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ThesisItem Open Access Molecular phylogenetic analysis of xanthomonas axonopodis pv. punicae isolates and bio-prospecting of selected botanicals and bio-agents against bacterial blight of pomegranate(UAS Dharwad, 2013) Basamma; V. I. BenagiPomegranate, Punica granatum L., regarded as the “Fruit of Paradise” is subjected to various disease problems. Among diseases, bacterial blight caused by Xanthomonas axonopodis pv. punicae (Hingorani and Singh, 1959) Vauterin et al. is one of the important diseases, which caused a great havoc in recent years. The present investigation was undertaken to assess the diversity among different isolates collected from different agroclimatic regions and to manage this disease effectively. Water soaked lesions on leaves were produced after six days of inoculation. All the eighteen isolates of Xap showed hypersensitive symptoms in Nicotiana tabacum leaves. Colony characters of eighteen Xap isolates varied from circular and entire colony shape; slightly to highly mucoid character. Among the varied temperature and pH levels tested, 28±10C and pH of 6.0 to 7.0 were found optimum. All eighteen isolates of Xap produced fuscan pigment on LB agar after nine days of incubation. Among the bactericides, K-Cycline was found effective against all isolates of Xap, which was significantly superior to rest of the bactericides. The multiplex polymerase chain reaction was performed using template containing DNA from Xap targeting 16S rRNA and gyrB gene by 16S rRNA and gyrB specific primers. High intensity of amplification of the gene specific products of 1537 bp and 491 bp corresponding to 16S rRNA and gyrB were obtained at annealing temperature of 57.5º C for 1 minute as compared to other temperatures. Aqueous soapnut extract, Pseudomonas fluorescens strain [326(4)] and Pink Pigmented Facultative Methylotroph (PPFM) strain 10L, CuSO4, ZnSO4, COC, Streptocycline showed inhibition zone. Average fruit yield of two locations recorded significantly higher fruit yield per tree in T9 (COC 0.3% + streptocycline 0.05%) and T5 (P. fluorescens strain 326 (4) 1.0% + soapnut 5.0%) which were on par with each other. The maximum C:B ratio was observed in COC + streptocycline (0.3+0.05%).ThesisItem Open Access Onion twister disease: etiology, their characterization, epidemiology and integrated management(UAS Dharwad, 2013) Suresh Patil; V. B. NargundIn recent years, twister disease of onion has become epidemic in coastal tract and other onion growing districts of Karnataka which caused heavy loss. Survey carried out during kharif and rabi/summer 2011-12 and 2012-13 revealed highest PDI 26.71 in Uttar Kannada district. Typical symptoms of the disease: twisting of leaf, neck with blight as well as dieback (anthracnose), scanty root system with galls and showing fungal growth was noticed. Artificial inoculations of onion seedlings with Colletotrichum gloeosporioides, Fusarium oxysporum, Meloidogyne spp. alone and in combinations expressed twister disease symptoms. Metabolomic changes like increased total sugars and growth hormones (IAA and GA) were seen. A two year pooled correlation analysis of weather parameters on incidence and severity showed that cumulative rainfall contributed more (0.97) to disease development followed by maximum temperature (0.41). Among the different planting dates, least incidence was recorded in 15th January. Molecular identification of fungi by amplification of ITS rDNA region was done, sequenced and confirmed as C. gloeosporioides, C. acutatum, Glomerella acutata, F. oxysporum and Gibberella moniliformis. Sequences were deposited in genbank. Specific amplification of CgInt region at 450 bp for C. gloeosporioides ; for C. acutatum CaInt region at 490 bp and for F. oxysporum at 550-570 bp was obtained. PCR – RFLP with HaeIII resulted in a characteristic pattern of three fragments. In vitro evaluation of fungicides revealed triazolels and combi