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Professor Jayashankar Telangana State Agricultural University, Hyderabad (Telangana State)
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ThesisItem Open Access BIOPOLYMER BASED MULTILAYER SEED COATINGS WITH TRICHODERMA, RHIZOBIUM OR BACILLUS AND COMPATIBLE FUNGICIDES AGAINST SEED AND SOIL BORNE DISEASES IN SESAMUM, GROUNDNUT AND SOYBEAN(PROFFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY, 2023-01-09) Vijay Kumar, S.; Rajeswari, B.Sesamum, groundnut and soybean are important oilseed crops grown in many parts of the world. Among different fungal diseases, Macrophomina phaseolina in sesamum and soybean, Sclerotium rolfsii in groundnut are common, widespread and causing significant yield losses. M. phaseolina in sesamum and soybean, S. rolfsii in groundnut were isolated from infected plant parts and purified by hyphal tip method. Pathogenicity test was conducted on susceptible sesamum cv. Swetha Til, groundnut cv. K-6 and soybean cv. JS-335. Pathogen was re-isolated from artificially inoculated plants and found similar in all aspects thus fulfilling Koch’s postulates. The efficacy of six bioagents viz., T. asperellum (TaDOR 7316), Trichoderma sp. (T16), T harzianum (Th), T. asperellum (Ta10), T. asperellum (Ta11) and T. harzianum (Th4d) were evaluated against M. phaseolina isolates of sesamum and soybean and S. rolfsii of groundnut. The results revealed that among six bioagents tested, T. harzianum (Th4d) was found effective in inhibiting the mycelial growth of M. phaseolina of sesamum (66.3 %), S. rolfsii of groundnut (71.9 %) and M. phaseolina of soybean (67.0 %), respectively. The efficacy of five fungicides viz., carboxin 37.5 % + thiram 37.5 %, tebuconazole 25 % EC, mancozeb 75 %WP, penflufen 13.28 % w/w + trifloxystrobin 13.28 % w/w FS and chlorothalonil 75 % WP were evaluated under in vitro by poisoned food technique against M. phaseolina isolate of sesamum, S. rolfsii of groundnut and M. phaseolina isolate of soybean. Among five fungicides tested, penflufen + trifloxystrobin, chlorothalonil and tebuconazole were found effective at all the concentrations with 100 % inhibition in M. phaseolina of sesamum. Similarly, penflufen + trifloxystrobin fungicide was found highly effective at all the concentrations tested and inhibited 100 % growth of S. rolfsii in groundnut and M. phaseolina of soybean when compared with control. In-vitro compatibility of carboxin 37.5 % + thiram 37.5 %, tebuconazole 25 % EC, mancozeb 75 %WP, penflufen 13.28 % w/w + trifloxystrobin 13.28 % w/w FS and chlorothalonil 75 % WP at 20, 50, 100, 250, 500, 750 and 1000 ppm concentrations were tested with three Trichoderma isolates i.e., T. harzianum (Th4d), T. asperellum (TaDor 7316), T. harzianum (Th) and Bradyrhizobium sp., B. subtilis. Among five fungicides, penflufen + trifloxystrobin was found compatible with T. harzianum (Th4d) at all the concentrations of 20, 50, 100, 250, 500, 750 and 1000 ppm with 90 mm growth was observed. In case of bacterial bioagents, penflufen + trifloxystrobin was found compatible with Bradyrhizobium sp. and recorded OD value of 1.188 at 1000 ppm concentration. Compatibility among three Trichoderma isolates with Bradyrhizobium sp. and B. subtilis indicated that Trichoderma was over grew on Bradyrhizobium sp. and without formation of inhibition. Whereas, the growth of Trichoderma isolates were inhibited by B. subtilis isolate. Characterization of chitosan films were done by Fourier-Transform Infrared Spectroscopy (FTIR) and variation was observed in presence of fungicides and bioagents. Scanning Electron Microscope (SEM) microphotographs showed rough texture of chitosan blend film and undulation were observed in chitosan blended film with T. harzianum (Th4d) and Bradyrhizobium sp. was due to presence of spores of both bioagents. Standardization of multilayer seed coating with combination of biopolymer chitosan, fungicide and bioagents with different concentrations were tested in groundnut cv. K-6 under in vitro by using small pots and kept in growth chamber at 25± 20C with 90 per cent relative humidity. Among different seed treatment combinations tested, the double layer seed coating consisting of chitosan + fungicide + T. harzianum Th4d and chitosan + Bradyrhizobium sp. showed higher germination percentage and seedling vigour index - I and seedling vigour index - II as compared to three layer and single layer seed coating in all the concentrations tested. Combination of effective fungicide (penflufen + trifloxystrobin) with potential biocontrol agents (T. harzianum (Th4d) and (Bradyrhizobium sp.) and seed coat biopolymer chitosan and their combinations were tested against M. phaseolina of sesamum and soybean and S. rolfsii of groundnut by rolled paper towel method. Among different treatments, T5 treatment with double layer seed coating with chitosan 5 ml + fungicide 7.5 ml + Th4d 0.1 g as inner layer and chitosan 5 ml + Bradyrhizobium sp. 0.5 g as outer second layer was significantly enhanced seed germination per cent in sesamum and soybean (86.7 %) and groundnut (100%) as compared with untreated control (without pathogen) recorded germination of 71 %, 70 % and 80 %, respectively. Whereas under inoculated conditions with pathogen, the per cent seed germination was significantly enhanced in sesamum (83.8 %), soybean (83.1 %) and groundnut (94.0 %) as compared to untreated control with 59.0 %, 66.2 % and 64.0 %, respectively. In pot culture studies, the maximum per cent germination in sesamum and groundnut was recorded in T5 treatment with 80.0 % and 94.0 %, respectively. Whereas in soybean, the highest germination per cent was recorded in T5 and T7 treatments with 82.5 % of germination which was on par with each other. Disease incidence was low in T5 treatment with 21.6 %, 11.7 % and 13.1 % in sesamum, groundnut and soybean, respectively. Storability and shelf studies in seed coatings with biopolymer chitosan, fungicide and bioagents and their combinations in sesamum, groundnut and soybean at four months storage period revealed that seed quality parameters viz., seed germination and seed vigour index-I and seed vigour index II were gradually decreased with increase in period of seed storage. Maximum seed germination per cent of 81.4 %, root length (11.9 cm), shoot length (4.7 cm), SVI-I (1351), SVI-II (5.4) and dry weight (0.066 g) in sesamum were recorded in T5 treatment at the end of four months period of seed storage. The data on shelf life (Log CFUs) of T. harzianum (Th4d) and Bradyrhizobium sp. on coated seeds of sesamum recorded more number of colony forming units in T3 treatment (chitosan 5 ml + fungicide 7.5 ml + Th4d 0.1 g) + (chitosan 5 ml + Bradyrhizobium sp. 0.5 g) with 4.92 and 2.94 Log CFU g seed-1 of both T. harzianum (Th4d) and Bradyrhizobium sp., respectively. At the end four months period of seed storage in groundnut, recorded maximum germination per cent of 86.7 %, root length (19.6 cm), shoot length (10.1 cm), SVI-I (2254), SVI-II (49.8) and dry weight (0.33 g) in T5 treatment. The results on shelf life of T. harzianum (Th4d) and Bradyrhizobium sp. on coated seeds