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.

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.

Phylogenetic relationship between different race representative populations of Fusarium oxysporum f. sp. ciceris in respect of translation elongation factor-1α, ß-tubulin, and internal transcribed spacer region genes.

Genetic diversity of 70 isolates of Fusarium oxysporum f. sp. ciceris originated from various states of India representing eight races causing wilt in chickpea (Cicer arietinum) was analyzed using translation elongation factor-1α (TEF-1α), ß-tubulin, and internal transcribed spacer (ITS) gene regions. TEF-1α, ß-tubulin, and ITS gene-specific markers produced ~720-, ~500-, and ~550-bp amplicons, respectively, in all the isolates of the pathogen. A phylogenetic tree constructed from the sequences generated in the present study along with the sequences of foreign isolates of Fusarium species available in NCBI database sharing more than 90 % nucleotide sequence similarity grouped the isolates into two major clusters. Most of the isolates of the present study showed more or less similar grouping pattern in case of the three gene sequences. Each group had the isolates representing different races as well as place of origin indicating low level of diversity among the isolates in respect of these gene sequences. Except TEF-1α, the groups generated by ß-tubulin and ITS gene sequences did not correspond to the state of origin and races of the pathogen. However, the groups of TEF-1α partially corresponded to the place of origin as well as races of the pathogen. The isolates did not show any race-specific grouping patterns; however, most of the isolates representing race 1 clustered separately.

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.

Resistance screening and in silico characterization of cloned novel RGA from multi-race resistant lentil germplasm against Fusarium wilt (Fusarium oxysporum f. sp. lentis).

Fusarium wilt caused by Fusarium oxysporum f. sp. lentis (Fol) is the most devastating disease of lentil present worldwide. Identification of multi-race fusarium wilt resistance genes and their incorporation into existing cultivars will help to reduce yield losses. In the present study, 100 lentil germplasms belonging to seven lentil species were screened against seven prevalent races of Fol, and accessions IC201561 (Lens culinaris subsp. culinaris), EC714243 (L. c. subsp. odemensis), and EC718238 (L. nigricans) were identified as resistant. The typical R gene codes for the nucleotide-binding site and leucine-rich repeats (NBS-LRR) at the C terminal are linked to either the Toll/interleukin 1-like receptor (TIR) or coiled coil (CC) at the N terminal. In the present study, degenerate primers, designed from the NBS region amplifying the P-loop to the GLPLA motif, isolated forty-five resistance gene analogues (RGAs) from identified resistant accessions. The sequence alignment identified both classes of RGAs, TIR and non-TIR, based on the presence of aspartate (D) and tryptophan (W) at the end of the kinase motif, respectively. The phylogenetic analysis grouped the RGAs into six classes, from LRGA1 to LRGA6, which determined the diversity of the RGAs present in the host. Grouping of the RGAs identified from Lens nigricans, LnRGA 2, 9, 13 with I2 revealed the structural similarity with the fusarium resistance gene. The similarity index ranged from 27.85% to 86.98% among the RGAs and from 26.83% to 49.41% among the known R genes, I2, Gpa2, M, and L6. The active binding sites present along the conserved motifs grouped the RGAs into 13 groups. ADP/ATP, being the potential ligand, determines the ATP binding and ATP hydrolysis activity of the RGAs. The isolated RGAs can be used to develop markers linked to the functional R gene. Furthermore, expression analysis and full-length gene isolation pave the path to identifying the molecular mechanism involved in resistance.

Genetic diversity analysis of Sclerotinia sclerotiorum causing stem rot in chickpea using RAPD, ITS-RFLP, ITS sequencing and mycelial compatibility grouping.

Sclerotinia sclerotiorum is one of the most devastating soil-inhabiting fungal plant pathogens infecting various crop plants including chickpea. Genetic diversity of 24 isolates of S. sclerotiorum representing 10 different states of India was determined by different molecular markers and mycelial compatibility grouping (MCG). The majority of the isolates showed more than 90% genetic similarity. Unweighted paired group method with arithmetic average cluster analysis of DNA profiles generated by 21 RAPD primers grouped the isolates into seven categories showing high magnitude of genetic homogeneity and showed partial correlation with geographical origin of the isolates. Identical ITS-RFLP profiles were generated in all the isolates. Limited variability was observed among the nucleotide sequences of ITS region of the isolates. The phylogenetic tree generated from bootstrap neighbor-joining analysis indicated that 50% of Indian populations were distinct and grouped separately. The isolates were variable in mycelial compatibility and they were grouped into seven MCGs, namely, MCG A, MCG B, MCG C, MCG D, MCG E, MCG F and MCG G.

Development of a sequence-characterized amplified region marker for detection of Ascochyta rabiei causing Ascochyta blight in chickpea.