products were effective in inhibiting growth of both fungal pathogens. Botanicals as consortia of plant extracts (neem, prosopis, clerodendron and Vinca rosea) and bioagent Trichoderma harzianum were also effective. Integrated management of twister disease revealed that, T5 (Adoptive + Nutrient module) recorded least PDI 20.50 and 19.86 with maximum yield of 67.33 t/ha and 39.36 t/ha at Dharwad and Kumta locations respectively.ThesisItem Open Access Development of bud blight viral disease resistant transgenic tomato(UAS Dharwad, 2013) Gurupada B. Balol; M. S. PatilTomato, an economically important crop in many countries is affected by many viral diseases including bud blight disease caused by the Groundnut bud necrosis virus, belongs to the family Bunyaviridae a member of group Tospovirus . Survey was carriedout to assess the GBNV in major tomato growing regions of Dharwad, Belgaum, Haveri, Kolar and Bangaluru rural districts during summer 2011. From the survey it is revealed that the highest incidence (68%) of bud blight was noticed in fields of Poovandahalli village in Kolar district, whereas least (12%) incidence of the disease was recorded at Singanalli village of Dharwad district. The coat protein (CP) gene of five GBNV isolates of Karnataka were cloned and characterized. The sequence similarity index revealed, that the GBNV- To- DWD coat protein gene sequence had 96.0 per cent homology with GBNV-To-BGM, 99.2 per cent homology with GBNV- To- HVR, 94.9 per cent homology with GBNV- T0-KLR and 94.8 per cent homology with GBNV-To-BAN-R isolates. Highest homology of 99.2 per cent was found between CP gene sequences of GBNV To- DWD and GBNV- To- HVR followed by 96.1 per between GBNV-To- BGM and GBNV-To-BAN-R. Therefore, GBNV- T0 -DWD isolate was selected for plant transformation studies. The transgenic tomato (DMT-2) lines were developed by following in planta transformation method. PCR analysis of the genomic DNA isolated from T1 transformants demonstrated that the transgene was integrated in the genome of the twenty four out of thirty transgenic tomato plants. ELISA using the antibody against the coat protein indicated that the transgenic plants expressed the coat protein at varying levels. Bioassay results also indicated the coat protein mediated resistance by the stability of the transgene.ThesisItem Open Access Variability in rhizoctonia solani kühn, the causal agent of sheath blight of rice and its management(UAS Dharwad, 2013) Nagaraju P.; M. K. NaikThe sheath blight of rice caused by Rhizoctonia solani (Kuhn.) is considered as major bottleneck in successful production of rice in irrigated tract of Tungabhadra Project area of north eastern zone of Karnataka. The study comprising survey on prevalence and incidence of sheath blight of rice, collection of sheath blight pathogenic isolates for exploring cultural, morphological and molecular variability, evaluation of rice genotypes, influence of planting density, nitrogen level, botanicals and biocontrol agents and IDM against sheath blight of rice was also attempted. The study identified pockets in Bellay, Koppal and Raichur districts under TBP area affected by moderate to severe incidence of sheath blight compared to Cauvery belt areas in Mysore, Shimoga and Mandya during kharif 2006 and kharif 2007. The diversity with respect to morphological and cultural characters of 32 isolates of R. solani on PDA revealed that the maximum radial growth of mycelia was noticed in isolates RS-14, RS-16 and RS-18 each with 89.33 mm. Based on the morphological and cultural characteristics, the isolates of R. solani were grouped into four and designated as G1, G2, G3 and G4 where as the molecular analysis of variability using RAPD primer indicated two major clusters such as C1 and C2 comprising 15 and 17 isolates, respectively. Among 139 rice genotypes screened under field condition, Aditya, Ajaya, Swarnadhan, Nidhi and Vikramarya were found resistant by registering 1 grade. In the