideales, according to Kiss et al., 1998. In the Dothideales, Ampelomyces ITS sequences and other ascomygetous fungus established two lineages (Arenal et al., 2000). The primary goal of this research was to determine the genetic variability in twenty isolates of Ampelomyces spp. from different ecosystems were evaluated with ten different RAPD markers. MATERIALS AND METHODS Isolation of mycoparasite. Ampelomyces isolates were isolated from powdery mildew-infested plants collected around Tamil Nadu. Effective 20 Ampelomyces isolates (Table 1) were selected based on mycoparasitic and plant growth promotion activity. All the cultures of ten isolates were maintained on liquid culture media (Potato Dextrose broth, PDB) for 7 days at 28±1ºC in a shaker and its DNA analyzed for genetic variability. Genetic analysis DNA extraction. Pure cultures of the Ampelomyces isolates were maintained on PDA slants and incubated at 25±1°C for 7 days under controlled temperature. Mycelia were aseptically transferred to flasks of potato-dextrose broth (PDB, HI Media) and incubated for 5 days at 25±1°C. The mycelia were filtered from the liquid medium for the extraction of DNA. The CTAB method (Moller et al. 1992) was used to extract genomic DNA (gDNA) from each isolate, which was then measured using a Nano Drop 1000 spectrophotometer (Thermo Scientific). Ampelomyces cultures were identified molecularly using the conserved ribosomal internal transcribed spacer (ITS) region. Using the universal primer pairs ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′- TCCTCCGCTTATTGATATGC-3′), we amplified the ITS regions between the small nuclear 18S rDNA and the large nuclear 28S rDNA, including 5.8S rDNA. Amplification was carried out on a Thermal Cycler (Applied Biosystems 9700) using 25 µl reaction mixtures comprising 2.5 µl of 10X buffer (10 mMTris–HCl, pH 8.8); 2.5 mM MgCl2; 2 mM each dNTP; 25 pmolmM primer (ITS-1 and ITS-4); 1U Taq DNA Polymerase; and 60–100 ng genomic DNA. The amplification cycle initial denaturation at 95 °C for 10 min followed by 35 cycles at 94 °C for 30 s, 60 °C for 45sec and 72°C for 1 min and a final extension at 72°C for 8 min. Separation of amplified PCR products on an agarose gel (1.5 percent w/v) in 1X TAE buffer at 75 V for 90 minutes. Studying the genetic variability among the isolates of A. quisqualis using RAPD marker. Ten RAPD primers were used to determine the genetic diversity of twenty A. quisqualis isolates (Tables 1 and 2). DNA 25 ng/l, 10X buffer (containing 2.5 mmol l-1 MgCl2) - 2 µl, 2 mmol l-1d NTP mixture - 2µl, 2 mol l-1 primer - 5 µl, Taq DNA polymerase 3U and sterile water - 8 l make up the PCR reaction. PCR amplification was performed in a thermocycler (Nexus PCR Mastercycler gradient) using the PCR conditions mentioned in Table 1 (Gaitan-Solis et al., 2002). The amplified PCR products were run on a 1.5 percent agarose gel to separate the amplified products by primer sequence. The gels were then separately visualised using UV transilluminators and photographed in the Gel Doc EZ Imager (Bio-Rad, Hayward, California). Cluster analysis. The genetic similarity of twenty Ampelomyces isolates was assessed, based on RAPD data, using S17 Bray-Curtis similarity (Jaccard 1908). Based on RAPD data, the genetic similarity of twenty Ampelomyces isolates was analysed using S17 Bray-Curtis similarity (Jaccard 1908). The unweighted pair group technique of arithmetical averages (UPGMA) algorithm was then used to create a dendrogram from the data. The presence/absence of bands in fingerprint profiles was analysed using the PRIMER 7 statistical software (Plymouth Routines in Multivariate Ecological Research, version 7.0.9; PRIMER-E, Plymouth, UK). The Bray–Curtis coefficient was used to calculate similarities between each pair of samples, resulting in a similarity matrix (Clarke, 1993). The similarity data was ordinated using non-metric multi-dimensional scaling (MDS). MDS ordination produces a map in which each sample's position is decided by its distance from all other points in the analysis. The plot's stress indicates how much distortion was created to allow the data to be represented in the specified dimensions. A stress value of more than 0.2 implies a plot that is close to random, less than 0.2 offers a useful two-dimensional representation, and less than 0.1 represents an ideal ordination with no significant risk of misinterpretation (Clarke, 1993). Within PRIMER 7, stress was calculated as stated by Kruskal (1964). The similarity matrix using the Bray– Curtis coefficient was also analysed by Hierarchical Cluster Analysis (HCA), a classification method that aims to group discrete clusters based on similarity, to visualise the relationship between different isolates of A. quisqualis using RAPD. Using PRIMER 7 software, a dendrogram was generated from the prioritised similarities using a weighted, group average linkage agglomerative technique. The effectiveness of RAPD molecular markers on assessing the genetic variability of isolates of E. cichoracearum The analysis included bands that were common to all isolates. By counting the number of bands formed by each primer in all twenty isolates, the characteristics of banding pattern, such as total number of bands, number of monomorphic, polymorphic bands per primer, and percent polymorphism, were calculated. In addition, the RAPD and ISSR markers assay efficiency index (AEI = Polymorphic bands/Total number of primers) was recorded. Anderson et al. 1993 devised a method to quantify polymorphism information content (PIC). Each locus' PIC value was estimated as follows: PICj= 1– ∑l=1to LP2lj Plj is the relative frequency of the lth allele for