of groundnut recorded more number of colony counts in T3 treatment with 2.86 Log CFU g seed-1 and 4.69 Log CFU g seed-1 , respectively. Maximum germination of 78.8 %, root length (24.9 cm), shoot length (15.0 cm), SVI I (3137), SVI-II (41.1) and dry weight (0.52 g) were recorded in T5 treatment. The results on shelf life (Log CFUs) of T. harzianum (Th4d) and Bradyrhizobium sp. on coated seeds of soybean recorded more number of colony counts in T3 treatment with 2.86 Log CFU g seed-1 and 4.79 Log CFU g seed-1 , respectively at the end of four months period of seed storage in soybean. The results on persistence of T. harzianum (Th4d) and Bradyrhizobium sp. in chitosan and fungicide film after application in sterilized red and black soils in absence of plants over 120 days period revealed that maximum colony forming units of T. harzianum (Th4d) were recorded in T3 treatment with 3.54 and 3.50 Log CFUs/g. Whereas in Bradyrhizobium sp. the maximum colony forming units were observed in T3 treatment with 5.34 and 5.27 Log CFUs/ g at 90 days after application (DAA). Root colonization efficacy of T. harzianum (Th4d) and Bradyrhizobium sp. in sesamum, groundnut and soybean crop in red and black soil types were assessed at 15, 30 and 45 DAS. The maximum number of T. harzianum (Th4d) and Bradyrhizobium sp. colony forming units were observed in red soils in sesamum and groundnut crops whereas black soils in soybean crop at 45 DAS. In sesamum and soybean crops, the influence of seed coatings with effective fungicide (penflufen + trifloxystrobin), potential biocontrol agent (T. harzianum (Th4d), Bradyrhizobium sp.) and seed coat biopolymer (chitosan) and their combination under glasshouse were studied. The highest germination per cent, plant height (cm), number of pods per plant, hundred seed weight (g), seed yield per plant (g), number of seeds per pod and seed yield per ha (q) and low disease incidence (%) were recorded in T5 seed treatment. Effect of seed coating on biochemical parameters viz., oil content, protein and fatty acid content in sesamum and soybean were differed. In groundnut, the influence of seed coatings with effective fungicide (penflufen + trifloxystrobin), potential biocontrol agents (T. harzianum (Th4d), Bradyrhizobium sp.) and seed coat biopolymer (chitosan) and their combinations were tested under field conditions. The results indicated that highest germination per cent was recorded in T5 seed treatment with 91.9 %, field emergence index (84.8 %), plant height (42.8 cm), nodules (279.0), number of pods per plant (40.7), hundred seed weight (38.5 g), seed yield per plant (22.40 g), number of seeds per pod (1.84), seed yield per ha (21.1 q), haulm yield (4760.0 kg), B:C ratio (1:2.41) and low disease incidence (5.2 %) and AUDPC (102.8). Effect of seed coatings on biochemical parameters viz., oil content, protein and fatty acid content in groundnut were significantly different. Among seed treatments the higher oil content, protein content were recorded in T5 treatment with 52.85 % and 23.6 %.ThesisItem Open Access STUDIES ON VARIABILITY AND INTEGRATED DISEASE MANAGEMENT OF WILT OF SAFFLOWER (Carthamus tinctorius L.) CAUSED BY Fusarium oxysporum f. sp. carthami(PROFFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY, 2022-07-02) Rajendra prasad, M.; Vidya Sagar, B.Safflower (Carthamus tinctorius L.) is one of the major Rabi oilseed crop cultivated in the Deccan Plateau region of India. Wilt of safflower caused by Fusarium oxysporum f. sp. carthami Klisiewicz and Houston (Foc) results in yield loss up to 93 per cent in susceptible varieties. Wilt samples of safflower were collected from different locations of Telangana and Andhra Pradesh and fifteen isolates of Fusarium oxysporum f. sp. carthami were isolated from the collected diseased samples and they designated as FOC 1 to FOC 15. Morphological variations among collected isolates were studied. Based on the observed length of microconidia the isolates could be divided into 5 distinct groups i.e. Group I to V, based on macroconidia length the isolates could be divided into 3 distinct groups i.e. Group I to III. Based on chlamydospore diameter, the isolates could be divided into four distinct groups (Group I to IV), based on the cultural characters they were categorized into eight distinct groups (Group I to VIII) and based on colony diameter the isolates grouped into seven distinct groups (Group I to Group VII). The variation was present among the isolates at molecular level and the isolates were grouped into different clusters. The results of molecular variability revealed that There was a relationship exists between clustering of isolates based on molecular variability with the location. Pathogenic variability of the fifteen isolates of Fusarium oxysporum f. sp. carthami was tested using susceptible safflower cultivar PBNS-12 under high inoculum pressure in glasshouse conditions. Wilt incidence was recorded in the range of 53.3 % (FOC-11) to 93.3 % (FOC-I). Forty one germplasm lines were collected from ARS, Tandur and were screened against Fusarium oxysporum f. sp. carthami. Based on the disease scale (0-9), they were categorized from highly resistant to highly susceptible. Seed mycoflora detected from the seeds of 41 germplasm lines by using standard blotter method and agar plate methods. In both the methods, eight fungal genera viz., Alternaria spp., Aspergillus flavus, Aspergillus niger, Chaetomium spp., Rhizopus spp., Curvularia spp., Macrophomina phaseolina and Fusarium spp. were recorded. Among the observed fungi A. niger was found predominant (40.28 % and 42.52 %), while M. phaseolina was least (2.39 % and 2.02 %). Effect of fungicides and biocontrol agents on seed germination, seedling vigour index and seed mycoflora was evaluated on four popular verities of safflower viz., Nira, ISF-764, PBNS-12 and Manjira by standard blotter method and paper towel method. Seed treatment with captan (0.25 %) was found superior followed by seed treatment with Trichoderma harzianum (1 %), in all the four popular varieties tested in the testes parameters. Susceptible variety PBNS-12 was used to find the effect of seed treatment with bioagents, organic amendments and carbendazim and their combinations on per cent seedling emergence, pre emergence and post emergence mortality in glass house conditions. Among all the treatments tested, T11-combination treatment of (T1 + T2 + T3 + T4 (seed inoculation with test pathogen followed by seed treatment with Trichoderma harzianum (10 g kg-1 seed) + T2 (seed treatment with Pseudomonas fluorescens (10 g kg 1 seed) + T3 (seed treatment with carbendazim (1 g kg-1 seed) + T4 (soil application with neem cake (10 g kg-1 soil) was found superior in all the parameters discussed. When tested for growth parameters, shoot length was recorded highest in T11 (59.2 cm), root length was recorded highest in T6 (10 cm) and Total length was recorded highest in T11 (68.2 cm). Highest fresh weight (272.10 g) and dry weight (105.35 g) was also recorded from the same combination treatment. In integrated disease management experiment, when the cost benefit ratio is compared, T16 (Seed treatment with carbendazim) was found effective with C:B ratio of 1:67 followed by T14 Seed treatment with Trichoderma harzianum + Seed treatment with Pseudomonas fluorescens (1:52) and T3 Seed treatment with Trichoderma harzianum (1:50). Among the above treatments, T16 recorded highest yield (1681 kg ha 1 ) highest germination (88.42 %) and lowest wilt incidence (15.12 %) followed by T14 (Seed treatment with Trichoderma harzianum + Seed treatment with Pseudomonas fluorescens) recorded yield of 1580 kg ha-1 , germination of 82.33 per cent and wilt incidence of 22.10 per cent respectively.ThesisItem Open Access STUDIES ON INTERACTION OF LEAF AND NECK BLAST CAUSED BY Magnaporthe oryzae (Hebert) Barr AND ITS MANAGEMENT(PROFFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY, 2022-11-21) Rajeswari, E.; Vidyasagar, B.