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ThesisItem Open Access Distribution and characterization of Burkholderia species causing panicle blight in paddy and its management(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-09) Sudha Nandni; Vishunavat, KarunaThe bacterial panicle blight of rice is an important bacterial disease which was identified for the first time at Pantnagar in the year 2010-2011, at field though, in low incidences and sporadically. Bacterial panicle blight (BPB) caused by Burkholderia glumae, is an emerging disease which has recently come out as a menace in rice-growing areas of northern India. Considering the importance of crop and continuous spread of the disease, present research was initiated to decipher the prevalence and characterization of the Burkholderia sp. in different regions of northern India and its management strategies during the year 2017-2018 and 2018-2019 at GBPUA&T., Pantnagar. The findings observed under the present investigation are summarized as follows. Symptomatic and asymptomatic seeds and infected plant parts collected from different geographical regions of northern India mostly, including states viz. - Uttarakhand, Haryana, Uttar Pradesh, Bihar and Assam exhibited symptoms of BPB on infected leaf, leaf sheath, base of boot leaf and panicles. A total of 28 bacterial isolates were recovered out of which only 21 isolates were found pathogenic on rice under glass house conditions. Colony characters revealed that 6 different coloured colonies viz. - White (glistening), Creamy white (smooth), Creamy (smooth), Creamy yellow (smooth), Pale yellow and (smooth), Shiny yellow (flattened) were present among the isolates. Biochemical studies revealed that the pathogenic isolates were positive for gelatin liquefaction, KOH Solubility and catalase test while negative for starch hydrolysis, oxidase reaction and arginine dihydrolase test. Molecular characterization of the isolates on the basis of their 16S rDNA sequences confirmed that (14) Burkholderia glumae, (5) B. gladioli and (2) B. plantarii were recovered from the infected samples and thus, showed the prevalence of different Burkholderia sp. causing BPB in the regions of northern India. Under the oil immersion lens of compound microscope (100X) through gram staining the bacterial isolates were found to be gram negative absorbing red colour of the counter stain and rod shaped with round ends. The bacterium was found to be seed-borne. Maximum recovery 93% of the bacterium was recorded from the freshly harvested lot which was gradually reduced to 26% after one year of storage under normal laboratory conditions (14-30°C). Twenty- two paddy varieties were screened for BPB resistance out of which early maturing varieties (105-125 days) Pant Dhan-11, Pant Dhan-16 and Pant Dhan-12, mid maturing varieties (120-130 days) Pant Dhan-19, Pant Dhan-10 and Pant Dhan-28 and late maturing varieties (130-140 days) Pant Dhan-18, Pant Basmati-2, Pant Sugandh Dhan-15, Pant Basmati-1 and Pant Sugandh Dhan-25 exhibited moderate resistant reaction while rest others showed moderate susceptible or susceptible reaction towards the pathogen. In vitro screening of antibiotics at five different concentrations against B. glumae exhibited that the 100 ppm concentration of all the antibiotics were most effective. The minimum disease incidence and disease severity was in copper oxychloride and Copper oxychloride + Norfloxacin.ThesisItem Open Access Variability and management of Fusarium oxysporum f.sp. lentis causing wilt of lentil(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-08) Arya, Anshul; Kushwaha, K.P.S.Lentil (Lens culinaris Medic.) is one of the oldest annual grain legumes consumed and cultivated in the world. The origin of lentil is South West Asia. India is the second largest producer of lentil after Canada. Wilt disease of lentil, caused by the soil and seed-borne fungus Fusarium oxysporum Schlecht.emend.Snyder &Hansen f.sp. lentis Vasudeva & Srinivasan is one of the most severe and prominent diseases of the lentil. The continuous prevalence of this disease makes it most important to manage during lentil cultivation. The present investigation was undertaken to study prevalence of wilt in selected lentil growing areas of Uttarakhand and Uttar Pradesh. The morphological and molecular characters of the twenty-two pathogen isolates