STUDIES ON INTERACTION OF LEAF AND NECK BLAST CAUSED BY Magnaporthe oryzae (Hebert) Barr AND ITS MANAGEMENT

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2022-11-21
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PROFFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY
Abstract
The present investigation was undertaken with an aim to know the interaction of leaf blast and neck blast pathogen population to host and the variability of isolates using cultural, morphological, pathological, molecular characteristics and avirulent gene distribution. Further efforts were also made to identify the effective R-gene for both leaf blast and neck blast resistance. Attempts were also made to manage the disease with non-chemical and biocontrol agent T. asperellum in integration. A survey on incidence of rice blast disease was conducted in 13 major rice growing districts of Telangana. The incidence varied from 8.24 per cent in Vikarabad district to 58.26 per cent in Warangal district. Disease incidence was recorded among all the prominent cultivars grown in their particular regions and it was observed that different cultivars showed different levels of incidence irrespective of the regions. Among the different cultivars, HMT Sona showed the highest incidence of 71.33 per cent whereas, cultivar Tellahamsa with 49.16 per cent incidence. The maximum isolation rate of the pathogen M. oryzae was recorded from Siddipet with 75 per cent followed by Mehbubnagar with 50 per cent. The disease was observed from nursery to grain hardening stage. A total of 40 monoconidial isolates of Magnaporthe oryzae were established. Out of forty isolates, 36 were leaf blast and 4 were neck blast isolates. The Principal Component Analysis (PCA) was performed for analyzing all forty isolates of M. oryzae growth pattern in 10 different media by considering different components (sectors, zonations, wrinkles, elevation, colony colour, Radial growth and growth rate) for studying cultural variability in M. oryzae population of Telangana. The components elevation, sectors, zonations and wrinkles have been observed to be in strong association among each other (F2: 23.59%). From the eigen value data, it was observed that three components (wrinkles, zonations and sectors) accounts for eigen values more than one and highest variance as indicated in PCA analysis. In present investigation it was ascertained that even in presence of different carbon compounds, host extracts as media source to the isolates their effect was insignificant i.e., irrespective of the carbon source and host extracts the above three parameters were not affected significantly. Oat meal agar was found best for supporting the growth of M. oryzae. However, ragi leaf extract agar media recorded as best for sporulation. Sucrose as carbon source and ammonium nitrate favours the growth of M. oryzae. Morphological variability among M. oryzae isolates was studied through the conidial morphology. Among the isolates, the overall size of the conidia was 14.43-26.80 µm × 3.68- 8.76 µm (Length × Width). A total of 25 rice host differentials with different combinations of resistance genes were tested in for rice blast resistance. Among 25 genotypes tested, NILA57, RamanadStr3, Shia-tia-tsao, Tetep, Tadukan and Dular were found resistant. Genotypes CO 39 and HR 12 were highly susceptible genotypes. A total of 18 races were identified among them the most frequently occurred race was ID-14 followed by IB-41. M. oryzae isolates were identified further to species level with ITS4 and ITS5 universal primers. Actin and TEF 1 α gene primers were also used for further accuracy of identification of conserved sequence, in order to support the morphological identification of M. oryzae isolates. In case of ITS region, all the isolates were appeared as identical by producing the amplified fragment of 560 bp. PCR amplification with actin gene and TEF 1 α gene produced the 336 bp and 550 bp amplicon with DNA from all fourty isolates. These results further confirmed by sequencing of ITS region and identified the isolates as M. oryzae, the pathogen is same that causes both leaf and neck blast. The genetic diversity of M. oryzae isolates was studied using a total of 8 SSR markers and 10 RAPD markers. The polymorphic information content value was high for SSR primers i.e. 0.85 whereas RAPD primers showed only 0.64. Hence the study of genetic diversity among M. oryzae isolates using RAPD primers was more useful than MGM primers. The RAPD analysis formed two main clusters at the Jaccard similarity coefficient of 0.50. 