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 yearsThesisItem Open Access STUDIES ON CHILLI (Capsicum annuum L.) VIRAL COMPLEX IN MAJOR CHILLI GROWING AREAS OF TELANGANA AND ITS MANAGEMENT(PROFFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY, 2022-07-02) Yamini Sousheel, N.; Bharati Bhat, N.Survey conducted in major chilli growing areas of Telangana viz., Warangal, Mahabubabad, Mulugu, Jayashankar Bhupalpally, Nagarkurnool and Khammam during Rabi 2019-20 and Rabi 2020-21 seasons revealed the natural occurrence of chilli viral complex in different mandals. In all the districts, the disease incidence ranged from 2 to 40 per cent being maximum in Khammam (41.59 per cent) followed by Warangal (32.80 per cent) and Mahabubabad (26.79 per cent) and minimum disease incidence of disease was recorded in Mulugu (19.18 per cent) and Nagarkurnool district (20.21 per cent). Severity of disease complex varied with the cultivars. Different types of symptoms viz., leaf curl, puckering, mosaic, shoe string and yellowing were observed in the fields surveyed. Occurrence of thrips was recorded in every field irrespective of cultivar grown and the season surveyed but comparatively the population was higher during Rabi 2019- 20 in different chilli growing areas of Telangana. Among them, Scirtothrips dorsalis was showing higher incidence during Rabi 2019-20 (24.25 per 5 terminals) than Rabi 2020-21 (20.75 per 5 terminals) followed by Frankliniella schultzei (19.5 per 5 terminals) during Rabi 2019 and 20 per 5 terminals during Rabi 2020-21 respectively. Whitefly population was also observed in all the fields surveyed, their population constituted between 4 to 20 whiteflies/plant (Rabi 2019-20) and 5 to15 whiteflies/plant (Rabi 2020-21). During both the years of study whitefly infestation was highest in Warangal (8.50 whiteflies/ plant; 6.53 whiteflies/plant) followed by Khammam (6.35 whiteflies/plant; 5.75 whiteflies/plant), Mahabubabad (7.25 whiteflies/plant; 5.26 whiteflies/plant), Jayashankar Bhupalpally (5.75 whiteflies/plant; 5.10 whiteflies/plant), Nagarkurnool (2.55 whiteflies/plant; 1.35 whiteflies/plant) and Mulugu (1.55 whiteflies/plant; 1.25 whiteflies/plant. It has been observed that there was not much difference in its occurrence in rainfed - direct sown and irrigated - transplanted crop grown in these districts surveyed. In the present study, twenty weed species were found in all the surveyed fields across locations, type of soils and seasons. Parthenium hysterophorus was found to be most prevalent in chilli fields surveyed and frequency of occurrence of P. hysterophorus was maximum (78 per cent) in all the six districts under survey over two years followed by Amaranthus tricolor, Cyperus rotundus, Portulaca oleracea, Tridax procumbens, Digitaria marginalis, Argemone mexicana, Phyllanthus niruri, Commelina bengalensis, Datura stramonium, Sida acuta and Cleome viscosa. DAC-ELISA was carried out using CMV antisera during Rabi 2019-20 and 2020-21, and results revealed that out of a total of 80 virus isolates collected from different fields of Telangana during Rabi 2019-20, 55 isolates tested positive in DAC ELISA tests. Results of Rabi 2020-21 revealed that out of 80 isolates tested, only 45 isolates were positive for CMV. PCR amplification techniques were used in the present study for the detection of leaf curl virus in the infected samples collected during survey. Upon amplification by PCR, 1000 bp size amplicon was obtained in 25 leaf curl infected chilli samples. Results confirmed the specific association of Gemini virus (ToLCV) is involved with the chilli viral complex. Similarly, during Rabi 2020-21 a total of 30 samples were subjected to PCR amplification using the specific primers. Upon amplification by PCR, 1000 bp size amplicon was obtained in 25 leaf curl infected samples. These results confirm the presence of leaf curl virus in the suspected samples. Electron microscopic studies of negatively stained purified and leaf dip preparations at 20,000X magnification revealed the presence of typical spherical particles with an average diameter falling in the range of 28-30 nm. The sap from leaf curl infected samples was partially purified and examined by transmission electron microscopy. Twinned and icosahedral virions about 20 nm in diameter were observed in the purified preparation, which were the typical morphology of virus particles in the genus Begomovirus. Infectivity assays revealed that many weed species in different families that commonly grow in or near chilli fields harbor the viruses. In most cases, the infected weeds were asymptomatic. Symptoms that could be associated with virus infection such as yellowing, leaf curling, and interveinal chlorosis were only observed in some species belonging to the genera of Amaranthus, Datura, Tridax, Parthenium and Euphorbia which tested positive. Twenty six plant species belonging to ten families were tested under artificial inoculation for their susceptibility to CMV and leaf curl under insect proof conditions. Out of twenty six species tested for their reaction to chilli mosaic and leaf curl viruses, eighteen species have expressed the visible symptoms. The host range studies clearly indicated that weed species occurring in chilli fields and in crop plants such as groundnut, cowpea, curcurbits etc. were found to be the most common CMV sources under field conditions. Appearance of CMV in fields, where no known sources of CMV in the vicinity due to the migratory aphids and thrips carrying and contributing to the long distance spread of the virus. Three different modules were formulated and tested for the integrated management of chilli viral complex at two different locations. The disease appeared in all the modules at 4 weeks after transplanting (WAT). In general, the disease incidence increased with the age of the plants and recorded maximum incidence at 9 WAT. The disease progress was gradual in all the modules and at all the periods of observation. However, per cent disease incidence (PDI) was maximum at 9 WAT and least at 4 WAT. Out of the three modules the disease incidence was significantly less in module 2 during both the years consecutively, which recorded 27.2 per cent during Rabi 2019- 20 and 25.2 per cent during Rabi 2020-21 respectively, which can be due to the treatments