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ThesisItem Open Access Isolation and characterization of bacterial endophytes with antagonistic potential against Rhizoctonia solani(Punjab Agricultural University, Ludhiana, 2021) Bikramjeet Singh; Pratibha VyasThe present study was carried out with the aim to select bacterial endophytes from Stevia rebaudiana with biocontrol potential against a broad host range phytopathogen Rhizoctonia solani. A total of 22 morphological distinct bacterial endophytes were isolated from the leaves of Stevia rebaudiana collected from the Herbal Garden, PAU Ludhiana and Hara Agro Centre Nursery, Ludhiana, Punjab. Of 22 bacterial endophytes, eight isolates (36%) showed antagonistic activity against R. solani with a growth inhibition ranging from 12.5 to 40% in dual plate assay. Three isolates SR1EB1 SR1EB11 and SR2EB5 exhibited more than 25% growth inhibition of R. solani. The inhibition in fungal biomass by the three antagonistic bacteria singly or in combinations varied from 61.3 to 86.6% in liquid medium. The effect of antagonistic bacterial endophytes on the ultrastucture of R. solani studied by scanning electron microscopy showed stripping of the fungal hyphae leading to the accumulation of debris in the presence of antagonistic bacterial isolates. In addition to antagonistic activity, the isolates also exhibited tricalcium phosphate solubilization, zinc solubilization, auxin production, ammonia production, cellulose hydrolysis and starch hydrolysis. All the three isolates were tested negative for the production of siderophores and hydrogen cyanide. Based on morphological features, biochemical tests and MALDI-TOF-MS analysis, the isolates were identified as Ochrobactrum gallinifaciens SR1EB1, Alcaligenes faecalis SR1EB11 and Bacillus licheniformis SR2EB5. Maize seeds inoculated with the antagonistic bacterial endophytes singly or in combinations showed vigour index ranging from 351 to 695. An increase of 37.1 to 64.4% in root length, 27 to 40% in shoot length and 20.5 to 47.1% in dry weight over uninoculated control in maize in soil infested with R. solani was observed with the three antagonistic bacterial endophytes and their combinations in pots. Disease control varied from 66.7 to 83.3% in bacterial treatments with R. solani. The study has led to the selection of three bacterial endophytes Ochrobactrum gallinifaciens, Alcaligenes faecalis and Bacillus licheniformis strains as potential biocontrol agents against Rhizoctonia solani. This is the first study on the bacterial endophytes from Stevia rebaudiana leaves with antagonistic activity against Rhizoctonia solani. As a future line of recommendation, the formulation containing the consortium of Ochrobactrum gallinifaciens, Alcaligenes faecalis and Bacillus licheniformis may be developed and tested in maize to control Rhizoctonia solani infection in field conditions.ThesisItem Restricted Bioaugmentation of microalgae for anaerobic co-digestion of paddy straw(Punjab Agricultural University, Ludhiana, 2021) Saldi, Sejal; Phutela, Urmila GuptaPresent study was aimed at bioaugmentation of microalgae by co-culturing with nitrogen fixing bacteria co-digestion of bioaugmented microalgal biomass with paddy straw (PS) to enhance biogas production. All five microalgal strains (Spirulina subsalsa, Anabena sp., Chlorosarcinopsis eremi, Chlorella sp. and Scenedesmus MKB) were co-cultured with Rhizobium bacteria for enhancing biomass productivity. Maximum increase of 5.84% was found inChlorosarcenopsis eremi showed followed by Spirullina subsalsa. Chlorosarcenopsis eremi was further co-cultured with different bacterial strains (Rhizobium sp., Pseudomonas sp., Pseudomonas gessardii A-13 and Pseudomonas aeruginosa A-14) at 5% and 10% (v/v) concentrations. Highest biomass productivity was observed in Chlorosarcenopsis eremi co-cultured with 10% Rhizobium which showed 20% increase in biomass followed by co-culturing with 10% Pseudomonas sp.