SARADA JAYALAKSHMI DEVI, RBHASKAR, B2019-05-102019-05-102018http://krishikosh.egranth.ac.in/handle/1/5810102677D5730The present investigation was undertaken with an aim to know the variability between leaf blast and neck blast pathogen populations. Studies carried out on the variability of isolates using cultural, morphological, pathological and molecular characteristics. In another study efforts were made to identify the promising lines for both leaf blast and neck blast resistance. Attempts were also made to manage the disease with biocontrol agent P. fluorescens, fungicides and their integration. Survey was conducted in nine major rice growing districts of Andhra Pradesh during 2015-16. The highest mean blast disease incidence 29.05% was recorded in Nellore district. The lowest mean PDI 20.79% was recorded in Srikakulam district. Regarding the mandals, highest mean blast incidence was recorded in Kovvur mandal of Nellore district with 38.99% in a range of 20.45% to 51.14%. The lowest incidence 15.41% with 10.94% to 21.02% range was noticed in Madugula mandal of Visakhapatnam district. The disease incidence was highly varied among the cultivars rather than the locations. The ascending order of the cultivars regarding the mean disease incidence was NDLR-8 (3%) < MTU-3626 (4.46%) < MTU-1121(5.89%) < NLR-34449 (11.09%) < RGL-2537 (12.86%) < MTU-1061 (16.83%) < MTU-1001 (19.74% ) < ADT-37 (23.81%)<NLR-145 (25.56%) < MTU-1010 (25.06% PDI)<BPT-5204(33.07%)<MTU-7029(59.44%). The leaf and neck blast samples were collected from Andhra Pradesh, Telangana, Karnataka and Tamilnadu. A total of 50 monoconidial isolates of Pyricularia oryzae were established. Out of fifty isolates, 33 were leaf blast and 17 were neck blast isolates. xviii The variability in cultural characteristics viz., radial growth, colony colour, growth pattern, elevation (flat/raised growth), sectored or non-sectored growth, zonation and wrinkles formation were studied among the isolates of P. oryzae isolates on OMA medium. Cluster analysis of the data on cultural characterization revealed three major groups (I, II and III) at the distance of 3.0. No difference in cultural characteristics between leaf and neck blast isolates was observed since the distribution of neck blast isolates in each group along with the leaf blast isolates. The correlation coefficient matrixes among cultural characters revealed that the most significant positive correlation was observed between sectoring and wrinkles formation (r2 = 0.43, n = 50, p< 0.05). Culturing of P. oryzae on OMA medium could not produce spores, hence efforts were made for induction of sporulation in different cultural conditions viz., different media, light regimes and stem and leaf bits of different hosts. Good amount of sporulation was observed in case of Finger millet Leaf extraction Agar (FLA) medium with 1.71×105 ml-1 followed by Rice leaf extract Agar (RLA) medium with 1.65×105 ml-1. In case of light regimes, on different media, no significant difference in sporulation was observed among three light regimes i.e. light (0.90 ×105 ml-1), dark (0.70 ×105 ml-1) and dark + light (0.70 ×105 ml-1). In another experiment, leaf and stem bits of eight hosts viz., Pennisetum purpureum, Paspalum sp., Brachiaria mutica, Digitaria sanguinalis, Echinochloa colonum, Panicum repens, Oryza sativa and Eluesine coracana were used for induction of sporulation and observed high amount of sporulation in Brachiaria mutica leaf and stem bits with 7.61×105 ml-1. Over all, the method i.e., culturing of P. oryzae on Brachiaria mutica leaf and stem bits found as best method for induction of sporulation rather than cultering on RLA or FLA or under different light conditions. Morphological variability among P.oryzae isolates was studied through the morphology of conidia. Among the isolates, the overall size of the conidia was 18.59 – 26.25 μm × 7.17 – 9.16 μm (Length × Width). In order to study the pathological variability between leaf blast and neck blast isolates, both were cross inoculated on a susceptible cultivar BPT-5204. Before that cross inoculation studies, artificial inoculation technique for neck blast infection has been standardized by using three methods viz., Injection, smearing and tying of inoculum bits. Out of these three methods, injection method gave 95.56% PDI whereas smearing method and tying method showed 33.33% PDI and 12.78% PDI, respectively. In cross inoculation studies, a pool of isolates consists of three leaf blast isolates i.e., Po-6, Po-19 and Po-13 and three