were studied along with the evaluation of ecofriendly management practices viz; Botanicals, Animal Products, Bioagents, Oils, Medicinal Mushrooms, Spent Mushroom Compost, Synthetic Defense Inducers, fungicides, and host resistance against the pathogen under in vitro and in vivo conditions during the Rabi season 2016-17 and 2017-18. The prevalence of the wilt disease was 100 percent along with a maximum 25 percent of disease incidence in all twenty-two lentil growing areas surveyed. The fungal pathogen and its isolates were isolated on the PDA medium from roots of infected plants showing peculiar disease symptoms like yellowing, withering, and drying. After morphological identification of the fungal pathogen, isolates were further genetically identified through the universal primers ITS1 and ITS4, as Fusarium oxysporum and accession no. MK452341 was obtained for the Pantnagar isolates from NCBI. Variation in pathogenic behavior of the isolates was also observed under glasshouse conditions and isolates were categorized as highly and extremely pathogenic in their behavior. Huge variation in the morphological characteristics of the isolates was observed based on the cultural growth, pigmentation, size, and shape of conidia. At the molecular level twenty-two isolates of the pathogen with ISSR (8 primers) and SSR (5 primers) primers showed significant genetical variation with 100 percent polymorphism. PIC value for the different ISSR primers was ranged from 0.65 to 0.93 and the PIC value for the SSR primers varied from 0.68 to 0.87 that showed high level of genetical variability among the isolates. The leaf extracts of Bael and Eucalyptus were found highly significant to inhibit the pathogen growth as well as reducing the disease incidence at 30 percent concentration under in vitro and in vivo conditions. The phytochemical Eucalyptol and 1,2 Diisopropenylcyclobutane were found responsible for the antifungal activity of the plant extracts of Eucalyptus and Bael, respectively, through GCMS analysis. Among the screened animal products, cow urine with 20 percent was performed best under in vitro and in vivo conditions. Badri cow urine was found superior over Sahiwal urine and gave 100 percent inhibition in the radial growth of pathogen, at 20 percent concentration under in vitro conditions. Seed treatment with cow urine was found an effective method for management of the disease in field conditions. The induction of defense-related enzymes PAL and Catalase were found maximum at 48 hours with 7 percent concentration of Badri cow urine. Panchgavya at 15 percent concentration gave 100 percent inhibition in the radial growth of the pathogen under in vitro condition. Induction of defense-related enzymes PAL and Catalase at 3 percent concentration of panchgavya was observed at 24 hours after challenged inoculation. Seed treatment with 5 percent concentration of panchagvya showed minimum disease incidence. Trichoderma harzianum (Strain Th14) gave 70 percent inhibition in the radial growth of the pathogen. PB3 at 1.0 percent induced the defense-related enzymes PAL, PO, PPO and catalase at 48 hours after challenged inoculation of the pathogen. The seed treatment with PB-2 @ 6g/kg seed and PB-2 at 0.5 percent concentration was found most suitable for soil drenching. The medicinal mushrooms (Ganoderma lucidum) was also found effective in inhibiting the radial growth of the pathogen and seed treatment with 30 percent extract of Ganoderma lucidum was showed minimum disease incidence. Mustard oil at 20 percent concentration inhibited the radial growth of pathogen under in vitro condition as well as 20ml/kg seed of it decreased the disease incidence under in vivo conditions. Application of 25 ton/ ha of Spent Mushroom Compost in the field was found effective in managing the disease. SA at 0.03 percent was found most effective in inducing the defense-related enzymes PAL, PO, PPO, and Catalase under glasshouse condition. Seed treatment with a combination of SA and BTH (1:1) at 0.1 percent concentration was found best for reducing the disease incidence. Tebuconazole was found most effective in reducing the radial growth (100% inhibition) of the pathogen under in vitro conditions at 0.05 percent concentration. Seed treatment with Tebuconazole or Propiconazole at the rate of 0.1 percent was found most effective in managing the disease under field