(increase of 18.9%). Co-cultured biomass of microalgae was co-digested with chopped paddy straw. Feedstock was analyzed for its proximate and chemical composition before and after anaerobic digestion. The biogas potential of co-cultured microalgae under ambient conditions (32°C-42°C) revealed that PS co-digested with co-cultured biomass of Rhizobium and Chlorosarcinopsis eremi in presence of poultry dropping yielded highest biogas (156.80 l/kg PS) which was 20.2% more than control followed by PS supplemented with Rhizobium broth showing an increase of 5.9% (148.50 l/kg PS). Co-cultured biomass resulted in more biogas production in short period of time as maximum harvested biogas (155% increase) was found in case of PS co-digested with co-cultured biomass of Rhizobium and Spirullina subsalsa in one month only. Proximate and chemical analysis showed decrease in total solids, volatile solids, total organic carbon, feedstock consumed while increase in ash, lignin, silica and nitrogen content. Seed germination studies were also conducted to analyze the effect of monocultured microalgae, co-cultured microalgae and Rhizobium broth on growth parameters of moong seeds. Seed germination parameters were improved in range of 1.74%- 40.6%. Highest enhancement of 40.6% in germination parameters was observed with soaking of seeds in co-cultured broth of (Rhizobium and Spirulina subsalsa). Hence, co-culturing of microalgae with bacteria has tremendous potential in increasing biomass productivity which can be further used for paddy straw management through biogas production.ThesisItem Restricted Putative bacterial consortium for mitigating wilt incidence in chickpea(Punjab Agricultural University, Ludhiana, 2021) Jakilanki, Mounica; Sharma, PoonamDespite the multifold importance, there was negligible increase in the chickpea production in the last decade due to Fusarium wilt caused by Fusarium oxysporum f. sp. ciceris (Foc). The present investigation focused on the development of bioinoculant formulations with dual plant growth promotion and biocontrol of Fusarium wilt in a sustainable manner. Mesorhizobium (10) and endophytic rhizobacteria (35) were screened for antagonism based on growth inhibition of Foc using dual culture and sealed plate assay. 93%, 76%, 64% and 53% isolates were active producers of protease, lipase, cellulase and chitinase, respectively. Biocompatible dual inoculants with no zone of inhibition were further screened for multifarious PGP traits viz. P-solubilization, production of IAA, gibberellic acid, ACC-deaminase, siderophore and biofilm. Efficacy of different liquid (LB, NB, LB+0.1%CMC, and NB+ 0.1%CMC) and carrier based (Talc and charcoal) formulations of dual inoculants was assessed up to 90 days. Highest viable count (8.668 log CFU ml-1) and optical density (0.8946 OD 600nm) was recorded in SG4 + FRW2 in NB + 0.1% CMC. Effect of bioinoculant formulations on growth, symbiotic traits, soil quality, yield and biocontrol of Fusarium sp. was recorded under wilt sick pot conditions in chickpea. Significant increase in growth and symbiotic traits was observed in SG4+ FRW2 in LB with 0.1% CMC as compared to inoculated control at 80DAS. An enhancement in peroxidase activity was observed in 0.1% CMC amended LB formulation LCRE9 + FRW2 (0.178 U min-1 mg-1 fresh weight of root) after the appearance of disease. Highest catalase (11.31 U min-1 g-1 fresh weight of root), SOD (11.06 U min-1 g-1 fresh weight of root) and total phenol content (85.42 Tannic acid equivalents g-1 fresh weight of root) was recorded in SG 4 + FRW 2 in LB with 0.1% CMC with 4% superior wilt control over recommended charcoal formulation (RB1 + LGR33). Highest grain yield was recorded in bioinoculant formulation SG4 + FRW2 in LB amended with 0.1% CMC (1.91 g plant-1). SG4 + FRW2 and LCRE9 + FRW2 along with liquid formulations can be further developed to improve the chickpea productivity and reduce the disease severity of Fusarium oxysporum f. sp. ciceris in an ecofriendly manner.ThesisItem Open Access Pathogenicity of nucleopolyhedrosis virus against Spodoptera litura (Fabricius)(Punjab Agricultural University, Ludhiana, 2021) Manreet Kaur; Joshi, NeelamNatural mortality