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ThesisItem Open Access Evalution of Plant Growth Promoting Rhizobacteria As potemtial Biofertilizer For Broccoli ( Brassica Oleracea L.)(Punjab Agricultural University, Ludhiana, 2022) Sharma, Kavia; Jupinder KaurThe present study was carried out with the aim to develop a plant growth-promoting rhizobacterial inoculant for improving the growth and yield of broccoli. A total of 24 bacteria were isolated from rhizosphere of broccoli collected from the fields of Punjab Agricultural University, Ludhiana and village Sangrur, Mansa, Barnala and Patiala. Biochemical characterization of these rhizobacterial isolates showed that most of these were oxidase, urease, indole, MR and catalase positive and were involved in starch hydrolysis and gelatin liquefaction whereas negative for citrate and VP test. These isolates were screened for multifarious plant growth-promoting traits viz. solubilization of phosphate and zinc, production of IAA, siderophore, ammonia, HCN and growth on nitrogen free medium. Out of 24 isolates, 17 isolates showed solubilization of phosphate on modified Pikovskaya agar with solubilization index ranging from 0.5-4.5 cm and 53.63-178.6 μg/ml in liquid medium. Eleven isolates showed solubilization of zinc oxide (solubilization index: 0.4-1.9 cm), five isolates showed solubilization of zinc carbonate (solubilization index: 0.3-1.2 cm) and zinc phosphate (solubilization index: 0.1-2.5 cm). Siderophore production was exhibited by fourteen isolates (0.3-1.4 cm). All the isolates were positive for auxin (without tryptophan: 2.04- 13.90 μg/ml; with tryptophan: 10.05-32.04 μg/ml) and ammonia production as well as ammonia excretion (1.803.65 μg/ml). The luxuriant growth on different nitrogen free medium was observed for all the isolates and higher HCN production was observed in three bacterial isolates. Out of these 24 isolates, two potential bacterial isolates RP4 and RP5 (which were named as CBF 2) were selected on the basis of PGP traits for evaluation of their potential to promote growth and yield of broccoli under pot as well as under field conditions. In green house experiment, these isolates when used synergistically showed a significant improvement in soil enzymatic activities (viz. dehydrogenase: 26.13 µg TPF formed h-1 g-1 of soil, alkaline phosphatase: 14.02 µg PNP formed h-1 g -1 of soil and urease: 370.80 µg urea hydrolysed formed h-1g -1 of soil) and plant growth parameters (plant height: 45.7 cm, root length: 6.3 cm, stem diameter: 3.9 cm, fresh weight of shoot :167.85 g, dry weight of shoot:35.70 g, number of leaves per plant: 26, days taken to visible curd:56 days, head compactness: 1.36 cm, number of heads/plant: 4 heads, mean head weight: 87 g and floret stalk length: 12.8 cm) over the control as well as other bioinoculants. The field evaluation of these bacterial isolates as consortium along with FYM and inorganic fertilizers was carried out using Palam samridhi variety of broccoli. The combined application of bacterial strains (RP4 and RP5) and FYM resulted in significantly higher soil microbial population and enzyme activities. Significantly higher available nitrogen (137.30 kg ha-1), available phosphorus (34.21 kg ha-1) and available potassium (170.15 kg ha-1) were also observed in treatment having FYM and bacterial inoculants (N75FYM+N25 + CBF 2). Significantly higher plant nutrient uptake of nitrogen (98.87 kg ha-1), phosphorus (24.84 kg ha-1), potassium (182.27 kg ha-1) was observed in (N75FYM+N25+ CBF 2) and higher uptake of micronutrients viz. zinc (75.5 mg/kg), iron (172.16 mg/kg) and manganese content (71.11 mg/kg) were found to be significantly higher in treatment (N100FYM+ CBF 2) having synergistic application of organic and bio-fertilizers. The various growth attributes of broccoli viz. plant height: 42.1 cm, root length: 11 cm, stem diameter: 4.9 cm, fresh weight of shoot: 214.20 g, dry weight of shoot:38.18 g, number of leaves per plant: 29, days taken to visible curd:44 days, head compactness: 1.11 cm, head yield: 153.15 q/acre, number of heads/plant: 6 heads, mean head weight: 88 g and floret stalk length: 12.8 cm was observed to be statistically higher in treatment T10 with combined application of FYM and bacterial isolates (N100FYM+ CBF 2). The study has led to the selection of a plant growth-promoting bacterial consortium as a potential bacterial inoculant for improving the growth, yield and nutrient content in broccoli.ThesisItem Restricted Bioprospecting production of fermented type II low fodmap (fermentable oligosaccharides, disaccharides, monosacchrides and polyols) sourdough(Punjab Agricultural University, 2023) Khanna, Rahul; Arora, RichaFermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs) in the food are short chain carbohydrates which are poorly absorbed and easily fermented in the large intestine by colon micro-organisms, thereby, producing acid and gas. These FODMAPs adversely affect human health leading to irritable bowel syndrome (IBS). In this