Genetic diversity of 89 isolates of Rhizoctonia solani isolated from different pulse crops representing 21 states from 16 agro-ecological regions of India, 49 morphological, and 7 anastomosis groups (AGs) was analyzed using 12 universal rice primers (URPs), 22 random amplified polymorphic DNA (RAPD), and 23 inter-simple sequence repeats (ISSR) markers. Both URPs and RAPD markers provided 100 % polymorphism with the bands ranging from 0.1 to 5 kb in size, whereas ISSR markers gave 99.7 % polymorphism with the bands sizes ranging from 0.1 to 3 kb. The marker URP 38F followed by URP13R, URP25F, and URP30F, RAPD marker R1 followed by OPM6, A3 and OPA12 and ISSR3 followed by ISSR1, ISSR4, and ISSR20 produced the highest number of amplicons. R. solani isolates showed a high level of genetic diversity. Unweighted pair group method with an arithmetic average (UPGMA) analysis grouped the isolates into 7 major clusters at 35 % genetic similarity using the three sets of markers evaluated. In spite of using three different types of markers, about 95 % isolates shared common grouping patterns. The majority of the isolates representing various AGs were grouped together into different sub-clusters using all three types of markers. Molecular groups of the isolates did not correspond to agro-ecological regions or states and crops of the origin. An attempt was made for the first time in the present study to determine the genetic diversity of R. solani populations isolated from different pulse crops representing various AGs and agro-ecological regions.

Four hundred seventy Rhizoctonia solani isolates from different leguminous hosts originating from 16 agro-ecological regions of India covering 21 states and 72 districts were collected. The disease incidence caused by R. solani varied from 6.8 to 22.2 % in the areas surveyed. Deccan plateau and central highlands, hot sub-humid ecoregion followed by northern plain and central highlands and hot semi-arid ecoregion showed the highest disease incidence. R. solani isolates were highly variable in growth diameter, number, size and pattern of sclerotia formation as well as hyphal width. The isolates obtained from aerial part of the infected plants showing web blight symptoms produced sclerotia of 1-2 mm in size whereas, the isolates obtained from infected root of the plants showing wet root rot symptoms produced microsclerotia (<1 mm). Majority of R. solani isolates showed <8 μm hyphal diameter. Based on morphological characters the isolates were categorized into 49 groups. Seven anastomosis groups (AGs) were identified among the populations of R. solani associated with the pulse crops. The frequency (25.6 %) of AG3 was the highest followed by AG2-3 (20.9 %) and AG5 (17.4 %). The cropping sequence of rice/sorghum/wheat-chickpea/mungbean/urdbean/cowpea/ricebean influenced the dominance of AG1 (16.3 %). Phylogenetic analysis utilizing ITS-5.8S rDNA gene sequences indicated high level of genetic similarity among isolates representing different AGs, crops and regions. ITS groups did not correspond to the morphological characters. The sequence data from this article has been deposited with NCBI data libraries with JF701707 to JF701795 accession numbers.

Tilletia indica Mitra is the causal agent of Karnal bunt of wheat, an important disease prevalent in several countries. The disease is internationally quarantined and the pathogen due to its heterothallic nature shows high variability. In the present study, we compared the pathogenic behaviour of various isolates of T. indica collected from different geographical locations of India and genetically characterized monosporidial (Ms) culture lines raised from these isolates of the pathogen. Pathogenic variability revealed existence of three pathotypes based on aggressiveness on a set of differential host genotypes. Monosporidial culture lines viz., 5 each from KB1, KB2, KB4 and KB5 and three lines of KB3 were established and analyzed genetically using 12 Universal Rice Primers (URPs). Amplification showed 98.44% polymorphism and primer URP 13R produced 100% polymorphic bands. Maximum similarity (83%) was between KB1MsB and KB1MsD as calculated by Jaccard's similarity coefficient, whereas, minimum similarity was between KB1MsC and KB4MsB; KB1MsE and KB3MsA (46%). Three groups were formed among all Ms culture lines. One major group consisted of 13 lines with approximately 70% similarity, the second group consisted of 7 culture lines showing 55% similarity and the third group consisted of 3 Ms lines. URPs were able to differentiate the Ms culture lines raised from different T. indica isolates and the results indicated heterogeneity in the pathogen population.