condition. Germplasms, DL16-5, DL16-7, VL152, IPL339, IPL340, PL237, RL7-3, RVL15-5, IPL227, IPL338, IPL332 were found highly resistant against the wilt disease of lentil. These germplasms can be utilized for the cultivation of lentil crop and also in the resistance breeding program as the donor parent.ThesisItem Open Access Pathotype variation of Albugo candida, the cause of white rust disease in rapeseed-mustard(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-09) Dev, Devanshu; Tewari, A.K.White rust disease of rapeseed-mustard is one of the most important disease which severely affects production and productivity of the crop. The obligate and high variable nature of the pathogen poses a challenge for the eco-friendly management of the disease. A limited work has been done in India/world on the variability of this pathogen. So, present investigation has been done on the pathotype variability of A. candida based on molecular, morphological and pathogenic characterization of 33 A. candida isolates belongs to 12 different states of India and from 09 Brassica spp. Molecular characterization using 12 SSR markers, among them 05 viz. DM 3, DM 16, DM 22, M31 and Pv65 showed high polymorphism. Molecular characterization using ITS and COX2 gene yielded 1250bp and 650bp band respectively and maximum parsimony tree showed 15 and 13 cluster from 33 A. candida isolates respectively. Morphological characterization based on the shape of sporangia, 33 A. candida isolates were categorized in 03 groups viz. globose, ovoid and irregular while based on size, isolates were grouped in Microsporangia (14-17μm), Mesosporangia (17.10-20μm) and Macrosporangia (>20μm) with largest size (22μm) of sporangia in Ac-Smr (Sirmaur, HP) and smallest (14.63μm) in Ac-Knp (Kanpur, UP). Based on pustule pattern on B. juncea cv. Varuna, isolates were categorized in 05 groups viz. Separate circular; Coalesced circular, Scattered and pin head; Restricted near veins and veinlets and Restricted near veins and veinlets separate or coalesced circular type pustules. On the basis of virulence on 19 Brassica genotypes, 33 A. candida isolates were categorized into 04 different groups viz. highly virulent, virulent, moderately virulent and least virulent. On the basis of aggressiveness, isolates were categorized into 04 different groups viz. highly aggressive, aggressive, moderately aggressive and least aggressive. Based on the cross infectivity on different Brassica spp., isolates were grouped in 19 different pathotypes. Based on the B. juncea cultivar (out of 7) and on different Brassica spp. 03 major groups has been identified viz. Group Ia was pathotype of B. juncea, Group Ib was pathotype of B. juncea cv. Cutlass, Group Ic was pathotype of B. juncea cv. Donskaja, Group Id was different B. juncea pathotype that showed disease reaction on B. oleracea, Group Ie was B. juncea pathotype that showed disease reaction on B. carinata cv. Kiran. Group II was pathotype of B. rapa var. Toria, Group III was pathotype of B. rapa. Finally, from 33 A. candida isolates a total of 21 pathotypes has identified on the basis of combined grouping on pathogenicity reactions on host differential and ITS and COX2 gene sequence analysis and new nomenclature has been given as per International standard, out of which 15 pathotypes showed more virulence to B. juncea that named as AC2-1 to AC-15, 06 pathotypes showed virulence to B. rapa that named as AC7-1 to AC7-6. The different A. candida pathotypes identified from different geographical regions of India could be utilized for the identification and deployment of resistant cultivar in major mustard growing areas.ThesisItem Open Access Characterization of Trichoderma isolates and their evaluation for induction of defense in Sorghum against Colletotrichum graminicola(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-08) Manzar, Nazia; Singh, Y.Many foliar diseases are known to attack sorghum crop. Among them anthracnose disease caused by C. graminicola has a potential to decrease yield upto many fold. The aim of present study was to manage the anthracnose of sorghum through Trichoderma spp. Twenty isolates identified as Trichoderma were isolated from rhizospheric soil of different locations in Uttarakhand. Nineteen isolates were having globose to ovoidal shape conidia and 1 isolate having globose to subglobose shape conidia and size ranging