management of disease with fungicides, botanicals and bioagents, Hexaconazole, Validamycin and Carbendazim, Tricure (Azadirachtin 0.03%), P. fluorescens (Pfr-l) were found effective in managing sheath blight. Application of 200 kg N per ha recorded higher disease severity (40.37%) and increased further with higher levels of nitrogen (350 kg/ha) when compared to plots with recommended nitrogen and zero nitrogen fertilizer applications. Among different planting densities, the Systematic Rice Intensification (SRI) method was found effective in reducing severity of sheath blight (5.00 PDI) when compared to other planting (16.12 PDI). In IDM trial, use of moderately resistant cultivar IR 64 with spray of hexaconazole @0.1% was found effective in managing sheath blight disease as compared to Samba mahsuri.ThesisItem Open Access Mechanism of slow leaf ruster, molecular characterization in bread wheat and variability in puccinia triticina eriks.(UAS Dharwad, 2013) Arunakumar G. S.; I. K. KalappanavarOff-season survey in Chikmagalur and Chitradurga districts of Karnataka revealed the absence of wheat crop. Three years normal season survey indicated the presence 20 different pathotypes. The pathotype 121R63-1 (77-5) was dominant and molecular profiling of 25 different isolates showed high genetic variability. UAS-326, UAS-315, VL-616, VL-924, HD-2189, HD-2932, HD-3091, NI-5439, HI- 977, HS-420, DBW-16, KRL-210, Pavon-76, RL-6077 and Parula were identified as slow leaf rusters and Agra Local, Lal Bahadur, Lok-1, Sonalika, C-306, DWR-162, PBW-343, DBW-17 and HS-240 were identified as fast leaf rusters. Molecular characterization of bread wheat showed 12 genotypes with Lr34/Yr18/Pm38, seven genotypes with Lr46/Yr29/Pm39 and none of the genotypes showed Lr67/Yr46. However, five genotypes (HD-2189, UAS- 315, VL-616, NW-4091 and RAJ-4270) carryed both Lr34/Yr18/Pm38 and Lr46/Yr29/Pm39 genes. Isozymes study revealed a higher variation of peroxidase and polyphenol oxidase at different growth stages under inoculated and uninoculated conditions. Pre-penetration processes of uredospore were not a good criterion for selection of slow leaf rusters. Loss of total grain protein content (GPC) was either increased or decreased or no significant changes observed when compared with protected and unprotected conditions among all the three group of genotypes. No significant difference was observed in the mean damaged starch of slow leaf rusters, whereas susceptible genotypes showed significant differences. Wet gluten and dry gluten content (%) was affected by leaf rust. Slow leaf ruster genotypes showed an average highest content of three micro-nutrients, viz zinc, iron and copper. The average yield loss was minimum in resistant and slow leaf rusters. However, it was very high in susceptible genotypes. A combi- product (Pyraclostrobin 13.3% + Epoxiconazole 5%) @ 0.1 % was found to be the best alternative to the propiconazole.ThesisItem Open Access Role of nematodes in soil health sustenance: relating their assemblages to soil types and cropping systems(UAS Dharwad, 2013) Kumari; S. LingarajuNematodes are functionally diverse. Most research on them is focused on plantparasitic nematodes that attack the roots of cultivated crops. Less attention has been given to nematodes that are not plant-feeders and play beneficial roles in soil environment. Therefore, the present study was taken up with the objective of determining nematode assemblages in different soil types and cropping patterns. Two agro-ecological Zones of Karnataka were selected (Zone III and IX), soil were vertisols and alfisols. The dominant cropping patterns were cotton, onion+chilli, paddy-paddy-paddy and paddy-paddy-paddy respectively. In addition to this, a field trial was taken was taken up at MARS, UAS Dharwad in two consecutive seasons, viz. Rabi-2011 and Kharif-2012 with major cropping pattern of this area (chickpea and soybean respectively) to know the influence of fertilizers