of Spodoptera litura due to entomopathogens on different crops viz. arvi, cabbage, cauliflower and summer moong bean was recorded during 2019. Natural mortality due to entomopathogens was very low. Maximum mean per cent mortality (3.77%) was recorded due to fungi followed by 2.33 and 2.31 per cent due to bacteria and viruses, respectively. Virus infected S. litura cadavers were processed for isolation of polyhedral occlusion bodies (POBs), which were observed as bright, round structures under phase-contrast microscope and as roughly polyhedral structures under Transmission Electron Microscope. Pathogenicity studies of three nucleopolyhedrosis virus suspensions viz. SpltNPV-native, SpltNPV-commercial and SpltNPV-NIPHM against second instar larvae of S. litura recorded maximum mortality at highest concentration (1.0ml/L) and all three suspensions which were statistically at par with each other at this concentration. The pathogenicity studies against fourth instar larvae recorded 78.33, 71.67 and 68.33 per cent mortality in SpltNPV-commercial, SpltNPV-native and SpltNPV-NIPHM suspensions, respectively at the same concentration, i.e., 1.0 ml/L. The LC50 values of the SpltNPV-native, SpltNPV-commercial and SpltNPV-NIPHM suspensions against second instar larvae were 0.584, 0.540, 0.625 ml/L respectively which increased to 0.696, 0.620, 0.756 ml/L against the fourth instar S. litura larvae. The LT50 at 1.0 ml/L was found to increase from 146.33, 137.51 and 155.88 h for SpltNPV-native, SpltNPVcommercial and SpltNPV-NIPHM suspension, respectively against the second instar larvae to 178.51, 162.07 and 187.67 h respectively against the fourth instar larvae of S. litura. Thus, the SpltNPV suspensions at the highest concentration were significantly better than all other treatments and the per cent larval mortality due to SpltNPV was indirectly related to the larval stage.ThesisItem Open Access Seasonal emergence of pathogenic bacteria in salads and remedial measures(Punjab Agricultural University, Ludhiana, 2021) Jawanda, Ishwerpreet Kaur; Sahota, Param PalModern food systems and ready-to-eat salads represent a complex dynamic network vulnerable to foodborne infectious outbreaks difficult to trace and control. Seasonal variations further aggravate the on-going foodborne patterns posing a fundamental public health challenge. Thus, in light of rising concern, a city-wide epidemiological surveillance study for the microbiological quality of salad samples with respect to seasonal variation (monthly average temperature) was conducted during the year 2019-2021 in Ludhiana, Punjab, India. A total of (N=350) salad samples: Greek salad (n=65), Fruit salad (n=74), Chicken salad (n=61), Pasta salad (n=74) and Sprouts salad (n=76), were collected from supermarkets, local stores, and online stores and the highest prevalence was recorded for Salmonella spp. (38.0%) followed by Yersinia enterocolitica (37.4%), E. coli (36.6%), Aeromonas hydrophila (35.4%), Staphylococcus aureus (35.4%), Listeria monocytogenes (34.0%), Klebsiella spp. (32.8%), Campylobacter spp. (30.8%), Bacillus spp. (27.6%) and Shigella spp. (26.3%), with a total of 197/350 (56.28%) bacteriologically unsafe produce. A notable seasonal trend with statistical tool was detected for Salmonella spp. (summer), Yersinia enterocolitica (winter) and Campylobacter spp. (monsoon) subjected to the red zone of concern which warrants detailed investigation. Klebsiella spp. (r=0.926), E. coli (r=0.8453) and Staphylococcus aureus (r=0.806) were found to exhibit a significant strong correlation with ambient temperature, whereas, inverse significant correlation was recorded for Yersinia spp. (r=-0.337) and Shigella spp. (r=-0.1727). Further, the shelf-life study depicted a significant difference at 4°C as compared to storage at 15°C and 37°C. The statistical test result of overall percentage of bacteriological contamination of salad samples by BIS Standard Methods and Bacteriological Food Testing Kit (BFTK) at 5% showed no significant difference accentuating an equal effectiveness of the two