from 3.09 μm to 3.80 μm in length and 2.95 μm to 3.73 μm width. SEM analysis revealed surface features like phialides arising in whorls and conidia having inconspicuous ornamentation were identified as T. asperellum and conidia smooth globose to subglobose in shape, ampulliform phialides, identified as T.harzianum. Molecular identification of 20 isolates of Trichoderma based on DNA sequence analysis of the ITS (1 and 4) and EF-1α gene to confirm species identity revealed that 19 isolates belong to Trichoderma asperellum and isolate T6 belong to T.harzianum. Among all the isolates tested in vitro against C.graminicola, T3 isolate of Trichoderma possessed maximum inhibition of radial growth (76.47%) of test pathogen. The mechanism of host defense induction in sorghum by Trichoderma isolates and accumulation of SOD, PPO and PO was observed in the first 24 hours after pathogen inoculation and highest at 48 hours after pathogen inoculation but gradually decreased at 72 hapi in all the experiment.T3 isolate showed the highest lignification in most of the root parts as compared to pathogen challenged sorghum and uninoculated untreated healthy control. Integration of seed biopriming, soil application enriched FYM and foliar application of Trichoderma isolates T3 and T19 was found highly effective in promoting root and shoot length and stem diameter of sorghum and minimum disease severity, minimum AUDPC and lowest mean infection rate.ThesisItem Open Access Elucidating Cu-Trichoderma interaction and Trichoderma-chitosan interaction in “Cu-Chi-Tri”, a novel consortium for potato late blight management(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-08) Bohra, Yogita; Kumar, J.Threat of emerging pesticide resistant races and excessive usage of pesticides calls for an effective and ecofriendly management strategy. A triple combination “Cu-Chi-Tri” consisting of reduced dosage of Cu(OH)2, an antimicrobial and defence inducing biopolymer chitosan and a Trichoderma TCMS 36 exhibiting copper-chitosan tolerance, was found effective against late blight in earlier researches. Therefore, this study was conducted to decipher the underlying operative mechanism for disease control by the ‘triple combination’. The cultural, morphological and molecular characterization established the identity of TCMS 36 to be Trichoderma asperellum. SEM studies on interaction of Cu(OH)2 and tolerant and sensitive Trichoderma isolates, TCMS 36 and TCMS 25 revealed mycelial shrinkage and collapse in the sensitive isolate. Elemental analysis through EDX-mapping revealed presence and uniform distribution of Cu in the mycelial matrix of TCMS 36. Moreover, dual combination containing 500 ppm Cu(OH)2 and 1 percent Trichoderma and the triple combination containing 500 ppm Cu(OH)2 along with 500 ppm chitosan in oligomeric form and Trichoderma TCMS 36 (1%) was found to support the maximum build-up of Trichoderma population in soil as evident from 58.21 fold and 46.88 fold increase in population under sterilized soil condition. TLC and UPLC-qTOF-MS studies determined the production of chitosan oligomers by T.asperellum TCMS 36 machinery as was evident from development of dimer and trimer spots in TLC plate that were absent in control and moreover by increased relative intensities of m/z ~162, ~180, ~323 in MS spectrum corresponding to monomer, dimer and trimer units. The expression profile of chitosanase gene of TCMS 36 revealed the maximum expression accounting to 50.56 fold relative increase on 3rd day by combination of both Cu(OH)2 and chitosan. The expression of chitosanase gene was found to reduce gradually from 3rd to 10th day thus indicating the need of repeating sprays at 10 days interval. The in-vitro and microscopic observations revealed anti-Phytophthora activity of chitosan in terms of extensive mycelial inhibition and morphological alterations, the larger oligomers (pentamers to heptamers) being more effective. The polymeric chitosan was found to form a chemical barrier between host and pathogen leading to reduced sporangial germination and sporangiophore production on leaf surface. However, Trichoderma depolymerized smaller oligomers (monomers to tetramers) were found to enhance the defense activities mediated by lipoxygenase and catalase higher than those activated by polymers and larger oligomers (greater than hexamers), hence, leading to a switch-over