Aradhika Tripathi

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.

Pathogenic and genetic variability in Tilletia indica monosporidial culture lines using universal rice primer-PCR

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.

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.

Determination of variability in monosporidial lines of Tilletia indica by RAPD analysis

Genetic variability in 23 monosporidial lines developed from five isolates of Tilletia indica causing Karnal bunt of wheat isolated from four wheat growing states of India was determined by using 19 rapid amplified polymorphic DNA (RAPD) markers. Amplification profile generated with all the 19 primers produced 3–16 numbers of bands of 1.5–5 kb size. High level of polymorphism (95.2%) suggested wide range of variability. Maximum Jaccards similarity coefficient (80%) was observed between KB2MsB and KB2MsC followed by KB5MsC and KB5MsE with 75% similarity, whereas it was minimum between KB3MsA and Kb4MsB (47%). The dendrogram derived from the fingerprint analysis with 19 RAPD primers by using UPGMA showed different levels of genetic similarity among monosporidial lines. At 35% genetic similarity, the monosporidial lines were grouped in two clusters. Some primers, viz., OPN-1, OPN-6, OPN-9, OPN-12, OPN-13, OPN-18, OPM-2, OPM-8, OPM-10, OPB-8, OPB-17 and OPB-20 showed 100% polymorphism. The RAPD fingerprint generated by OPN-1 and OPM-3 were analysed and showed high range of variation in genetic make-up of monosporidial lines.

ANALYSIS OF VIRULENCE AND ITS REGION-BASED GENETIC VARIABILITY AMONG THE INDIAN POPULATIONS OF FUSARIUM OXYSPORUM f. sp. CICERIS CAUSING CHICKPEA WILT

Virulence of 36 representative isolates of Fusarium oxysporum f. sp. ciceris causing chickpea wilt, collected from 12 major chickpea-growing states of India was analysed on a new set of 10 differential chickpea cultivars, namely, C 104, JG 74, CPS 1, BG 212, WR 315, KWR 108, GPF 2, DCP 92-3, Chaffa and JG 62 to determine races of the pathogen. Differential cultivars for each races were identified and, based on differential reactions, the isolates were grouped into eight races. Except for the isolates from Delhi, Haryana, Bihar, Uttar Pradesh and Madhya Pradesh, the race groups corresponded to the area of their origin. Genetic diversity of 14 isolates representing 8 races of the pathogen was determined using internal transcribed spacer region of rDNA- restriction fragment length polymorphism (ITSRFLP) and ITS sequencing. ITS1, 5.8S rDNA and ITS2 region was amplified and digested with seven restriction enzymes. Some of the isolates showed area-specific ITSRFLP patterns, whereas others from the same area showed distinct pattern. Thus, the ITS-RFLP pattern was partially corresponding to the area of origin of the isolates indicating the presence of highly variable populations in chickpea-growing areas of the country. The phylogenetic analysis of ITS sequences (JF727559- JF727572) of the isolates included in the present study also grouped them into eight categories and these groups were partially corresponding to the fungal races. Thus, the Indian populations of the pathogen were highly variable in respect of their virulence and genetic characteristics.

Development of conventional and real time PCR assay for detection and quantification of Rhizoctonia solani infecting pulse crops

Abstract A specific and sensitive conventional and real-time PCR assays were developed for the detection and quantification of Rhizoctonia solani populations infecting pulses. Five different sets of R. solani specific molecular primers ARSF1&R1, ARSF2&R2 and ARSF3&R3, ARSF4&R4 and ARSF5&R5 were designed from the internal transcribed spacer sequences of R. solani isolates associated with pulse crops representing 7 anastomosis groups (AG1, AG2-2, AG2-2LP, AG2-3, AG3, AG4 and AG5). The designed primers yielded variable amplicons ranging from 174 to 563 bp specific to R. solani populations. The markers proved to be highly sensitive to R. solani and were able to detect the pathogen from both mycelium and infected mung bean plants at various concentrations of genomic DNA ranging from 0.025 to 1.0 ng using conventional PCR. The markers were also found suitable for real-time PCR assay with enhanced sensitivity up to 1.24 pg genomic DNA. The efficacy of markers for early detection of R. solani infection in mung bean plants was also tested and the marker ARSF4&R4 was the most sensitive in detecting the pathogen at 24 h after artificial inoculation under field and at 6 h after artificial inoculation under in vitro conditions. The radioactive and fluorescein amidite labelled probes for markers ARSF4&R4 and ARSF5&R5 were designed and tested for cross-reactivity and sensitivity by Southern blotting and real-time PCR analyses, respectively. Both probes proved to be highly sensitive and specific only to R. solani. Both quantitative PCR and probe-based diagnostics have been developed for R. solani detection in pulses.