Expression profiling and characterization of key RGA involved in lentil Fusarium wilt Race 5 resistance.

Fusarium wilt is a major threat to lentil production in India and worldwide. The presence of evolving virulent races has imposed the necessity of reliable management practices including breeding for resistance using unexplored germplasms. The magnitude of resistance by the plant is determined by rapid recognition of the pathogen and induction of defence genes. Resistance gene analogues have been key factors involved in the recognition and induction of defence response. In the present study, the expression of key RGA previously cloned was determined in three resistant accessions (L65, L83 and L90) and a susceptible accession (L27). The expression was assessed via qPCR at 24, 48 and 72 hpi against virulent race5 (CG-5). All the RGAs differentially transcribed in resistant and susceptible accession showed temporal variation. RGA Lc2, Lc8, Ln1 and Lo6 produced cDNA signals during early infection (24 hpi) predicting its involvement in recognition. LoRGA6 showed significant upregulation in L65 and L83 while downregulating in L27 and the full length of LoRGA6 loci was isolated by 5' and 3' RACE PCR. In-silico characterization revealed LoRGA6 loci code for 912 amino acids long polypeptide with a TIR motif at the N terminal and eight LRR motifs at the C terminal. The tertiary structure revealed a concave pocket-like structure at the LRR domain potentially involved in pathogen effectors interaction. The loci have ADP binding domain and ATPase activity. This has further paved the path for functional analysis of the loci by VIGS to understand the molecular mechanism of resistance.

Quadruplex and q-PCR based diagnostic assay to delineate the major quarantine and other seed-borne fungal pathogens of soybean.

Soybean is one of the most important crops grown worldwide and accounting for significant global trade including transgenic soybean. The crop is attacked by several seed-borne fungal pathogens and some of them are of quarantine concern for India. Keeping in view of the risks associated with movement of soybean seeds, sensitive and reliable molecular diagnostics have been developed for precise and simultaneous detection of three pathogens of quarantine concern for India namely, Diaporthe phaseolorum (stem blight), D. longicolla (seed decay), Peronospora manshurica (downy mildew), along with Macrophomina phaseolina causing dry root rot. The targeted pathogens after isolation from imported transgenic and non-transgenic soybean seeds were identified. Quadruplex and qPCR assays were developed targeting the sequences of different genes such as Histone-3 for detection of D. longicolla and M. phaseolina. The markers DlHisF2&R2 and MpHisF1&R1 produced 265 and 309 bp amplicons for D. longicolla and M. phaseolina, respectively. Actin gene based marker DpActF1&R2 was developed for D. phaseolorum which provided 113 bp amplicon whereas, COX2 based marker PmCoxF2&R2 was developed for P. manshurica with amplified product of 152 bp. During qPCR analysis, these markers proved highly specific and sensitive for detection of these pathogens up to 0.1 pg of template DNA. Quadruplex PCR protocol was also developed by combining these specific markers which could distinguish all the targeted pathogens simultaneously in a single reaction. The developed diagnostic protocols are extremely valuable for quarantine clearance and to ensure the safe transboundary exchange and healthy conservation of germplasm in the National Genebank.

Development of PCR-based assays to diagnose the major fungal pathogens infecting pulse crops, potential for germplasm health certification and quarantine processing.

Plant diseases cause serious economic losses of agriculture production worldwide. Rapid, accurate and reliable diagnostic methods are required to alleviate the detection of fungal plant pathogens to prevent their spread and achieve effective management. This study was aimed to develop fast, reliable and highly sensitive diagnostics to detect fungal plant pathogens for quarantine processing, safe exchange and conservation of germplasms of pulse crops. Multiplex and real time PCR assays were developed for detection of Rhizoctonia solani, Macrophomina phaseolina, Ascochyta rabiei, Alternaria alternata, A. tenuissima, Fusarium oxysporum f. sp. ciceris, Sclerotium (Athelia) rolfsii, Sclerotinia sclerotiorum, Pseudocercospora cruenta and Cercospora canescens causing various diseases in pulse crops. Twenty-two sets of primers from various genomic regions such as cytochrome oxidase subunit (COX 1), internal transcribed spacer region (ITS), translation elongation factor-1 alpha (TEF-1α), large subunit (LSU), small subunit (SSU) and ß-tubulin as well as two SCAR primers from RAPD profile were designed. The developed markers proved to be species-specific and validated against other fungal plant pathogens associated with pulses for cross-reactivity. The markers proved highly sensitive during conventional and qPCR analysis. Duplex PCR assays for R. solani and M. phaseolina; C. canescens and P. cruenta; A. alternata and A. tenuissima; and a quadruplex PCR assay for A. rabiei, S. sclerotiorum, S. rolfsii and F. oxysporum f. sp. ciceris were developed and validated for simultaneous detection of these pathogens in a single reaction. The assays developed in the present study were able to detect and identify major fungal plant pathogens causing disease in pulse crops.