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 M. oryzae isolates was studied by using mating type specific SSR primers. Most of the isolates were proved as MAT1-1. We have identified 8 haplotype groups where, haplotype 2 was predominant (n=27) in the population. M. oryzae isolates were grouped into 8 haplotypes. Majority of the isolates (n=27) were under the Hap-2 group, found to be older haplotype with broad geographical distribution and quite older than other haplotypes as evidenced with its multiple connections with other haplotypes in the study area followed by Hap-4 (n=5) and Hap-1 (n=3) respectively. Haplotype 2 (Hp-2) has the broader genetic base in the entire study area with its swift distribution in the areas of Siddipet, Suryapet, Mehbubnagar, Karimnagar, Medak and Rangareddy. The broader genetic base of DNA sequence polymorphism analysis of Haplotypes was illustrated in the present study using PopART (Population Analysis Reticulate Tree). In the present investigation, Tajima’s D test (-2.660) Fu’s Fs statistics (2.931) supported presence of higher rare alleles in the population. To identify the diversity of the 14 avirulence genes, specific primer pairs were used in this study. The highest frequency of avirulent gene prominent in M. oryzae population was Avr Pi54 (97.5%) followed by Avr Pizt with (95%) and Avr Pi9 with (92.5%). The avirulent genes Avr Pik, ACE1, PWL2, Avr Pib, Avr Pii were able to amplify with a frequency of 90 per cent, 90 per cent, 80 per cent, 80 per cent and 17.5 per cent respectively. The avirulent genes with lowest prominence were PWL1, Avr Pita and Avr C039 with frequency of 7.5 per cent, 5 per cent and 5 per cent respectively. Five artificial inoculation methods, Spraying, smearing, cotton string, cotton wool and Injection were evaluated and out these, injection method gave 96.00 per cent PDI whereas, cotton wool method and string method showed 58 per cent PDI and 24 per cent PDI, respectively on HR 12 cultivar. In cross inoculation studies, a pool of isolates consists of two leaf blast isolates i.e., Mo-31, Mo-28 and two neck blast isolates i.e., Mo-3 and Mo-6 were used. The results indicated that all the isolates (both leaf and neck blast isolates) showed both leaf blast and neck blast infection. In cross inoculation studies on other cereal hosts the rice blast pathogen can infect ragi rather than wheat. In the present study, the interaction between 29 monogenic lines, carrying at least one major R gene, with 16 races commonly found. The monogenic lines carrying Piz5, Pi1, Pi2 and Pi54 provided broad spectrum resistance and their resistance frequencies were 62.5 per cent, 97.5 per cent, 97.5 per cent and 77.5 per cent respectively. The races of M. oryzae from Telangana were able to overcome 12 resistance genes (Pib, Piks, Pik, Pik-h, Pik-m, Pik-p, Pi1, Pi7(t), Pish, Pita, Pita-2, Pita-3 and Piz-t). This information is useful for development of durable resistant variety to blast disease in Telangana. In present investigation, non-chemical agents viz., neem oil, panchagavya, beejamrutham, vermiwash and buttermilk were tested against M. oryzae using Poisoned food technique. All the non-chemical agents at different concentrations were significantly superior to control in checking the mycelial growth of the fungus. Among the tested agents, panchagavya and neem oil showed 81.67 and 79.63 per cent inhibition of mycelium growth. The antagonistic microflora shown a antagonism percentage of 64.71 to 72.22 against M. oryzae under in vitro conditions, upon seed treatment they exhibited highest seed vigor index ranging from 1561.35 (B7) to 2183.68 (TAIK 1) when compared with control. Further, they were identified through internal transcribed spacer (ITS) and 16S rRNA gene sequences analysis for bacteria and fungi respectively and they were identified as Bacillus cereus, Bacillus xiamenensis, Bacillus subtilis and Trichoderma asperellum. Bio-efficacy tests were carried out in field conditions during the two consecutive years Kharif 2019-20 and Kharif 2021-22 by spraying different combinations of the non-chemical agents. From the two years data, the treatments T2 (Seed treatment with Beejamrutha+ Three foliar sprays of neem oil at 40, 60 DAT and panicle initiation) T5 (Seed treatment with T. asperellum + Three foliar spray of neem oil at 40, 60 DAT and panicle initiation) and T8 (Seed treatment with T. asperellum + Three foliar sprays of Panchagavya) were proved superior for control of blast disease with higher yield. The results showing significant improvement in the seedlings treated with different concentrations of non-chemical agents in terms of disease incidence reduction, lower AUDPC and lower r value during two consecutive years
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