the locus j, and it is averaged across all alleles (L) throughout all lines. The PIC calculates a locus discriminating power by taking into consideration not just the number of alleles expressed, but also the relative frequencies of those alleles. With numerous loci in equal frequency, PIC values might vary from 0 (monomorphic) to 1 (very discriminative). EMR (E) = np (np/n) where np denotes polymorphic loci and n denotes the total number of loci (Sornakili et al., 2017). The Marker Index (MI) was calculated by multiplying the PIC by the number of polymorphic bands per assay unit, and the Effective Multiplex Ratio (EMR) was calculated by multiplying the proportion of polymorphic loci by the number of polymorphic loci for each assay RESULTS AND DISCUSSION The present study is the first extensive investigation of quantitative aspects of a mycoparasite A. quisqualis in different host range from different districts of Tamil Nadu. Genetic variability analysis of isolates of A. quisqualis by RAPD marker. DNA markers have turned into a very useful tool for studying the taxonomy of species. The random amplified polymorphic DNA (RAPD) approach, which is based on the polymerase chain reaction (PCR), is one of the most frequently used molecular techniques for the development of DNA markers (Levi et al., 2001). It enables rapid assessment of genetic variability across taxonomic groups and has been used to investigate inter- and intraspecific variability among isolates of many fungal species utilized in biological control (Tiganoet al., 1995). In this present study, RAPD analysis was performed on twenty isolates of A. quisqualiswas amplified using 10 RAPD primers. The ten primers amplified products ranging in size from 0.2 to 1.5 kb. The vast majority of ten RAPD primers showed a moderate level of polymorphism. Three primers viz., OPA5, OPA7 and OPF6 produced fragment profiles showing at least one amplicon common to all isolates of A. quisqualis (Fig. 2). Two-dimensional Non-metric Multi-Dimensional Scaling (MDS) plot using the Bray-Curtis similarity index ordinating the samples using the data from RAPD discriminated A. quisqualis from each isolates (Fig. 2a). The different isolates of A. quisqualis were evenly distributed in the MDS plot with less Kruskal’s stress (0.09). The isolates A. quisqualis AQTNAU-DST16, 18 and 20 are slackly different placed in the MDS plot, which shared different ordination. Six isolates of A. quisqualis viz., AQTNAU-DST10 to 15 shared the same similarity of coordination in the plot. This results commensurate with Rajalakshmi et al., (2016) described the (MDS) plot using the Bray-Curtis similarity index for different isolates of C. globosum were with Kruskal’s stress values of 0.07. The isolates of A. quisqualis (AQTNAU-DST04-07, AQTNAU-DST17 and AQTNAU-DST19) show separate cluster similarity of 58%. Isolates of AQTNAU-DST11 and AQTNAU-DST12 shows 86% similarity followed by AQTNAU-DST01 and AQTNAU-DST03 showed 83% similarity cluster. Genetic diversity using AFLP markers in this fungus was also explored earlier (Aggarwal et al., 2003), showing that five C. globosum isolates formed two distinct clusters, one comprising isolates Cg6, Cg7 and Cg8, and the other encompassing isolates Cg1 and Cg5. To depict genetic relationships in the form of a dendrogram, a genetic similarity estimate (S17 Bray Curtis coefficient) based on RAPD banding pattern was performed for cluster analysis. In this Dendrogram, HCA-based clustering of isolates showed 55-85 % similarity. The studied isolates were clearly classified into two groups based on the dendrogram. Cluster A, the first group, consisted of three isolates: AQTNAU-DST01, AQTNAU-DST02, and AQTNAU-DST03. Cluster B, which contained two sub clusters including AQTNAU-DST08 to AQTNAU-DST16, 18 and 20were clubbed and formed sub cluster 1 whereas AQTNAU DST 04 – 07, 17 and 19 clustered together to form sub cluster 2. The present study's findings corroborate those of Delye et al., (1997), who described the diversification of Ampelomyces. The RAPD markers were able to distinguish all isolates at 94% similarity level. Similar results were also obtained in previous researchers (Zimand et al., 1994; Hermosa et al., 2001; Grendene et al., 2002). Effectiveness of RAPD marker in assessing the genetic variability of A. quisqualis isolates. PIC scores of each primer ranged from 0.56 (OPA-05) to 0.91 (OPG-11), with an average of 0.67. The EMR value ranged from 3.20 (OPA-05, OPA-07 and OPA-09) to 7.11 (OPG-05), with an average of 4.36 indicating hyper variability among the individuals studied. To determine the overall usefulness of a given marker system, the marker index (MI) was calculated for RAPD with an average value of 2.95 was determined per primer (Table 3). The estimates of MI were found to be highest for primer OPG-05 (5.05) and the lowest with the primer OPA-05 (1.79). Singh and Sengar et al., 2015 showed that the analysis of RAPD indicated RAPD gave maximum PIC values of 0.9925. RAPD primer OPF-13 gave the maximum accessions coverage depending on the value of PIC in the rice genome. Rakhonde et al. (2015) reported that genetic variability (RAPD primers) among eighteen Indian isolates of Fusarium oxysporum f. sp. ciceri causing wilt in chickpea. The PIC value ranged from 0.504 to 0.926 across eighteen isolates of Fusarium oxysporum f. sp. ciceri indicating high degree of variation in respect to genetic similarity. This findings are in accordance with El Komy et al., (2015) studied the genetic diversity of Trichoderma sp by RAPD fingerprinting and the data exhibited a wide range of genetic similarity coefficients (0.443 to 0.944) with an average of 0.76.