of defense mechanism from being antimicrobial to a defense inducer. The lipoxygenase and catalase were activated earliest by smaller oligomers (DP<5) and were most prolonged (high activity upto 96 hpi) by the ‘triple combination’ containing chitosan monomers to hexamers. The totality of different mechanisms was summed up with reference to disease severity that was found to be minimum for Cymoxanil+Mancozeb (14.82 %) followed by Metalaxyl M+Mancozeb (20.37 %) which was followed by triple combination containing Trichoderma depolymerized chitosan CS661 as chitosan (25.93 %) 91 DAS, that was much effective than dual combinations (50-59.26 %) and the defense inducer BABA (48.15 %) thus establishing a resilient synergy for sustainable management of late blight disease. Moreover, no residual Cu was found in soil 130 DAS with treatments containing 500 ppm Cu(OH)2 whereas, Cu residue corresponding to 0.79 percent weight of soil was found 130 DAS with treatments containing 1000 ppm Cu(OH)2, thus establishing environment resilience of the triple combination. The efficacy of triple combination Cu-Chi-Tri with both polymeric and oligomeric chitosan variant needs to be validated in larger scale at farmer’s field in different potato growing regions. Moreover, future study needs to focus on large scale validation and development of product for the farmers and also on understanding the role of copper in enhancing the expression of chitosanase gene in T.asperellum TCMS 36.ThesisItem Open Access Detection and characterization of Dickeya dadantii, the stalk rot pathogen of sorghum and its management(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-02) Yadav, Sujata Singh; Singh, YogendraSorghum bicolor (L.) Moench is one of the prime food and fodder crops of the world. It has an important role as a source of carbohydrates. The yield potential of this crop is affected by a number of diseases. Stalk rot caused by Dickeya dadantii is one of the most destructive diseases of sorghum crop in India. Since, much less information is available about the different aspect of this destructive disease. In the present investigation, isolate of D. dadantii obtained from LRC, G.B.P.U.A&T, Pantnagar was characterized morphologically, biochemically and at molecular level. The isolate was oxidase negative, catalase positive, Citrate utilization positive and molecular characterization showed the ̰ 200-base pair amplification with specific primers. This indicated that the isolated bacteria was D. dadantii. The pathogenicity of test bacterium was established by Koch’s postulates under glasshouse condition and it was proved to be pathogenic and inoclution techniques were evaluated for creating artificial bacterial stalk rot under glasshouse conditions. Highest stalk rot severity (89.75 %) was found in case of Immersion of wounded roots in bacterial suspension followed by stem injection (80.12%), hand atomizer or leaf whorl (74.45%), and tooth-pick (70.08%) method of inoculation. Partial rotting was encountered with midrib injection (61.80%) and cotton wool method (54.76%) over control. Screening of chemicals against the test pathogen under in vitro and in vivo conditions. The antagonistic potential of Trichoderma, Pseudomonas isolates/species and B.subtilis were evaluated against the D.dadantii by dual culture assay. Trichoderma viride performed best which gave 88.3% inhibition of radial growth followed by Th-R (86.0 %), Th-19 (84.5%), T. viride+P. flourescens (79.4 %) and Th-14 (77.9 %), whereas least inhibition was obtained with TCMS-39 (37.9%). In the sensitivity test of D. dadantii to bactericides and chemicals, maximum zone of inhibition was observed with Streptomycin and Cifotaxime (3.80 cm) followed by followed by Streptocyclin (3.50 cm), Tertracyclin (3.30 cm), Oxolinic Acid (2.90 cm), Kaunamycin (2.00 cm), Gentamycin (1.81cm) at 400 ppm concentrations. An effort was made to check the effect of Plant extract, Panchagavya and Cow urine at different concentration against the Stalk rot. Highest Inhibition zones were recorded by Eucalyptus globules, Bael (Aegle maemelos), Garlic, Neem and Guava ranging from 2.1- 1.8 cm against the pathogen at all four concentration (5, 10,15, 20 %). GC-MS analysis was carried out in two samples viz. Eucalyptus globules, Bael (Aegle maemelos). The 27 (Eucalyptus) and 30 (Bael) volatile phytochemical compounds found and