like spray of thiomethoxam@0.2g/l at 30, 45 and 60 days, barrier crop (fodder maize 3 rows), trap crop (marigold) and sticky traps. Where module-3 was least effective due to lack of treatments like trap crop, barrier crop and systemic insecticides. The correlation analysis for whitefly population with weather parameters during Rabi 2019-20 and 2020-21 indicated that there existed positive and significant correlation of whiteflies with minimum temperature, morning humidity and wind velocity; but negative correlation with evening relative humidity and sunshine hours. Regression analysis between whitefly population and weather parameters indicated that minimum temperature and wind velocity accounted for 39 and 46 per cent total variation in whitefly population in both the seasons respectively. A detailed study was made on the effect of the viral complex on the yield parameters and the data was recorded in different modules for both the seasons. There was significant reduction in plant height, pod per plant and also the length of the pods in different modules over the control. In the present study there was significant increase in the severity of the disease (viral complex) and there was corresponding decrease in the yield, yield attributes of the chilli crop. Further the disease complex also influenced the yield contributing factors by mainly reducing the number of the pods per plant and also the length of the pods. The results indicated that there was significant difference between the CBR of different modules in correspondence with the disease incidence which had remarkable influence on the yield and yield attributes hence on the CBR. The present investigation revealed that module 2 not only reduced the number of application of pesticides to cut down the cost of production but also enhanced the yield of fruits with improved benefit cost ratio.ThesisItem Open Access IMPACT OF CLIMATE CHANGE ON THE OCCURRENCE OF GROUNDNUT STEM ROT DISEASE AND RE-ORIENTATION OF DISEASE MANAGEMENT STRATEGIES(PROFFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY, 2022-09-22) Vamshi, J.; Uma devi, G.Groundnut (Arachis hypogaea L.) is an important oilseed crop grown in India. Of the various diseases inciting groundnut, the stem rot caused by S. rolfsii is a potential threat to groundnut production. The present investigations were carried out to assess the diversity of S. rolfsii causing stem rot of groundnut, biochemical and physiological changes occurring in groundnut in response to climate change and pathogen interaction and to identify the potent fungal and bacterial biocontrol agents for effective management of disease. The roving survey conducted during kharif, 2019 and rabi, 2019-2020 in major groundnut growing areas of Telangana recorded 12.1 to 31.24 per cent and 10.11 to 25.66 per cent incidence of stem rot respectively. During kharif-2019, the mean disease incidence of stem rot in major growing areas of Telangana ranged from 16.93 to 21.80 per cent. Warangal district recorded the highest per cent mean disease incidence of (20.31%), followed by Wanaparthy (19.86%) and Nagarkurnool (19.18%). During rabi 2019-2020, the mean disease incidence of stem rot in major growing areas of Telangana ranged from 14.23 to 19.38 per cent. Warangal district recorded the highest per cent mean disease incidence of (17.53%), followed by Wanaparthy (17.03%) and Nagarkurnool (14.82%). Correlation of incidence of the disease and weather parameters for kharif, 2019 indicated that temperature, relative humidity and rainfall showed positive correlation, whereas evaporation showed negative correlation. In rabi, 2020 correlation of incidence of the disease and weather parameters indicated that temperature, relative humidity and evaporation showed positive correlation and rainfall showed negative correlation. All the 30 isolates of S. rolfsii in the study were found pathogenic. Further, there was great variation among the isolates for virulence levels on their host plants, hence the isolates were grouped into two categories viz., highly virulent (24) and less virulent (6). Further, positive correlation was found between the amount of oxalic acid produced in vitro and virulence of the isolates. Thus, the highly virulent isolates produced significantly highest amount of oxalic acid (2.00-2.61 mg/ml) whereas less virulent isolates produced least amount (0.42-0.79 mg/ml). Culturally the 30 isolates of S. rolfsii were found diverse. The growth rate of isolates ranged from 0.76 to 1.35 mm/hr. All the 30 isolates of S. rolfsii under study produced sclerotia on PDA medium. The isolates showed diverse growth pattern with respect to colony type. Most of the isolates produced colonies which were raised at ends (14) followed by flat type (12) and raised type (4). As per the mycelial growth type, most of the isolates were highly profuse in growth (18) and few were profuse in growth (12). Likewise, the 30 isolates were also found diverse morphologically. Wide variation was found with respect to number of sclerotia produced per petri dish by 30 isolates of S. rolfsii ranged from 58 to 536. Similar type of variation was found with respect to size of sclerotia produced by isolates tested and was ranged from 0.17 mm to 2.34 mm. Additionally, most of the isolates (26) produced the sclerotia of more than 1 mm in size and few (4) produced sclerotia of less than one mm in size. PCA extracted three main components from the population. Component 1 (growth rate) describes for highest (1.23) Eigen value with variance of 32.86 per cent while the lowest (0.06) Eigen value was observed in component 7 (scleroial size) with variance of 1.70 per cent. Out of four fungicides screened, 100 per cent inhibition of S. rolfsii was observed with thiram and tebuconazole at both recommended and half the recommended dosages. Wheareas, azoxystrobin recorded 89.07 per cent and 83.14 per cent inhibition at recommended concentration and at half the recommended concentration respectively followed by carbendazim which recorded 76.29 per cent and 34.29 per cent inhibition at recommended and half the recommended concentration respectively. The fungal bioagent Trichoderma viride (T2) and bacterial bioagent Bacillus cereus (B5) was found most effective against virulent isolate of S. rolfsii (SrPWp) and are known to be highly compatible with fungicide azoxystrobin with each other. Virulence of Sclerotium rolfsii decreased at increased levels of carbon dioxide levels in both TMV-2 and ICGV-14082 cultivars. The activity of total phenols, oxalic acid, ascorbic acid, catalase, peroxidases, polyphenol oxidase and chlorophyll content index was increased in stem rot infected groundnut plants as carbon dioxide levels increased. The biochemical activity was found highest in inoculated plants on 4th and 6th day post inoculation of pathogen (dpi) and gradually decreased on 8th and 10th dpi. Stomatal density and guard cell length in groundnut leaves were decreased as carbon dioxide levels increased. The biocontrol activity of fungal and bacterial bioagents increased as well as the effectiveness of fungicides increased as carbon dioxide levels