neck blast isolates i.e., Po-7, Po-33 and Po-38 were used. The results indicated that all the isolates (both leaf and neck blast isolates) showed both leaf blast and neck blast infection. In order to identify the races of P. oryzae, a total of 25 monosporic isolates represents the collected geographical locations of Andhra Pradesh, Telangana, Karnataka and Tamil Nadu were selected and each was inoculated on international host differential set. The race ID-14 was recorded as most frequently occurred race and it was followed by ID-13, IG-2, IB-44, IC-32, IB-46, IA-69, IC-29, IA- 106 and IA-77. Molecular identification of P. oryzae isolates with ITS4 and ITS5 universal primers and Actin gene primers were used In order to support the morphological identification of P. oryzae isolates. In case of ITS region, all the isolates were xix appeared as identical by producing the amplified fragment of 560 bp. PCR amplification with actin gene specific primer produced the 336 bp amplicon with DNA from all 50 isolates. These results further confirmed the identification of the isolates as P. oryzae the pathogen is same that causes both leaf and neck blast. The genetic diversity of P. oryzae isolates was studied using a total of 12 MGM markers and 10 RAPD markers. The polymorphic information content value was high for RAPD primers i.e. 0.78 whereas MGM primers showed 0.52 only. Hence the study of genetic diversity among P. oryzae isolates using RAPD primers was more useful than MGM primers. The RAPD analysis formed four main clusters (I–IV) at the Jaccard similarity coefficient of 0.67. This grouping revealed that, all the isolates were clustered in accordance of geographical location but not in the specificity of leaf blast and neck blast isolates, because all the neck blast isolates were clustered in all groups along with the leaf blast isolates. Mating type distribution of P. oryzae isolates was studied by using mating type specific SSR primers. Most of the isolates were proved as MAT1-1. To identify the diversity of the three avirulence genes i.e., PWL-2, Avr-Piz-t and Avr-Pii, three specific primer pairs were used in this study. PWL-2 gene (614 bp) was present in 78% of the isolates whereas the gene Avr-Piz-t gene (642 bp) and Avr-Pii gene (534 bp) were present in 45 (90%) and 18 (36%) isolates, respectively. Hundred rice lines were evaluated for both leaf blast and neck blast resistance at both phenotypic and genotypic level during 2016 and 2017 seasons. In phenotypic screening, 46 lines were recorded as resistant for both leaf blast and neck blast. But in genotyping, among these 46 lines, only 25 lines were identified with resistance genes alone and with different combinations. Among these 25 lines, most of the lines were found with Pi1 + Pi54 + Pb1 (7 lines) followed by Pi1 + Pi54 (3 lines), Pi1 + Pi2 + Pi54 (3 lines), Pi1 + Pb1 (3 lines), Pi1 (2 lines), Pi2 (2 lines), Pb1 (2 lines) Pi54 (1 line) and Pi54 + Pb1 (1 line). Protein profiling of resistant and susceptible cultivars revealed a total of three differentially expressed proteins in resistant variety Tetep i.e., 30 kD, 48 kD and 55 kD sizes were observed upon imposition of blast disease. Ten Pseudomonas fluorescens isolates were evaluated for their antagonistic activity against P. oryzae by dual culture technique in vitro. Results indicated that, Pf 2 had highest per cent inhibition of mycelial growth (91.11%). The bio efficacies of fungicides i.e., Triazoles, Strobilurins and combination of triazoles and strobilurins along with one bio-agent P. fluorescens Pf 2 were evaluated against the leaf blast disease in a field laid out in Uniform Blast Nursery (UBN) pattern during kharif 2015-16. The least PDI was found in Trifloxystrobin+Tebuconazole @ 0.08% (13.33%) followed by Tricyclazole@0.06% (14.41%) and Pyraclostrobin@0.04% (18.14%) and no significant difference was found among themselves. The fungicide Tricyclazole was evaluated in vitro for its compatibility with the P. fluorescens Pf 2. No effect of tricyclazole was observed on bacterial growth. In vivo efficacy of Tricyclazole and P. fluorescens Pf 2 alone or in combinations were assessed against blast disease during Rabi 2015-2016 and Rabi 2016-2017 on a local highly susceptible cultivar NLR 34242 at ARS, Nellore. From the two years data, the treatments T1 (TST + 4 sprays of Tricyclozole); T8 (TST + T + T + P.f + P.f sprays) and T9 (P.fRD + T + P.f + T + P.f sprays) were proved as better for control of both leaf and neck blast disease with higher yield.en-USnullThesis