Sunil C. Dubey

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.

Morphological and pathogenic variability of Indian isolates of Fusarium oxysporum f. sp. ciceris causing chickpea wilt.

Wilt of chickpea (Cicer arietinum) caused by Fusarium oxysporum f. sp. ciceris is prevalent in almost all chickpea growing areas of the world and its incidence varied from 14.1 to 32.0% in the different states of India surveyed. The isolates were highly variable in their colony growth pattern, size of colony and pigmentations. The size of microconidia varied from 5.1–12.8 × 2.5–5.0 μm, whereas macroconidia ranged from 16.5–37.9 × 4.0–5.9 μm with 1–5 septations. One hundred and twelve isolates were grouped into 12 categories on the basis of their radial growth, size of macroconidia and growth pattern. Majority of the isolates were highly pathogenic causing more than 50% wilt in chickpea cultivar JG 62. Virulence analysis of 56 representative isolates on a set of 18 cultivars of chickpea, including 10 international differentials, grouped them into three categories. Chickpea cultivar KWR 108 differentiated all isolates of Punjab, Haryana and Delhi states and a few isolates of Rajasthan from others by showing resistant reactions and were placed in the first group. The rest of the isolates of Rajasthan state showed susceptible reactions on KWR 108 placed in a second group. Cultivar CPS 1 distinguished the isolates of Jharkhand state and placed them into a third group. An international set of cultivars recommended for race differentiation were not able to distinguish all the isolates into known races of the pathogen, therefore cultivar KWR 108 should be included in the existing differential set of cultivars.

ITS-RFLP fingerprinting and molecular marker for detection of Fusarium oxysporum f.sp. ciceris

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.

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 (ITS-RFLP) 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, 5.8rDNA 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.

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.

Integrated management of Fusarium wilt by combined soil application and seed dressing formulations of Trichoderma species to increase grain yield of chickpea

Wilt caused by the fungus Fusarium oxysporum f. sp. ciceris adversely affects the productivity of cultivated chickpea. For the management of this disease, seed and soil application formulations developed from another fungus, Trichoderma species, were evaluated. In pot experiments, T. harzianum-based formulations Pusa 5SD for seed dressing and Pusa Biopellet (PBP) 10G and Pusa Biogranule (PBG) 5 for soil application, and T. viride-based formulations Pusa 5SD for seed dressing and PBP 4G and PBG 4 for soil application, were found to be highly effective against the disease. A combination of PBP 4G (T. viride) for soil application and Pusa 5SD (T. harzianum) for seed treatment together with a fungicide, carboxin, provided the highest seed germination, shoot and root lengths and grain yield with the lowest incidence of wilt in chickpea under field conditions. Individually, soil application of PBP 4G, and seed treatment with Pusa 5SD were effective in reducing the incidence of wilt and increasing the grain yield of chickpea, but their effectiveness was greater when applied as a combination. Thus, combined application of the formulations of two different species of Trichoderma in two modes of application is recommended for the management of chickpea wilt.

Evaluation of seed dressing and soil application formulations of Trichoderma species for integrated management of dry root rot of chickpea

Abstract The efficacy of the newly developed seed dressing and soil application formulations of Trichoderma viride, T. virens and T. harzianum were evaluated individually and in combinations under pot and field experiments for the management of dry root rot (Rhizoctonia bataticola) of chickpea (Cicer arientinum). In pot experiments, T. harzianum based seed dressing formulation, Pusa 5SD, and soil application formulations, Pusa Biogranule 5 (PBG 5) and Pusa Biopellet 10G (PBP 10G), were found to be effective in reducing dry root rot incidence in chickpea and increasing the seed germination, shoot and root lengths of the crop. Under field experiments, a combination of soil application of T. harzianum based PBP 10G and seed treatment with Pusa 5SD+carboxin was found to be the best by providing the highest seed germination, shoot and root lengths and grain yield and the lowest dry root rot incidence in chickpea.

Seed treatment and foliar application of insecticides and fungicides for management of cercospora leaf spots and yellow mosaic of mungbean (Vigna radiata).