showed their antibacterial properties. Cow urine, maximum zone of inhibition was showed at 40 % (2.1 cm) concentration followed by 30% (1.9), 25% (1.6), 20% (1.1), 15% (0.7), 10% (0.3) and 5% (0.1). Panchagavya, maximum zone of inhibition was showed at 10% (2.2 cm) followed by 7 % (1.8) 5% (1.5) and least inhibition zone was found at 3% (1.0) respectively. Quantitative analysis of enzymatic activity of Sorghum plant was done as influenced by Cow urine, Plant extract, Panchagavya, Induced elicitors and Bio-control agents. Highest PAL, TPC, PPO, PO and CAT were recorded different in each treatment. LAMP –PCR was used to detect D. dadantii with the help of species specific (Indigoidine) gene. This was found to be highly reliable, technique to confirming the presence of D.dadantii.ThesisItem Open Access Induction of mutation in Trichoderma strains for enhancing biocontrol potential and compatibility with fungicides(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Dwivedi, Meenakshi; Vishunavat, K.Biocontrol serves as an effective alternate approach for plant disease management under changing food habits and commercialization of agriculture. Trichoderma has become one of the most successful fungal biocontrol agents (BCAs) in past few decades. The progress in achieving commercial, large-scale usage of biological pest control has been rather slow, major roadblocks being instability under diverse environmental conditions, questionable shelf-life and inconsistent performance, and limited compatibility with many fungicides commonly used in agriculture. BCAs need to be tolerant to chemicals which would be a prerequisite for their application in an integrated pest management (IPM) strategy and could be produced through induction of chemical and physical mutation in popularly used biocontrol species of Trichoderma. The present study was attempted to induce mutation by chemical (ethylmethane sulphonate, EMS) and physical (UV rays and _ rays) mutagens in two Trichoderma strains viz. T. asperellum (Th 14) and T. harzianum (Th 3), to develop mutants tolerant to the fungicides (thiophanate methyl and propiconazole) incompatible with the parent strains, that also possessed comparatively enhanced biocontrol potential. Mutants were developed to tolerate thiophanate methyl (up to 200μg ml-1) and propiconazole (up to 1000 μg ml-1). Mutants Th 3 M1 and Th M6 were found comparatively superior in rhizosphere competence in three crops viz. urd bean, mung bean and chilli. Upon evaluation of growth promoting potential in urd bean, mung bean and chilli, mutants Th 3 M1 (8067, 8600 and 11166 PVI), Th 3 M2 (7233, 7633 and 100500 PVI) and Th 3 M6 (7500, 8033 and 9966 PVI) were found significantly better. On the basis of production of indole acetic acid (IAA), mutants Th 3 M1 and Th 3 M6 were found significantly superior with 40.44 and 40.41 IU ml-1, respectively. The parent strains and all the mutants exhibited 100 per cent parasitisation of R. solani and M. phaseolina at 7 days after contact (DAC) and 12 DAC, respectively, whereas only mutants mutants Th 3 M1, Th 3 M3 and Th 3 M6 were able to completely parasitize F. oxysporum capsici at 15 DAC. Mutants Th 3 M1 and Th 3 M6 performed exceedingly well at 52°C with cfu count of 2.90 and 2.85 log10 cfu ml-1, respectively, as compared to the parent strains and other mutants. In quantitative assay for cellulase, mutants Th 3 M1 and Th 3 M6 were found superior with 163.00 and 123.00 IU ml-1 cellulase activity, respectively, as compared to the parent strains and other mutants. High cellualse activity of the mutants Th 3 M1 and Th 3 M6 could be correlated with their high rhizosphere competence potential. Quantitative assay revealed that mutants Th 3 M6 was best with 122.00 IU ml-1 chitinase activity, followed by Th 3 M1 (98.00 IU ml- 1) and Th 3 M3 (94.33 IU ml-1) as compared to the parent strain Th 3 (48.66 IU ml-1) and other mutants. Among all these secondary metabolites identified during analysis, the maximum peak area of chromatogram was covered by tributyl phosphate followed by benenamine, 2-methyl and quinoline. Their quantity was elevated in the mutants Th 3 M1 and Th 3 M6 as compared to parent strain Th 3. Hence, the present investigation highlighted that induction of mutation led to enhancement as well as diminution of biocontrol characteristics of Trichoderma strains (Th 14 and Th 3). Through chemical