and manures (singly and in combinations) on nematode assemblages. Also influence of soil physical and chemical properties were studied during the course of present investigation employing the soil samples which were collected during surveys and during field experimentation. Differences in nematode assemblages were observed during the study in different soil types with respect to trophic groups across different agro-ecological zones and during field study. MWC had positive correlation with nematode assemblages. BD on total nematode abundance was negative and showed significant positive correlation. Whereas pH showed negative correlation and effect of EC was almost negligible. Under field study, both FYM and vermicompost singly and in combination increased the numbers of bacterivores and fungivore nematodes and lowered that of plant parasitic nematodes. Different indices viz., NCR, NGR, H’, combined maturity index (MI) were calculated - a slight change across the sampling period was noticed. Results suggested that a bacterial decomposer pathway dominated, mediated by bacterivorous nematodes. In a microcosm experiment it was observed that there was increase in total available nitrogen from 12 to 14 (kg/ha) over control in presence of bacterivorous nematodes.ThesisItem Open Access Studies on Soybean Rust (Phakopsora pachyrhizi Syd.) and Chickpea Rust [Uromyces ciceris-arietini (Grognot) Jacz. and Boy.](UAS Dharwad, 2012) Sachin A. Khedekar; V.B. NargundThe full potential of the soybean and chickpea is far from exploitation due to many biotic and abiotic stresses. These include rust caused by Phakopsora pachyrhizi Syd. in soybean and Uromyces ciceris-arietini (Grognot) Jacz. and Boy in chickpea. The investigations included survey, variability of pathogen, aerobiology, screening of genotypes, yield loss estimation and integrated disease management for soybean rust. Further survey and morphological variability of chickpea rust was also attempted. Soybean rust was more severe in irrigated black soil in northern Karnataka. During rabi 2010-11, the rust of chickpea appeared very late in the season in late sown irrigated condition at Arabhavi. Chickpea rust appearance and severity is related to rainfall during the month of December. Malic acid content on the leaf is responsible for resistance mechanism in chickpea for rust. Ugarkhurd isolate was found to be different among 23 different isolates of P. pachyrhiz with respect to size, shape and colour. RAPD data distinguished the 23 isolates of soybean rust pathogen into two major clusters. ITS sequences of Dharwad and Bailhongal isolate were highly similar. Aerobiological studies indicated fluctuation in uredospore deposition at different stages of the crop growth. Perpetuation of P. pachyrhizi in the uredinial stage occurs during the offseason. Detached leaf assay revealed only cowpea as collateral host. Severity (PDI) of the disease increased with delayed sowing and thereby drastic reduction in grain yield and 100 seed weight. Among the genotypes DSb 21 exhibited highly resistant reaction with high yield potential. Integrated disease management studies identified the effective biorationals and fungicides. Adaptive Module is the most preferred module over chemical and biointensive modules in integrated management of soybean rustThesisItem Open Access Epidemiology and Management of Leaf Spot of Cotton Caused by Alternaria spp.