context, sourdough technology can be exploited to reduce the FODMAP levels in food and alleviate the symptoms of IBS. In the present study, three yeast cultures, viz. Saccharomyces cerevisiae, Clavispora lusitaniae and isolate YB1 were compared for dough raising capacity, where yeast isolate YB1 was screened for maximum dough raising capacity (9.20 %). The screened isolate was characterised as Meyerozyma guillermondii by 18SrRNA gene sequencing and analysed by BLAST. Furthermore, the fermentation parameters for maximum dough raising capacity were optimised as inoculum size 13.6 % (2.4 x cfu/ml) and fermentation time 25.9 h using response surface methodology. After optimisation, fermentation of all- purpose flour using YB1 isolate resulted in reduction of total sugars (84.1 %), reducing sugars (79.8 %), glucose (60.2 %), fructose (88.8 %), lactose (39.3 %), sucrose (98 %) and raffinose (30.5 %). Apart, from sugar reduction, the fermented sourdough was found to have enhanced total antioxidants by 42.3 % and decrease in concentration of anti- nutritional compound i.e. phytic acid (16 %) after 25 h. Colour change by the fermentation process was found to be non- significant. Rheological characteristics of the sourdough, viscosity and shear strain decreased by 60 and 50 % respectively, thereby, increasing the porosity of the product. A total of 34 volatile compounds were detected using GC-MS with major contribution of butanoic acid, 2,3- butanediol tetracontane, n-hexadecanoic acid, octadecanoic acid which enhances aroma and shelf life of the fermented food. The present study demonstrates the potential of yeast isolate YB1 for production of low FODMAP sourdough fermented products using all-purpose flour as a promising alternative food for IBS patients.ThesisItem Embargo Evaluation of microalgal-bacterial consortia for waste water treatment(Punjab Agricultural University, 2023) Poonam; Sharma, Shiwani GuleriaThis study investigated the bioremediation abilities of microalgae and bacteria for the treatment of domestic waste water. Two microalgal viz. PL1 and PL2 and eight bacterial strains viz. B1(L1), B2(L1), B3(L1), B4(L1), B1(L2) B2(L2), B3(L2) and B4(L2) were isolated form indigenous population i.e L1 & L2. Both the microalgae isolates had a long exponential phase and continued to remain in this phase till the end of experimental growth period i.e. 30 days. Log phase of all bacterial isolates were observed at 60 hours of incubation except B1(L1) (48 hours). After screening, two microalgae i.e. PL1 & PL2 and four bacterial isolates were selected for efficient wastewater treatment. B1(L1), B3(L1), B2(L2) and B4(L2) were observed to be efficient in wastewater treatment than other bacteria under study. Coexistence of each strain with other was seen without any trace of growth inhibition which represented their compatibility. From the total of 29 tested consortia, most potential consortium was selected based on the treatment efficacy after 15 days of treatment. Developed consortium no. 29 i.e. PL1+PL2+B4(L2) was selected for domestic wastewater treatment as it showed significant percent reduction among all combinations i.e. BOD (82.9%), COD (77.77%), EC (53.8%), TDS (74.3%),Total nitrogen (71.05%) and Total Phosphorus (69.25%). Higher percent reduction in BOD (88.90%), COD (82.35%) was recorded with selected consortia 29 when treatment provided with source of aeration in 30 days of treatment. SEM observations revealed that several bacterial cells were attached to microalgal cells over 10 days of incubation and it led to the conclusion that microalgae PL1 & PL2 and bacteria B4(L2) can live together and efficiently treat wastewater. Molecular identification of members of consortia 29 was performed. It was revealed after 18S rRNA sequencing PL1 was identified as Chlorella variabilis, PL2 was tentatively identified as Desmodesmus and after 16S rRNA sequencing B4(L2) was identified as Paracoccus sp.. Sequence of PL1 and B4(L2) was submitted to NCBI GenBank with accession no. OR122520.1 and OR467496, respectively. Thus, present study concluded that microalgae and bacteria have more potential to treat wastewater when they were applied in form of consortia. The selected developed consortia no. 29 showed highest percent reduction in all physiochemical parameters with source of aeration in 30 days of wastewater treatment. Nevertheless, the assessment of polluted water composition should be completed prior to the designing and application of microalgae or cyanobacteria based remediation technology, since the pollutant removal efficiency is found to be essentially species dependent.ThesisItem Restricted Effect of slow release urea nano fertilizer on soil microflora and yield of direct seeded rice (Oryza sativa L.)