and physical mutagenesis, stable mutants Th 3 M1 and Th 3 M6 were obtained to tolerate high concentrations of fungicide propiconazole (1000 and 500μg ml-1). Mutants Th 3 M1 and Th 3 M6 were also found most promising in their performance in various parameters such as rhizosphere competence, growth promoting potential, mycoparasitic potential, conidial thermo-tolerance (upto 52°C), production of cellulase and chitinase enzymes and production of volatile secondary metabolites, as compared to parent strain Th 3. Mutants Th 3 M1 and Th 3 M6 could prove to be very crucial biocontrol component in integrated disease management strategies.ThesisItem Open Access Study of the mechanisms underlying synergy in triple combination of copper-chitosan-Trichoderma based novel fungicide against late blight disease of potato (Solanum tuberosum L.)(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Singh, Akansha; Kumar, J.A ‘triple combination’ comprising of low dose of fungicide (CuOH), plant defence activator compound (Chitosan) and copper tolerant biocontrol agent (Trichoderma asperellum TCMS 36 (MH593785) wasdeveloped at Biocontrol laboratory, GBPUA&T, Pantnagar and field tested over years. In the present study, the copper tolerance mechanism of Trichoderma TCMS 36 was investigated and was found that Trichoderma TCMS 36 showed Copper tolerance mechanism by modulating fatty acid composition of its membrnane in copper containing environment. Specifically, derivatives of linoleic acid, oleic acid, palmitic acid and phosphate ester, which determine the membrane stability of Trichoderma in copper toxic environment. Simultaneously, in ‘Triple combination’ chitotsan may chelate the copper ions making Cu-Chitosan chelated complex, which was not absorbed/adsorbed by the T.asperellum. Hence, high modulation in membrane fatty acid composition was recorded in dual combination of Trichoderma+copper while in Triple combination as Chitosan interfere copper adsorption by Trichoderma not much modulation in fatty acid composition was required. Four important beneficial secondary metabolites with high concentration were produced in case of ‘Triple combination’ (Diphenyle sulphone 12.36%; 6-Pentyl alpha pyrone 61.12%; Tetracontane 6.01% and Pentacosane 3.36%) while in dual combination of Copper + Trichoderma three metabolites (Diphenyle sulphone 10.39%, 6-Pentyl alpha pyrone 69.96%, and Tetracontane 2.98%) were observed. Resistance inducing marker enzymes (APX, GR, CAT and LOX) and non enzymatic antioxidants (Ascorbate, reduced Glutathione and total Flavanoids) were significantly high in triple combination and BTH as compared to single and dual combination within 24-72 hrs post inoculation. Hypersensitive reaction through ROS (H2O2) production was observed in triple combination and BABA treated leaves while excessive oxidative stress damage was found in control. Meanwhile, PR-2 (β1,3-Glucanase) protein and antimicrobial secondary metabolites including phytoalexins production were also significantly higher in Triple combination (1.433 μmol/mim/mg and 12.58 peak area% ) as compared to control ( 0.723 μmol/mim/mg and 2.5 peak area%). Disease severity (91.66%) and pathogen invasion were maximum in control and minimum in ‘Triple combination’ treated leaves (4.53%) in detached leaf assay at 72 hr. post inoculation. Field trial at Pantnagar (plain) and at village Gaja (hills) revealed that triple combination containing ready to use Chitosan-661-500ppm gave better results (4.8% to 8.33%) that were at par with commercially available standard fungicides Mancozeb (5% to 7%) as compared to control (95% to 98.99%). Resistance inducer BTH and BABA managed the disease severity up to a certain limit (25% to 33.33%) under field condition. Synergestic action of all theses mechanisms made the ‘Triple combination’ more effective than single or dual combination. The results revealed that ‘triple combination’ so designed could be a viable alternative to chemical fungicides for late blight disease management as well as for increasing the tuber yield of potato.ThesisItem Open Access Exploration of different methods for detection of Clavibacter michiganensis subsp. michiganensis (Smith) Davis, the cause of bacterial wilt and canker of tomato (Lycopersicon esculentum M.) and its management(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-01) Tripathi, Ruchi; Vishunavat, K.