increased. The bioagents Trichoderma viride and Bacillus cereus isolates performed well in glasshouse and field evaluation (at two locations, Patancheru and Rajendranagar), wherein, the bioformulation of treatment T10 (Trichoderma viride and Bacillus cereus as ST + SA + reduced rate of azoxysrobin) was found most effective in controlling stem rot of groundnut followed by treatment T8 (Trichoderma viride as ST + SA + reduced rate of azoxystrobin) and treatment T9 (Bacillus cereus as ST + SA + reduced rate of azoxystrobin) under glasshouse and field conditions. Further, these treatments recorded substantial amount of growth and yield attributing parameters and recorded highest pod yield and B:C ratio. Overall, the study revealed the considerable variability among field isolates of S. rolfsii. Further, the bioagent Trichoderma viride (T2) and Bacillus cereus (B5) have the potential to be used in the field for effective management of stem rot of groundnut.ThesisItem Open Access “INTEGRATED DISEASE MANAGEMENT OF FUSARIUM WILT OF TOMATO CAUSED BY Fusarium oxysporum f. sp. lycopersici (Sacc.) SYNDER AND HANSEN(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY, 2021-01-01) KISHORE KUMAR, CH. S.; BHARATI BHAT, NTomato is an important annual vegetable crop and known as poor man’s apple due to availability of vitamin C, minerals (Fe and Cu) and antioxidants. India ranks second to China in growing tomato with an area, production and productivity of 0.789 M ha, 21.24 Mt and 25 Mt ha-1 respectively. In India, tomato is the third important vegetable crop grown after potato and onion. The major tomato growing states in India are Odissa, Madhya Pradesh, Karnataka, Chhattisgarh, Andhra Pradesh and Telangana. In Telangana, tomato is cultivated in an area of 4,148 ha, with production 1171.50 Mt and productivity of 12 mt ha-1 .The major tomato growing districts in Telangana are Adilabad Rangareddy , Sangareddy, etc. Tomato is very often affected by several diseases incited by pathogens such as fungi, bacteria, viruses and nematodes. Among the fungal diseases, Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici (Fol), is one of the most destructive diseases across the world causing severe economic losses, wherever tomato is grown. Roving surveys were conducted in major tomato crop growing areas of Telangana state viz., Adilabad, Sangareddy and Rangareddy during the year 2017- 18 (kharif and rabi). A total of twenty seven villages from three districts were surveyed and recorded the wilt disease incidence ranging from 2.30 to 21. 33 per cent during kharif and 8.6 to 32.60 per cent during rabi, 2017-18. Among the three districts surveyed, Ranga Reddy district recorded maximum mean per cent disease incidence of 13.02 and 28.61 per cent during kharif and rabi, 2017-18, respectively. Fifteen isolates of Fusarium oxysporum f. sp. lycopersici collected from different tomato growing areas of Telangana were studied for their cultural, morphological, pathogenic and molecular variability. All the isolates showed wide variation in respect of mycelial colour and pigmentation, growth pattern, colony diameter, size and number of septations of macro and micro conidia and sporulation. Based on pigmentation, Fol isolates were categorized into 4 groups. Pathogenic variability studies revealed that Isolate Fol - 4, collected from Adilabad district was found to be more virulent in causing disease incidence and disease severity when inoculated to susceptible tomato cv. Pusa Ruby. Further, all the fifteen isolates were characterized at molecular level with species specific primer ITS 1( 5’TCCGTAGGTGAACCTGCGG-3′ ) and ITS 4 (5’-TCCTCCGCTTATTGATATGC-3′) and by using ISSR primers which revealed the relationship among the fifteen isolates with varied degree of coefficient. The most feasible method for managing Fusarium wilt disease of tomato is by growing resistant cultivars along with usage of effective fungicides. Twenty five germplasm lines were screened against Fol-4 under sick soil conditions and none of them were found to be resistant against Fusarium wilt disease especially at advanced age of the plant. Only six germplasm (EC 914 - 087, 088, 090, 100, 107, and 108) exhibited moderately susceptible reaction and seventeen germplasm as susceptible whereas, two germplasm were highly susceptible. Chemical methods i.e., fungicides with different mode of action were tested under in vitro conditions against Fol, which is the most effective method in managing the disease. It was found that (tebuconazole + trifloxystrobin) at 1000 ppm was very effective in inhibiting the radial growth of the Fol which was further tested at field level. Among the bio agents screened against Fol under in vitro conditions, Trichoderma viride (Tv 2) was found to be very effective in inhibiting the radial growth of the Fol., which was further evaluated under field conditions. A field experiment was conducted during rabi, 2018-19 and kharif, 2019-20 to evaluate the best integrated disease management strategies against the Fusarium wilt disease of tomato. The results revealed that among the different integrated disease management strategies comprising of fourteen treatments, integration of cultural (Soil application of neem cake @ 250 kg ha -1 + FYM @ 1000 kg ha -1) + chemical method (foliar application and soil drenching with (tebuconazole + trifloxystrobin) @ 0.1 per cent and biological method (soil application of Trichoderma viride (Tv2) @ 10 kg ha-1 was found to be effective in managing the Fusarium wilt incidence in the both the seasons, which recorded lowest disease incidence of 8.00 per cent and the highest yield of 31.53 t ha-1 with C : B ratio 1: 2.30 during rabi, 2018-19 and 6.66 per cent disease incidence with 30.60 t ha-1with C: B ratio of 1 :2.22 during kharif, 2019-20. Under the best disease management module tested at field level, it was found that integrating with cultural, chemical and biological methods had effectively managed the Fusarium wilt disease which can be recommended to the farming communityThesisItem Open Access INTEGRATED DISEASE MANAGEMENT OF FUSARIUM WILT OF TOMATO CAUSED BY Fusarium oxysporum f. sp. lycopersici (Sacc.) Synder and Hansen(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURE UNIVERSITY, 2021-01-01) KISHORE KUMAR, CH.S; BHARATI, NTomato is an important annual vegetable crop and known as poor man’s apple due to availability of vitamin C, minerals (Fe and Cu) and antioxidants. India ranks second to China in growing tomato with an area, production and productivity of 0.789 M ha, 21.24 Mt and 25 Mt ha-1 respectively. In India, tomato is the third important vegetable crop grown after potato and onion. The major tomato growing states in India are Odissa, Madhya Pradesh, Karnataka, Chhattisgarh, Andhra Pradesh and Telangana. In Telangana, tomato is cultivated in an area of 4,148 ha, with production 1171.50 Mt and productivity of 12 mt ha-1 .The major tomato growing districts in Telangana are Adilabad Rangareddy , Sangareddy, etc. Tomato is very often affected by several diseases incited by pathogens such as fungi, bacteria, viruses and nematodes. Among the fungal diseases, Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici (Fol), is one of the most destructive diseases across the world causing severe economic losses, wherever tomato is grown. Roving surveys were conducted in major tomato crop growing areas of Telangana state viz., Adilabad, Sangareddy and Rangareddy during the year 2017- 18 (kharif and rabi). A total of twenty seven villages from three districts were surveyed and recorded the wilt disease incidence ranging from 2.30 to 21. 