DNA barcode, multiplex PCR and qPCR assay for diagnosis of pathogens infecting pulse crops to facilitate safe exchange and healthy conservation of germplasm.

The DNA barcodes were developed from ITS region for the identification of fungal plant pathogens namely, Alternaria alternata and A. tenuissima both causing leaf spots, Ascochyta rabiei causing Ascochyta blight, Fusarium oxysporum f. sp. ciceris causing wilt, Macrophomina phaseolina causing dry root rot, Rhizoctonia solani causing web blight and wet root rot, Sclerotium (Athelia) rolfsii causing collar rot, Sclerotinia sclerotiorum causing stem rot and Cercospora canescens and Pseudocercospora cruenta both causing leaf spots in pulse crops. Barcode compliance for A. alternata (DBTPQ001-18), A. tenuissima (DBTPQ002-18), A. rabiei (DBTPQ003-18), F. oxysporum f. sp. ciceris (DBTPQ004-18), M. phaseolina (DBTPQ005-18), R. solani (DBTPQ006-18), S. rolfsii (DBTPQ007-18), S. sclerotiorum (DBTPQ008-18), C. canescens (DBTPQ009-18) and P. cruenta (DBTPQ029-20) have been generated based on the Barcode of Life Data System (BOLD) system. In addition to ITS, other genomic regions were also explored and on the basis of sequence variation they were ranked as TEF-α > SSU > LSU > ß-tubulin. These genes could be considered for secondary barcode and phylogenetic relatedness. ITS-based markers for the detection of A. alternata (BAA2aF and BAA2aR) and R. solani (BRS17cF and BRS17cR) were developed which provided 400 bp and 220 bp amplicons, respectively. While, for F. oxysporum f. sp. ciceris, COX1-based marker (FOCox1F and FOCox3R) was developed which amplified 150 bp. The markers proved highly specific and sensitive with detection limit of 0.0001 ng of template DNA using qPCR and simultaneously detected these three pathogens. The DNA barcodes and diagnostics developed are suitable for quick and reliable detection of these pathogens during quarantine processing and field diagnostics.

Expression of defense-related genes in mung bean varieties in response to Trichoderma virens alone and in the presence of Rhizoctonia solani infection.

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.

Diversity of Rhizoctonia solani associated with pulse crops in different agro-ecological regions of India.

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.

Molecular diversity analysis of Rhizoctonia solani isolates infecting various pulse crops in different agro-ecological regions of India.

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.

Genetic diversity analysis and development of SCAR marker for detection of Indian populations of Fusarium oxysporum f. sp. ciceris causing chickpea wilt.

Genetic diversity of the isolates of Fusarium oxysporum f. sp. ciceris causing chickpea wilt collected from 12 states representing different agro-ecological regions of India was determined through randomly amplified polymorphic DNA (RAPD) markers. The UPGMA cluster analysis grouped the isolates into eight categories showing high magnitude of genetic diversity. Each group had the isolates from different states present in various agro-ecological regions of India. Therefore, the groups generated through the RAPD analysis were not corresponding to area of the origin of the isolates. The RAPD primers, namely, OPA 7 and OPA 11 produced Foc specific fragment of ≈1.3 kb and ≈1.4 kb, respectively in all the isolates. These fragments were eluted, purified, cloned in pGEM-T Easy vector and sequenced. Primers were designed with sequence information of these two fragments using primer.3 software. Two sets of sequence characterized amplified region markers (SC-FOC 1 and SC-FOC 2) developed from the sequences of these fragments were found to be specific to Foc and produced an amplicon of 1.3 and 1.4 kb, respectively. These set of markers were validated against the isolates of the pathogen collected from different locations of India representing various races of the pathogen. They are non-specific to the other Fusarium species, Rhizoctonia solani and R. bataticola.