methods, the latter being rapid and cost-effective. Finally a promising intervention against the pathogenic microflora of salads: washing the produce with sodium hypochlorite (100ppm/5min) was found effective and the bioprocess for development of shelf stable, microbiologically safe and nutritionally enriched reproducible technology of lactic-acid fermented turmeric salad dressing (Lactic salad dressing) was optimised: [Turmeric 10%: ginger extract 7.5%: lemon juice 25%: xanthan gum 0.5%]; dilution ratio (1:1); condiment concentration (2.0%), pasteurization at 52ºC for 15 seconds and fermentation with pure culture consortia of ten established PAU lactic acid bacterial strains as starter culture (15.0%) at 37ºC for 24 hours. The microbiological and physicochemical parameters (90 days) of Lactic salad dressing were; ºBrix 1.5, titrable acidity 1.15 %, pH 2.15, total sugar 15.38 mg/100ml, reducing sugar 4.70 mg/100ml, antioxidant activity 58.11%, total polyphenols 37.12 mg GAE/100mL, total flavonoids 43.78 mg/100mL maintaining cell counts of 6.99 log CFU/mL. The fermented salad dressing is endowed with bioactive compounds of turmeric and a broad array of lactic culture metabolites, organic acids, antioxidants, polyphenols and flavonoids. The dressing when seasoned on salads (@1000mg/mL) showed high antimicrobial activity against Staphylococcus aureus MTCC3906, Listeria monocytogenes MTCC657, Klebsiella pneumoniae MTCC109, Escherichia coli MTCC443 and S. enterica serovar typhimurium MTCC 733. The current bio-invention, thus, is an economical and alternative plant based biological therapy: for replenishing gut microbiota in the era of microbial contamination and antibiotic resistance.ThesisItem Restricted Microbial degradation of paddy straw by submerged state fermentation(Punjab Agricultural University, Ludhiana, 2021) Verma, Ishita; Katyal, PriyaPaddy straw is the most abundant yet ineffectively managed agricultural residue leading to resource wastage and generation of environmental pollution. The action of microbes could help to curb the waste and evolve it to resourceful products. The present study was conducted to evaluate the lignocellulolytic efficiency of microbes for the degradation of paddy straw under submerged fermentation. Six bacterial, one actinomycete and one fungal culture were isolated, purified and screened qualitatively and quantitatively for lignocellulose degrading potential. Three standard lignolytic cultures (Delftia sp. PP4_S3, Bacillus sp. and Pseudomonas sp.) were also used as positive control. A consortium of the isolated and standard culture was used to decompose untreated paddy straw resulting in maximum enzyme activities of endoglucanase (1 U/ml), exoglucanase (1.31 U/ml), β-glucosidase (2.42 U/ml), xylanase (12.63 U/ml) and manganese peroxidase (5.43 U/ml) after 28 days of incubation along with laccase (4.36 U/ml) and lignin peroxidase (6.71 U/ml) after 21 days of incubation. By the action of the consortium, untreated paddy straw showed maximum reduction of 35.31% in cellulose, 22.5% in hemicellulose and 44.72% in lignin after 28 days of incubation. Consortium when used along with 5% urea pretreated straw under submerged fermentation reported maximum enzyme activities for endoglucanase (5.96 U/ml), exoglucanase (6.32 U/ml), β-glucosidase (8.63 U/ml), xylanase (31.78 U/ml) and manganese peroxidase (16.8 U/ml) after 28 days of incubation. Laccase (12.63 U/ml) and lignin peroxidase (27.34 U/ml) activities were found to achieve maxima after 21 days. Pretreatment of paddy straw with 5% urea followed by microbial degradation, reported maximum loss of 29.87% in cellulose, 43.48% in hemicellulose, 35.64% in lignin and 43.65% in dry matter. Field emission scanning electron microscope (FE-SEM) micrographs revealed sufficient sloughing off of cellulose and hemicellulose with profuse growth of microbes without any silica degradation.ThesisItem Restricted Effect of polymer seed coatings on microbiological status, and growth characteristics of muskmelon (Cucumis melo L.)