Field experiments were conducted during the rainy seasons of 2006 and 2007 at the Indian Agricultural Research Institute, New Delhi for the management of yellow mosaic (Mungbean Yellow Mosaic Virus) and cercospora leaf spots (Cercospora canescens and Pseudocercospora cruenta) of mungbean. Insecticides and fungicides as seed dressings, with or without foliar sprays, were evaluated. Amongst the treatments, a combination of seed treatment with thiamethoxam (Cruiser™) at 4 g kg−1 and carbendazim (Bavistin™) + TMTD (Thiram™) at 2.5 g kg−1 (1:1 ratio) followed by foliar applications of thiamethoxam (Actara™) 0.02% and carbendazim 0.05% at 21 and 35 d, respectively after sowing produced the highest seedling establishment, shoot and root lengths, number of pods, plant biomass, 1000-seed weight, and grain yield in mungbean with the lowest intensity of cercospora leaf spots and mungbean yellow mosaic. Vector (whitefly) populations were also the lowest in this treatment during all stages of the crop. This treatment was cost-effective, as it provided the highest return per Rupee of input. It was second best for the number of Rhizobium root nodules per plant.

Development of molecular markers and probes based on TEF-1α, β-tubulin and ITS gene sequences for quantitative detection of Fusarium oxysporum f. sp. ciceris by using real-time PCR

Fusarium wilt (Fusarium oxysporum f. sp. ciceris) causes significant yield losses in chickpea worldwide. Faster, reliable and more specific molecular detection techniques were developed for the detection of Fusarium oxysporum f. sp. ciceris (Foc). The sequences obtained from multiple alignments of target genes, namely, translation elongation factor-1α (TEF-1α), β-tubulin, and internal transcribed spacer (ITS), were used to design Foc-specific markers/probes. One set of TEF-1α-based molecular marker, namely, SPα-F and R, two sets of β-tubulin-based markers, namely, SPβ1-F and R, and SPβ2-F and R, and one set of ITS gene, namely, SPT-F and R, were developed for the detection and quantification of Foc from diverse samples. The specificity and sensitivity of the designed molecular markers were evaluated through conventional and real-time PCR assays which differentiated the Foc from closely related species of Fusarium and other plant pathogens. In conventional PCR, the minimum detection limits of the markers ranged from 12.5 pg to 100 pg for genomic DNA of Foc and 0.5 ng to 10 ng for infected plant samples. In real-time PCR assay, the minimum detection limits of the markers ranged from 0.001 pg to 0.25 pg for genomic DNA of Foc and from 0.04 pg to 1.5 pg for the infected plant samples. Thus, the markers designed in the present study were found to be specific for Foc and can be used consistently for the detection and identification of Foc isolates. The probes developed from the two sets of markers, namely, SPα and SPβ2, also showed specificity with Foc.

Conventional and real-time PCR assays for specific detection and quantification of Fusarium oxysporum f. sp. ciceris in plants using intergenic spacer region-based marker

Abstract Fusarium wilt of chickpea, caused by Fusarium oxysporum f. sp. ciceris (Foc) is one of the most important fungal diseases worldwide. The detection of the pathogen at reasonable time period is of great importance, which requires rapid and sensitive detection methods. The intraspecific divergence sequences found in the intergenic spacer region (IGS) were selected and utilized with the aim to develop a molecular marker specifically to identify the Foc. A marker set, ISR52 F1 and R1 developed, was tested for their specificity as well as sensitivity using conventional as well as real-time polymerase chain reaction (PCR). The specificity of the marker was tested against Foc, other Fusarium species which are closely related to Foc as well as with artificially infected host plant samples. The detection limits of conventional PCR assay was up to 100 pg of infected plant DNA. It proved possible to amplify the IGS region in different portion of a Foc infected host plant by this PCR method. Furthermore, the real-time assay showed more sensitivity and was able to detect the pathogen in infected chickpea plant samples at the DNA concentration of 5 pg. A single melting peak obtained at 87.5°C showed the specificity of the marker towards Foc. Thus, real-time PCR assay proved their potentiality for same-day diagnosis of fungal infection and can be used as a rapid and effective procedure for routine detection and identification of Foc in chickpea samples.