Ascochyta blight of chickpea is caused by Ascochyta rabiei (Pass.) Labr. which is primarily seedborne. For rapid detection and precise identification of A. rabiei, a sequence-characterized amplified region (SCAR) marker was developed for detection of genomic DNA and infected plant DNA. An SSR primer amplified monomorphic band was cloned in pGEM®-T easy vector and sequenced. The best primer pair was selected and validated on A. rabiei. The specificity and sensitivity of the SCAR-based marker designated as MBAR was evaluated using conventional PCR and real-time PCR. The marker produced consistently an amplicon size of 196 bp in all A. rabiei isolates tested. The sensitivity of the marker was 0.1 ng of genomic fungal DNA and 0.5 ng of plant DNA by conventional PCR and 0.5 pg of A. rabiei DNA and 1.0 pg of plant DNA by real-time PCR. This is the first SCAR marker having high specificity and sensitivity towards A. rabiei. The marker may be useful in detecting the pathogen before the disease appearance and in plant quarantine program to detect the pathogen in seed lots.

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea.

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITSRFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8 rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.

Phylogenetic relationship among Indian population of Fusarium oxysporum f. sp. lentis infecting lentil and development of specific SCAR markers for detection.

The present study was taken up to understand the phylogenetic relationship using ITS and TEF markers among 22 isolates of Fusarium oxysporum f. sp. lentis (Fol) causing lentil wilt belonging to eight races isolated from different geographic locations of India and to develop specific markers for its detection. The nucleotide sequences of ITS region varied from 490 to 560 bp whereas, 670-725 bp for TEF 1α. The phylogeny analysis revealed that the isolates were more than 98% similar based on the neighbour joining analysis and were grouped into two major clusters in both ITS and TEF. The first major cluster of ITS had twenty isolates whereas for TEF, there were 15 isolates. Two sets of SCAR markers MS1 (162 bp) and MS2 (125 bp) were designed and synthesised. These markers were used against race representative Fol isolates for amplification. While, MS 1 marker was able to detect the genomic DNA up to 0.1 ng, MS 2 could detect the Fol genomic DNA up to 0.05 ng. The specificity of these two markers to detect Fol and their inability to amplify most common lentil pathogens (Rhizoctonia solani, R. bataticola, Sclerotium rolfsii, Sclerotinia sclerotiarum, and Aschochyta rabiei) makes them a reliable tool for detection. The phylogenetic analysis is helpful in the understanding of variability in Fol populations and the SCAR markers help in rapid and reliable detection of an important pathogen of lentil.

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.

Analysis of differential transcript expression in chickpea during compatible and incompatible interactions with Fusarium oxysporum f. sp. ciceris Race 4.

The present study reports the transcriptome analysis of resistance (WR315) and susceptible (JG62) genotypes of chickpea in response to Fusarium oxysporum f. sp. ciceris (Foc) race 4 using the method of suppression subtractive hybridization. Altogether, 162 chickpea-expressed sequence tags (ESTs) were identified from two libraries and analyzed to catalog eight functional categories. These ESTs could be assembled into 18 contigs and 144 singletons with 10 contigs and 68 singletons from compatible and 8 contigs and 70 singletons from incompatible interaction. The largest category consisted of ESTs which encode for proteins related to hypothetical proteins (22.8%), followed by energy and metabolism (20.3%)-related genes, defense and cell rescue-related genes (17.9%) and signal transduction-related genes (16%). Among them, 17.1 and 18.7% were defense-related genes in compatible and incompatible interaction, respectively. These ESTs mainly includes various putative genes related to oxidative burst, pathogenesis and secondary metabolism. Induction of putative superoxide dismutase, metallothionein, 4-coumarate-CoA ligase, heat shock proteins and cysteine proteases indicated oxidative burst after infection. The ESTs belonged to various functional categories which were directly and indirectly associated with defense signaling pathways. Quantitative and semi-quantitative polymerase chain reaction exhibited differential expression of candidate genes and detected higher levels in incompatible interaction compared to compatible interaction. The present study revealed partial molecular mechanism associated with the resistance in chickpea against Foc, which is the key to design a strategy for incorporation of resistance via either biotechnological means or introgression of resistance genes.

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.

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.

Integration of soil application and seed treatment formulations of Trichoderma species for management of wet root rot of mungbean caused by Rhizoctonia solani.

BACKGROUND: The efficacy of seed dressing and soil application formulations from the isolates of Trichoderma viride (IARI P1; MTCC 5369), T. virens (IARI P3; MTCC 5370) and T. harzianum (IARI P4; MTCC 5371) were evaluated individually and in combination in pot and field experiments during the rainy seasons of 2005, 2006 and 2007 for the management of wet root rot (Rhizoctonia solani) and improvement in the yield of mungbean. RESULTS: A seed dressing formulation, Pusa 5SD, and soil application formulations, Pusa Biogranule 6 (PBG 6) and Pusa Biopellet 16G (PBP 16G), based on Trichoderma virens, were found to be superior to other formulations in reducing disease incidence and increasing seed germination and shoot and root lengths in mungbean. In field experiments, a combination of soil application with PBP 16G (T. virens) and seed treatment with Pusa 5SD (T. virens) + carboxin was superior to any of these formulations individually in increasing seed germination, shoot and root lengths and grain yield and reducing wet root rot incidence in mungbean. Seed treatment was more effective than soil application for all the evaluated parameters. The combined application of Pusa 5SD and carboxin was also superior to individual treatment. CONCLUSION: The efficacy of the evaluated formulations against wet root rot of mungbean proved that the integration of soil application of PBP 16G and seed treatment with Pusa 5SD + carboxin is highly effective for the management of wet root rot, increasing plant growth and grain yield of mungbean.