A specific and sensitive conventional and real-time PCR assays were developed for the detection and quantification of

Genetic diversity of 11 representative isolates of Fusarium oxysporum f.sp. ciceris causing chickpea wilt was determined through internal transcribed spacer (ITS) region of the ribosomal DNA-restriction fragment length polymorphism (ITS-RFLP). ITS1+5.8s+ITS2 regions of the isolates were amplified with a set of primers ITS1 and ITS4 and amplified products were digested with 4 restriction enzymes (AluI, MboI, RsaI, MseI). Six different kinds of ITS-RFLP patterns were obtained. The ITS region of these isolates was sequenced and deposited to NCBI GeneBank. The nucleotide sequence homology of ITS region grouped the isolates into 5 categories. Primers were designed with sequence information using Primer 3 software. F. oxysporum f.sp. ciceris specific markers (FOC F2 and FOC R2) based on ITS region were developed for the first time for detection of the pathogen. The markers produced an amplicon of 292 bp; they were validated against the isolates of the pathogen collected from different locations of India.

Web blight/wet root rot caused by Rhizoctonia solani is one of the major constraints for mung bean (Vigna radiata) production. Growing of resistant varieties and use of biocontrol agents are the feasible options available to manage the disease. The present study was conducted to determine the variation in the expression of various defense-related genes in susceptible and resistant mung bean varieties in response to biocontrol agent Trichoderma virens and R. solani interactions. The primers were designed using sequences of defense-related genes, namely PR 10, epoxide hydrolase (EH), catalase and calmodulin available in NCBI database and evaluated against cDNA obtained from both susceptible and resistant mung bean plants at 1-4 days post-inoculation (dpi) with the test pathogen R. solani and biocontrol agent T. virens using conventional PCR and qPCR analyses. R. solani inoculation upregulated the mean expression of PR 10 and calmodulin in susceptible and resistant varieties, respectively, whereas downregulated in the rest of the treatments. Quantitative PCR analysis showed that except catalase in the susceptible variety, which is downregulated, the expression of PR 10, EH, catalase and calmodulin was upregulated in both resistant and susceptible varieties in response to T. virens alone and in the presence of R. solani. In general, the expression of PR 10 and calmodulin was highest at 1 dpi whereas EH and catalase expression were maximum at 4 dpi. The application of T. virens suppressed the development of disease in the presence of R. solani in both susceptible and resistant varieties with more pronounced effect in resistant variety. Thus, the application of biocontrol agent T. virens upregulated the expression of defense-related genes and reduced disease development.

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Karnal bunt caused by Tilletia indica is a quarantine disease responsible for qualitative and quantitative losses in wheat. The present study aimed to determine the expression of defense-related genes involved in early stages of infection in response to T. indica by using the differential display reverse transcriptase-PCR method (DDRT-PCR) in susceptible (WL 711) and resistant (HD 29) cultivars of wheat. The DDRT-PCR banding profiles generated by the random primers in combination with oligo-dT primers of resistant and susceptible wheat plants were evaluated at 0 h, 12 h, 24 h, and 48 h after inoculation. Out of 9 sets of primers, 7 sets of primers produced remarkable induced transcripts. The highest up-regulated transcripts were observed in the resistant cultivar (HD 29), whereas the down-regulated transcripts were found in the susceptible (WL 711) cultivar. Several sets of primers were designed from the defenserelated gene sequences available in the GenBank database, and of these, 4 primer sets amplified the desired specific bands. Based on the putative functions, 3 defense-related genes, namely puroindoline protein PINB (AT_KB2), β-1,4-glucanase (AT_KB3), and chitinase (AT_KB7), and 1 housekeeping gene, actin (AT_KB1), were characterized.