(2012) Govindappa N. Hosagoudar; S.N. ChattannavarCotton, does not need much emphasis as being the principal raw material for a flourishing textile industry. The research studies revealed that the maximum Alternaria leaf spot disease severity was recorded in Gulbarga, Yadagiri, Dharwad and Raichur districts. Among 17 isolates, in morphological studies mean colony diameter ranged between 60 mm (Hirebagevadi) to 84 mm (Bagalkot). The mycelial width was larger in Kalmala (4.76 μm) and was least in Hattigudoor (2.53 μm). The dimensions of conidia of Kalmala isolate showed maximum size of 43.98-76.89 x 8.43-18.70 μm and Annigeri isolate showed maximum beak length of 16.30 μm. The temperature studies revealed that, the Hanumanamatti isolate (230.20 mg) recorded maximum dry mycelial weight. The highest dry mycelial weight was recorded at 30oC (252.38 mg) followed by 25oC (237.76 mg). The dry mycelial weight was significantly superior at pH 7 (230.21 mg), followed by pH 8 (215.18 mg). RAPD analysis revealed the maximum genetic similarity of 93 per cent between Gabbur (A7) and Sunkeshwarhal (A8) isolates, whereas the least similarity (40 per cent) was between Hattigudoor (A9) and Yaragatti (A13). The isolates were made into i.e., A cluster and B cluster. Stepdown regression led to the equation as Y = – 235.94 + 9.89X1 – 11.84X2 + 1.89X3 + 0.64X4 – 1.23X6 with R2 value of 0.78 including variables maximum temperature, minimum temperature, RH (morning), RH (evening) and rainy day. Fungi viz., Alternaria spp., Fusarium spp., Helminthosporium spp., Aspergillus spp., Penicillium spp., Rhizopus spp. and Mucor spp. were detected in infected seeds and survival studied for more than 14 months in refrigerator condition. The maximum per cent avoidable yield loss was recorded in Propiconazole (0.1%) at 35, 55, 75, 95, and 115 DAS. Yield reduction was 32.38 per cent in Bunny Bt caused by Alternaria leaf spot. Seed Treatment (ST) of Vitavax power (0.3%) + Foliar Spray (FS) with Propiconazole (0.1%) significantly lowered Alternaria leaf spot per cent disease index. However, the ST of Vitavax power (0.3%) + FS with Hexaconazole (0.1%) recorded highest incremental benefit: cost of 5.24. The T. viride (TV-97), seed treatment (0.8%) + foliar spray (0.8%) and Propiconazole, FS (0.1%) and both the treatments recorded highest and same incremental benefit: cost of 4.34.ThesisItem Open Access Molecular Characterization and Serodiagnosis of Vascular Pathogens Affecting Tomato(UAS Dharwad, 2011) Sumangala Koulagi; S. LingarajuSoil borne pathogens affecting tomato (Fusarium oxysporum f. sp. lycopersici, Ralstonia solanacearum and Meloidogyne incognita) were collected from different tomato growing regions of Karnataka. F. o. f. sp. lycopersici isolates, viz. Fol-1, Fol- 4, Fol-6 Fol-9, Fol-11, Fol-13, Fol-15 and Fol-21 showed abundant aerial mycelium and sporulation with maximum colony diameter (75 to 90.0 mm): These were highly virulent. Among twenty four isolates of R. solanacearum, Rs-1, Rs-4, Rs-7, Rs-8, Rs- 9, Rs-12, Rs-16, Rs-19, Rs-21, Rs-22, Rs-23 and Rs-24 isolates were highly virulent. RAPD analysis of F. o. f. sp. lycopersici revealed five major clusters. Maximum genetic similarity (73 %) was between Gubbi (Fol-5) and Doddaballapur isolates (Fol- 4), whereas least genetic similarity was observed between Chintamani (Fol-17) and Garag (Fol-2) isolates. The similarity co-efficient of R. solanacearum isolates ranged from 0.19 to 0.61. Maximum genetic diversity of 61 per cent was between Hosalli (Rs-7) and Doddaballapur (Rs-9) isolates whereas least similarity (0.19 per cent) was observed between Kolar (Rs-22) and Garag (Rs-2) isolates. PCR performed with the primer combination of Forward primer (5 -TGTATAAGTTTAATCGTTTTAACGA- 3 ) and 18s reverse primer (5 -GTATGTACCAACTATTTAGTAGGT- 3 ) produced only the single expected fragment of 1.3 kb for all isolates of M. incognita. DASELISA was more sensitive: it was precise enough to detect the F. o. f. sp. lycopersici antigen up to 51200 dilutions. Purified antigen of F. o. f. sp. lycopersici concentrations of 3.2 μg and 50 conidia/well could be detected by DAC –ELISA technique: The technique was capable of detecting R. solanacearum at 103 cells/ml; collected isolates of R. solanacearum showed positive reaction using DAC- ELISA techinique. This technique was also able to detect R. solanacearum directly from soil and plant sample infected with the bacterium. Four isolates M. incognita collected from different regions showed positive reaction by DAC- ELISA assay.