33 per cent during kharif and 8.6 to 32.60 per cent during rabi, 2017-18. Among the three districts surveyed, Ranga Reddy district recorded maximum mean per cent disease incidence of 13.02 and 28.61 per cent during kharif and rabi, 2017-18, respectively. Fifteen isolates of Fusarium oxysporum f. sp. lycopersici collected from different tomato growing areas of Telangana were studied for their cultural, morphological, pathogenic and molecular variability. All the isolates showed wide variation in respect of mycelial colour and pigmentation, growth pattern, colony diameter, size and number of septations of macro and micro conidia and sporulation. Based on pigmentation, Fol isolates were categorized into 4 groups. Pathogenic variability studies revealed that Isolate Fol - 4, collected from Adilabad district was found to be more virulent in causing disease incidence and disease severity when inoculated to susceptible tomato cv. Pusa Ruby. Further, all the fifteen isolates were characterized at molecular level with species specific primer ITS 1( 5’TCCGTAGGTGAACCTGCGG-3′ ) and ITS 4 (5’-TCCTCCGCTTATTGATATGC-3′) and by using ISSR primers which revealed the relationship among the fifteen isolates with varied degree of coefficient. The most feasible method for managing Fusarium wilt disease of tomato is by growing resistant cultivars along with usage of effective fungicides. Twenty five germplasm lines were screened against Fol-4 under sick soil conditions and none of them were found to be resistant against Fusarium wilt disease especially at advanced age of the plant. Only six germplasm (EC 914 - 087, 088, 090, 100, 107, and 108) exhibited moderately susceptible reaction and seventeen germplasm as susceptible whereas, two germplasm were highly susceptible. Chemical methods i.e., fungicides with different mode of action were tested under in vitro conditions against Fol, which is the most effective method in managing the disease. It was found that (tebuconazole + trifloxystrobin) at 1000 ppm was very effective in inhibiting the radial growth of the Fol which was further tested at field level. Among the bio agents screened against Fol under in vitro conditions, Trichoderma viride (Tv 2) was found to be very effective in inhibiting the radial growth of the Fol., which was further evaluated under field conditions. A field experiment was conducted during rabi, 2018-19 and kharif, 2019-20 to evaluate the best integrated disease management strategies against the Fusarium wilt disease of tomato. The results revealed that among the different integrated disease management strategies comprising of fourteen treatments, integration of cultural (Soil application of neem cake @ 250 kg ha -1 + FYM @ 1000 kg ha -1) + chemical method (foliar application and soil drenching with (tebuconazole + trifloxystrobin) @ 0.1 per cent and biological method (soil application of Trichoderma viride (Tv2) @ 10 kg ha-1 was found to be effective in managing the Fusarium wilt incidence in the both the seasons, which recorded lowest disease incidence of 8.00 per cent and the highest yield of 31.53 t ha-1 with C : B ratio 1: 2.30 during rabi, 2018-19 and 6.66 per cent disease incidence with 30.60 t ha-1with C: B ratio of 1 :2.22 during kharif, 2019-20. Under the best disease management module tested at field level, it was found that integrating with cultural, chemical and biological methods had effectively managed the Fusarium wilt disease which can be recommended to the farming communityThesisItem Open Access OCCURRENCE AND DISTRIBUTION OF AFLATOXIN AND IT'S BIOLOGICAL MANAGEMENT IN GROUNDNUT(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURE UNIVERSITY, 2021-01-01) SOMASHEKHAR, K; RAJESWARI, BGroundnut (Arachis hypogaea L.) is one of the most important food crop in India which contains 46 per cent oil and source of nutrition for human and animals. Aflatoxin contamination is a serious problem and has a tremendous impact on the global groundnut industry as well as posing public health risks. The present investigation was carried out to know the occurrence of aflatoxin in soil and prevalence of pre-harvest aflatoxin contamination, detection of aflatoxigenic nature of Aspergillus flavus isolates and evolving an effective aflatoxin management strategy using resistant groundnut genotypes and Streptomyces bioagent strains. A roving survey was conducted during Rabi, 2018-19 and Kharif, 2019 in major groundnut growing areas of Telangana state and recorded A. flavus kernel infection with a range of 0 to 70 per cent and 0 to 60 per cent, respectively. Among different districts, highest mean A. flavus kernel infection was recorded in Mahabubnagar district during both Rabi, 2018-19 (23.60 %) and Kharif, 2019 (26.25 %) whereas lowest kernal infection was observed in Wanaparthy (10.61 %) and Nagarkurnool districts (14.55 %) during Rabi, 2018-19 and Kharif 2019, respectively. The average A. flavus population was highest in soils of Mahabubnagar district during both Rabi, 2018-19 (7.8 × 103 cfu/g) and Kharif, 2019 (6.4 × 103 cfu/g) whereas lowest average A. flavus population was recorded in soils of Warangal district (1.5 × 103 cfu/g) and Nagarkurnool district (2.8 × 103 cfu/g) during Rabi, 2018-19 and Kharif, 2019, respectively and it was negatively correlated with soil exchangeable calcium content whereas soil pH was not related to soil population of A. flavus. A total of 48 A. flavus isolates were collected from major groundnut growing locations of Telangana state and showed variation in their cultural and morphological characters. A. flavus isolates showed variation in colony color from dark green (6 Nos.) to light green (42 Nos.) and margin of colonies varied from green color (31Nos.) to white (17 Nos.) with smooth (28 Nos.) to irregular margin (20 Nos.). Only 14 isolates produced sclerotia with variation in sclerotial pattern of concentric (8 isolates), irregular (2 isolates) and peripheral pattern (4 isolates). Variation was also observed in number of sclerotia per plate (32 to 534) and size of sclerotia (266.00 µm to 421.33 µm). The maximum radial mycelial growth of 85 mm was observed in seven isolates. The least radial mycelial growth was recorded in isolates of AFMB-8 and AFMB-9 (60 mm). A. flavus isolates exhibited wide range of variability in potency of toxin production from 4.90 to 3629.90 µg kg-1as determined by indirect competitive ELISA. The isolate AF 11-4 (ICRISAT) was found highly toxigenic (3629.9 µg kg-1) followed by AFNG-5 (3584.6 µg kg-1) and AFWRG-4 (3352.5 µg kg-1). Coconut cream agar test showed that 37 A. flavus isolates were found positive for aflatoxin production and 12 isolates were showed negative results