(Punjab Agricultural University, Ludhiana, 2021) Jashapreet Kaur; Kalia, AnuThe present study aimed to study the effect of the seed priming and coating (CMC) with metal oxide nanoparticles (NPs) and PGPR (Burkholderia seminalis). Metal oxide NPs (FeO and CuO) were synthesized by wet chemistry approach and exhibited semi-spherical (5-45 nm) and elongated tubular nanorods (30-60 nm long and 5-10 nm wide) morphologies respectively. The in vitro filter paper studies using different concentrations (0, 5, 10, 20, 40 mgL-1) of FeO NPs revealed that FeO NPs (5 mg L-1) significantly increased the vegetative growth parameters of the muskmelon seedlings. Further, a germination peg-tray assay was conducted to evaluate the effect of seed priming/ coating treatment by use of FeO (5 mg L-1) and CuO (0, 5, 10, 20, 40 mg L-1) NPs alongwith the PGPR culture. A similar trend was recorded for different vegetative parameters in germination peg tray assay. The one-monthold muskmelon seedlings were transferred to the field to evaluate the effect under field conditions. Various concentrations of bulk/ NPs alone had overall significant impact on the vegetative and fruit quality traits. Soil enzyme activity and photosynthetic pigments exhibited significant results for all treatments. Dual/ triple interactions of seed coating/ priming treatments and culture inoculation significantly altered the soil chemical, nutrient and micro-nutrient status. Various concentrations of bulk/ NPs alone and their dual interactions with treatment/ culture had significant impact on the shoot nutrient content. Seed treatments, culture inoculation, as well as dual/ triple interactions among these factors had significant impact on the root nutrient and Fe content. Culture inoculation had no significant impact on the Cu content. Seed coating/ priming, culture inoculation, various concentrations of bulk salts/ NPs and their dual or triple interactions significantly altered the soil microbial count. CMC polymer significantly altered the fungal, bacterial and P-solubilizing bacterial count. Seed priming had significant impact on the free-living nitrogen-fixing bacteria and numerically altered the count of actinobacteria. Inoculation of culture had significant effect on the bacterial, actinobacteria and free-living nitrogen fixing bacteria and it numerically altered the fungal count. Further, from various concentrations of bulk/ NPs, CuO NPs at 40 mg L-1 had significant highest impact on the fungus and Psolubilizing bacteria. The CuO NPs at 20 and 5 mg L-1 had significantly highest impact on free living N bacteria and bacteria respectively.ThesisItem Restricted Nanomaterial-mediated gene delivery in wheat (Triticum aestivum L.)(Punjab Agricultural University, Ludhiana, 2021) Harkamal Kaur; Kalia, AnuThe present study aimed for the synthesis and functionalization of different NMs and conjugation of plasmid DNA (pGFPGUSplus) onto the functionalized NMs. These NM-plasmid DNA conjugates have been evaluated as nano-delivery vehicles to deliver the desired plasmid DNA in wheat (Triticum aestivum L.). The UV-Vis absorption spectroscopy analysis showed a shift in the peak of the NMplasmid DNA conjugates in comparison to the plasmid DNA absorption peak and the pristine NMs absorption peak, which showed the intercalation of the plasmid DNA with the respective NMs. The FT-IR spectroscopy of the respective NMs and NM-plasmid DNA complexes showed the presence/weakening of functional groups before and after conjugation of plasmid DNA onto the NMs. Transmission electron microscopy (TEM) analysis revealed the formation of nano-scale (>100 nm) pristine NMs and NM-plasmid DNA complexes that formed aggregates after conjugation. DNase treatment and gel electrophoresis of the respective NMs and NM-plasmid DNA complexes confirmed the binding and accurate loading of plasmid DNA onto the NMs. The NM-plasmid DNA conjugates were used to transform two different strains of untransformed gram negative rods i.e. Escherichia coli (DH5alpha) and Agrobacterium tumifaciens (EHA105). The Fourier Transform-Infra Red spectroscopy studies depicted the shift in