for aflatoxin detection. The results of ammonia vapor test revealed that 42 isolates of A. flavus turned to plum red color upon ammonia hydroxide treatment indicated the presence of aflatoxin and seven isolates did not turn to plum red color indicating atoxigenic or less toxigenic nature of the isolates. PCR amplification indicated that all the selected A. flavus isolates showed presence of all three aflatoxin genes (aflR, norA and ver1) except isolate AFWNP-1 showed absence of norA gene. The highest average aflatoxin content was observed in soil samples of Nagarkurnool district (3.07 µg/kg) and Wanaparthy district (4.02 µg kg-1) during Rabi, 2018-19 and Kharif, 2019 respectively whereas least average aflatoxin content was observed in the soil samples of Nalgonda district (1.32 µg/kg and 1.27 µg kg-1) during both Rabi, 2018-19 and Kharif 2019, respectively. With respect to prevalence of aflatoxin in groundnut kernels, Mahabubnagar district recorded as high aflatoxin prone area (> 30 µg kg-1) whereas Warangal district and Nagarkurnool district recorded as aflatoxin free area (< 15 µg kg-1). In vitro seed colonization studies indicated that three groundnut genotypes viz., ICGV 17001, ICGV 181004 and ICGV 181002 showed resistant reaction and 16 groundnut genotypes found moderately resistant reaction for seed colonization against A. flavus AF 11-4 (ICRISAT isolate) and lowest aflatoxin accumulation was found in the groundnut genotype ICGV 171021 (5.45 µg kg-1) and five groundnut genotypes recorded lower levels of aflatoxin content (< 15 µg kg-1) in in vitro seed colonization studies. Screening of groundnut genotypes under sick field conditions during Rabi, 2018-19 indicated that groundnut genotypes viz., ICGV 16024, ICGV 16027, ICGV 181013 and ICGV 181036 were found free from A. flavus infection in . Kernel infection was found lowest (1.67 %) in the groundnut genotypes viz., ICGV 171002, ICGV 171011, Sunoleic 95-R. Pre-harvest aflatoxin content in groundnut genotype, ICGV 171011 was 0.37 µg kg-1 and it was less than 10 µg kg-1 in 15 tested groundnut genotypes. Groundnut genotype, ICGV 171001 showed resistance to in vitro seed colonization, per cent kernel infection and pre-harvest aflatoxin contamination under both in vitro and field conditions. The results of the dual culture plate technique among the tested Streptomyces bioagent strains and pathogen indicated that strain KAI-26 showed highest per cent mycelial growth inhibition of 60 per cent and it was on par with strains viz., MMA-32, CAI-13, CAI-155, KAI-27 and CAI-24. Culture filtrates of strain KAI-26 at 25 % concentration was found best with highest mycelial growth inhibition of 60.39 per cent. The results on crude secondary metabolites assay indicated that strain CAI-13 showed highest per cent mycelial growth inhibition of 55.74 per cent and was on par with the strain KAI-26 (53.33 %). Groundnut seeds (cv. JL 24) treated with Streptomyces strain KAI-26 showed least seed colonization severity (Scale 1.3) and least aflatoxin accumulation (386.09 µg kg-1) and it was found on par with the aflatoxin accumulation in seeds treated with Streptomyces strains CAI-13 (423.03 µg kg-1) and CAI-24 (537.05 µg kg-1). In vitro evaluation of Streptomyces strains for plant growth promotion indicated that Streptomyces strain KAI-26 increased plant germination percentage (82.67 %), shoot length (15.87 cm), root length (12.13 cm) and seed vigour index (2405) and found on par with strain CAI-13 in susceptible groundnut cv. JL 24. Based on dual culture plate technique, culture filtrate, crude secondary metabolite assay and in vitro seed colonization studies, Streptomyces strains CAI-13, CAI-24, KAI-26 and MMA-32 were further selected for compatibility. The results showed that strains CAI-13, KAI-26 and MMA-32 were compatible with each other and used for preparation of consortium. Evaluation of bioformulation of effective strain KAI-26 and its consortium under glasshouse conditions indicated that combined application of seed treatment and soil application of bioformulation of consortium recorded least kernel infection (10 %) and aflatoxin content in groundnut susceptible cv. JL 24 (4.36 µg kg-1). Field evaluation studies indicated that combined application of bioformulation of consortium through seed treatment and soil application was found superior in promoting plant growth and yield attributes and recorded lowest soil population of A. flavus at fields of ICRISAT (15.33×102 cfu/g soil) and fields of RARS, Palem (8.33×102 cfu/g soil) and found on par with combined application of seed and soil application of effective strain KAI-26 (17×102 cfu/g soil and 11.33×102 cfu/g soil) at both locations of ICRISAT and RARS, Palem. The kernel infection was lowest in the treatment involving combination of seed and soil application of consortium (11.67 %) and found on par with soil application of consortium (13.33 %) at ICRISAT and combined application of seed treatment and soil application of effective strain KAI-27 (14.17 %). Soil application of Streptomyces strain KAI-26 was found next best treatment which recorded 17.50 per cent kernel infection. Similarly, combination of seed and soil application of consortium recorded lowest kernel infection (8.67 %) which was on par with soil application of consortium (10 %) and combined application of seed treatment and soil application of effective strain KAI-26 bioformulation (11.33 %) at RARS, Palem. Combined application of seed treatment and soil application of consortium was found effective with lowest pre-harvest aflatoxin contamination (13.45 µg kg-1) and was on par with soil application of consortium (15.26 µg kg-1) and seed treatment with consortium (18.75 µg kg-1). The combined application of seed treatment and soil application of an effective strain KAI-26 was found next best treatment (20.45 µg kg-1) at ICRISAT location. At RARS, Palem, the lowest pre-harvest aflatoxin contamination was recorded in combined application of seed treatment and soil application of consortium (7.00 µg kg-1) and was on par with soil application of consortium (8.99 µg kg-1) and seed treatment with consortium (11.01 µg kg-1) and also with combined application of seed treatment and soil application of an effective strain KAI-26 alone (12.29 µg kg-1). Soil application with an effective strain KAI-26 alone was found next best treatment (13.88 µg kg-1).ThesisItem Open Access STUDIES ON THE EFFICACY OF BIOAGENTS AND THEIR METABOLITES AGAINST BACTERIAL LEAF BLIGHT (Xanthomonas oryzae pv. oryzae) DISEASE OF RICE (Oryza sativa L.)(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY, 2022-11-12) DIVYA MISHRA; RAJESWARI, BRice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major threat to the rice cultivation in almost all the major rice growing regions of the country. Detailed investigations were carried out on symptomatology, cultural and morphological characters of the pathogen, isolation of rhizosphere microflora and studied cultural, morphological and biochemical parameters and tested their antagonistic activity against the pathogen under in-vitro, net house and field conditions in two rice varieties Telangana Sona and TN1. Molecular identification of bacterial blight pathogen Xoo, Trichoderma and Bacillus isolates by PCR technique. Secondary metabolites from potential isolates were extracted and tested their efficacy against the pathogen under in-vitro. Attempts were made to develop effective microencapsulated formulations, study their structure and test their efficacy against Xoo in rice variety Telangana Sona under glasshouse and field conditions. The disease is characterized by kresak phase, leaf blight phase and yellowing of leaves. The pathogen associated with the disease was identified as Xanthomonas oryzae pv. oryzae and the pathogenicity was confirmed under glasshouse condition. For isolation of bioagents, rhizosphere soil samples were collected from different rice growing regions of Telangana State (Hyderabad, Khammam, Nalgonda and Suryapet). A total of ten isolates of Trichoderma and five isolates of Bacillus were isolated by serial dilution plate method. Cultural, morphological and biochemical characterization of Trichoderma and Bacillus isolates were studied under in-vitro conditions. Initial in vitro screening of the collected isolates indicated that growth rate of Trichoderma isolate T6 (2 days) and Bacillus isolate B1 (18 hours) were found fast as compared to the other fungal and bacterial isolates. Quantitative estimation of biochemical parameters showed that Trichoderma isolate T6 produced maximum amount of IAA (69.73 mg/ ml), PS (153.16 μg/ml), siderophores (92.70 % siderophore units), chitinases (32.52 min-1 mg-1 Protein) and β-1,3-gucanases (1.93 nmol/s/ml). In case of Bacillus isolates, B1 isolate showed highest amount of IAA (10.97 mg/ ml), PS (65.87 μg/ml), siderophores (40.35 % siderophore units) and biofilm (1.48 OD600nm) production. To compare with the isolated antagonists, Trichoderma asperellum (TAIK1) and Bacillus subtilis (BIK3) of IIRR were included. Antagonistic activity of Trichoderma isolates was evaluated against Xoo under in-vitro conditions using dual culture plate technique. In case of Bacillus isolates, efficacy of extracellular metabolites was tested to control the growth of pathogen. Trichoderma isolate TAIK1 followed by T6 and Bacillus isolate BIK3 followed by B1 showed maximum inhibition of pathogen. Microscopic and molecular identification of Xoo, T6 and B1 was done by using compound microscope, scanning electron microscopy (SEM) and specific primers in Sanger sequencing. Nucleotide sequencing, NCBI BLAST and phylogenetic analysis confirmed the identity of pathogen as Xanthomonas oryzae pv. oryzae (Xoo), T6 as Trichoderma atroviride and B1 as Bacillus subtilis. The two potential fungal isolates (TAIK1 and T6) and two bacterial isolates (BIK3 and B1) were used for evaluation of their efficacy against pathogen in the rice varieties, Telangana Sona and TN1 under net-house conditions. The plants treated with combination of Trichoderma isolates TAIK1 and T6 showed maximum per cent increase in shoot and root growth over control. However, plants treated with combination of Bacillus isolates BIK3 and B1 showed maximum per cent disease reduction over control in both the rice varieties. Varietal comparison showed that the performance of bioagents and their combination were found significant in rice variety Telangana Sona than TN1. Most potential isolates from net house experiment results viz., TAIK1 and BIK3 were tested against Xoo under field conditions in the rice varieties, Telangana Sona and TN1. Accordingly, seeds of Telangana Sona treated with TAIK1 showed maximum per cent increase in shoot and root length and highest per cent disease reduction over control. Extraction of secondary metabolites was done from TAIK1 and BIK3. Crude extract was purified through TLC to obtain different fractions of bioagents metabolites. Fraction 2 of TAIK1 was recorded to inhibit total growth of Xoo in MWA media at a concentration of 150 ppm. Nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HR-MS) confirmed that the dominant compound in fraction 2 was 6-pentyl-2H-pyran-2-one. Compatibility studies of TAIK1 and BIK3 with antibiotic streptomycin at different concentrations (50, 150, 250 and 500ppm) revealed maximum tolerance of TAIK1 at 50 ppm and no tolerance of BIK3 with streptomycin. Microencapsulated formulation of TAIK1 and BIK3 was developed through emulsion method using carriers like sodium alginate, gelatin and calcium chloride. Studies with compound microscope, scanning electron microscope (SEM) and dynamic light scattering (DLS) confirmed the final formulation structure (globose for TAIK1 and cylindrical for BIK3) and size (42 to 500 nm for TAIK1 and 20 to 408 nm for BIK3). The time taken for complete release of bioagents in the suspension from micro-encapsulated formulations was minimum of 30 mins. Minimum fluctuations in population density (measured as colony forming units) was found in micro-encapsulated formulations when compared with other formulations viz., talc, liquid and sorghum based. The encapsulated formulations were diluted with water and used as seed treatment in rice variety Telangana Sona. Due to paucity of large-scale production of microencapsulated formulations these formulations were mass multiplied in broken rice grain and used for soil application under field conditions. Plant growth promoting parameters viz., shoot length, root length and total dry matter and yield parameters viz., flag leaf length, panicle length, yield per hill and test weight were highest in the plants treated with combination of micro-encapsulated bioagents (TAIK1 and BIK3). In case of plants treated with the micro-encapsulated bioagents and challenge inoculated with pathogens, plants which were treated with micro-encapsulated TAIK1 and streptomycin @ 50 ppm was found most effective in control of Xoo as compared to other treatments. Significant increase in production of defense related enzymes viz., PAL, PO, PPO and SOD were observed after 24 h of pathogen inoculation. The peak enzyme production was recorded at 72 h and started declining after 120 h. The enzyme activities were found to be maximum in plants treated with micro-encapsulated TAIK1 and streptomycin (@50ppm). Evaluation of photosynthetic parameters viz., photosynthetic rate (PN), stomatal conductance (gs), transpiration rate (E) and internal CO2 concentration (Ci), photosynthetic pigments viz., Chl a, Chl b and carotenoids and chlorophyll fluorescence measured as maximal quantum yield (Fv/Fm), effective quantum yield (ΦPSII), photochemical quenching (qP) and electron transport rate (ETR) showed significant improvement in plants treated with micro-encapsulated bioagent formulations. Maximum activity of these parameters were observed in plants treated with combination of microencapsulated bioagents. However, non-photochemical quenching coefficient (qN) and yield of non-photochemical quenching (YNPQ) were higher in control treatment (plants inoculated with pathogen only). The present study identified the micro-encapsulated formulations of TAIK1 and BIK3 as potential formulation to control the spread of Xoo in rice variety Telangana Sona and improve the plant growth