bending/stretching vibrations of amino or phosphate groups present in bacterial polysaccharides after tethering of NM-plasmid DNA conjugates onto the bacterial cells. The TEM study of the transformed bacterial cultures showed the binding of NPs around and inside the bacterial cell. The transformed bacterial cultures under fluorescence microscopy showed green fluorescence due to the delivery of plasmid DNA inside the cells with the aid of NMs. The total soluble protein content analysis revealed production of different amounts of total intracellular and extracellular soluble proteins which were increased in a time dependent manner. LDH NPs showed lesser cytotoxicity towards bacterial cells along-with better viability rate than other NPs. The bacterial cell viability assay revealed the impact of different NMs on the viability count of the bacterial cells. Further, the NM-plasmid DNA conjugates were used for transformation of the wheat callus cell suspensions and entire callus tissues. All the callus cells transformed with different NM-plasmid DNA complexes exhibited green fluorescence on fluorescence microscopy. Agrobacterium-mediated infiltration was also performed in entire wheat callus tissues to compare the efficacy of Agroinfiltrations and nano-delivery vectors. The Layered Double Hydroxides (LDH) and Graphene oxide (GO) nanosheets showed maximum fluorescence as compared to other NM-plasmid DNA conjugates as these particles were able to penetrate deep inside the callus tissues while others also showed the ROS effect. Upon bath-sonicating the wheat callus tissues with the LDH-plasmid DNA and GOplasmid DNA complexes, the LDH nanosheets showed an increase in the intensity of the green fluorescence with an increase in sonication incubation period. Therefore, LDH nanosheets delivered the desired plasmid DNA inside the callus tissue cells more effectively without damaging to callus cells/hampering the cellular systems.ThesisItem Open Access Optimization of pretreatment for quality maintenance of fresh cut salad vegetables under refrigeration conditions(Punjab Agricultural University, Ludhiana, 2021) Gurliin; PoojaFive different disinfectants viz. sodium hypochlorite, potassium metabisulfite, citric acid, acetic acid and benzoic acid were evaluated over fresh cut salad vegetables (cucumber, carrot and tomato) to check their bactericidal efficacy. Amongst five disinfectants evaluated, sodium hypochlorite showed maximum microbial reduction of 49.99-56.51% Total Plate Count, 47.0566.66% Yeasts & Moulds and 51.27-70.83% coliforms with respect to control on fresh cut cucumber, carrot and tomato. Optimization of sodium hypochlorite concentration revealed maximum decrease in microflora @ 200 ppm. However, 100 & 150 ppm concentration levels also showed decrease in microbial reduction upto acceptable limits with non-significant difference at both concentrations. Considering residual chlorine level in fresh cuts and microbial reduction, 100 ppm concentration was optimized for the study. Further, optimization of pretreatment temperature, pH and contact time was statistically designed and analyzed using response surface methodology. Numerical optimization of results revealed a temperature of 6.5, 10.0 and 6.5°C; pH 6.0, 7.0 and 7.0; contact time 23.0, 8.0 and 30.0 minutes was found to be optimized for fresh cut cucumber, carrot and tomato disinfectant pretreatment with a desirability level of 94, 94 and 92%, respectively. Validation of results was done at 3Kg scale under all optimized conditions. Shelf life analysis of pretreated (under all optimized conditions) and untreated fresh cut vegetables stored under refrigeration conditions (5-7°C) revealed enhancement in shelf life of treated fresh cut vegetables from 6, 9 and 6 days to 9, 12 and 9 days of fresh cut cucumber, carrot and tomato, respectively. During shelf life period, all physicochemical parameters of pretreated fresh cut vegetables differed non-significantly along with microbial load under acceptable limits and a good sensory score.
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