(Punjab Agricultural University, 2023) Yashika; Kalia, AnuThe present work aimed to study the effect of a slow release urea nanofertilizer on soil microflora and yield of direct seeded rice (Oryza sativa L.). A soil column study indicated slow-release pattern of the urea from the nano-urea product varied for the soil samples having three different textures (low, medium and heavy).The leachate analysis revealed release of ammonical and nitrate nitrogen from the synthesized product. Further, the effect of the nanourea fertilizer on soil microflora study identified that the viable cell counts for a variety of soil microbes were not negatively affected on application of different doses of nanourea fertilizer. The soil urease, protease, and dehydrogenase enzyme activities exhibited significant increase in nano urea (100% RDN) treatment in light textured soil. A field experiment was performed to observe the effect of nano urea fertilizer on the growth and yield of direct seeded rice. An increase in the plant height, shoot and root fresh weight and dry weight was recorded for different doses of Nitrogen applied through nano urea and conventional urea N-fertilizer level for plants sampled after every 30 days after sowing till three months of the growth. The maximum plant growth was recorded for treatments which received 100% of the recommended N-fertilizer. Likewise, the yield attributing traits particularly the number of filled grains and thousand grain weight increased on application of 100% Recommended dose of N through nano urea. The soil chemical parameters were recorded to be non-significantly altered for plant and grain nutrient content (NPK content) at nano-urea fertilizer (100% RDN). Non-significant effect was recorded for the soil microbial viable cell count on different doses of nano-urea fertilizer. These results indicate the use of nano-urea fertilizer as a possible alternative for conventional urea fertilizer.ThesisItem Restricted Prospecting plant growth promoting and endophytic bacteria for improving growth and yield of sunflower (Helianthus annuus L.)(Punjab Agricultural University, 2023) Kiranjot Kaur; Vyas, PratibhaThe current study was carried out with the aim to develop a plant growth-promoting bacterial inoculant for improving the growth and yield of sunflower. A total of 64 morphologically distinct bacteria were isolated from leaves, stem, roots and rhizosphere of sunflower collected from the fields of Punjab Agricultural University, Ludhiana. The isolates were screened for multifarious plant growth-promoting traits viz. solubilization of phosphate and zinc; production of auxins, siderophore, ammonia, hydrogen cyanide, and growth on nitrogen free medium. Thirty-three bacterial isolates showed solubilization of phosphate with solubilization index ranging from 2.7 to 3.8 by rhizospheric bacteria and 2.4 to 5.4 by endophytic bacteria on modified Pikovaskaya agar. In liquid medium, phosphate solubilization ranged from 3.6 to 171.6 μg/ml by the rhizospheric bacteria and 1.1 to 889.5 μg/ml by endophytic bacteria. Thirty isolates showed zinc solubilization, 25 isolates showed siderophore production while 40 isolates showed luxuriant growth on different nitrogen free media. All the isolates were positive for auxin and ammonia production but negative for hydrogen cyanide production. Based on multiple plant growth-promoting traits, nine potential bacterial strains were tested for growth promotion of sunflower in pots. The isolates showed a significant increase in growth, chlorophyll content and N, P and K contents of plant and soil samples with the bacterial inoculations over the uninoculated control. Three isolates showing the highest growth promoting activity were identified by phenotypic features, biochemical tests and MALDI-TOF-MS analysis as Pseudomonas aeruginosa SRB1, Advenella kashmirensis SLE1 and Brevendimonas dimunata SLE15. The field evaluation of three potential bacterial strains Pseudomonas aeruginosa SRB1, Advenella kashmirensis SLE1 and Brevendimonas dimunata SLE15 and their consortium was carried out with and without 75 and 100 per cent recommended dose of fertilizers for sunflower. The highest growth promotion, nutrient content and yield enhancement was recorded with the treatment AkSLE1 + BdSLE15 + PaSRB1 with 100 per cent RDF which was statistically not different from that obtained with the treatment AkSLE1 + BdSLE15 + PaSRB1 with 75 per cent RDF, indicating the potential of these three PGPB strains in saving around 25 per cent N, P and K fertilizers for sunflower. The study has led to the selection of a plant growth-promoting bacterial consortium as a potential bacterial inoculant for improving the growth, yield and nutrient content in sunflower.ThesisItem Restricted Value Addition Of Volvariella Volvacea (bull.) Singer Through Mushroom Paste Formulations In Culinary Recipes(Punjab Agricultural University, 2023) Haifa; Jaspreet KaurPresent investigation was conducted to develop paste formulations of Volvariella volvacea (Paddy Straw mushroom) for its shelf life enhancement. Fruiting bodies of mushrooms harvested at egg stage were grinded to make salted, spiced and their fermented pastes. The salted paste formulation consisted of 2.5% salt, 0.5% sugar, 2ml refined oil, 1.5ml vinegar and 0.1% each of ginger and garlic while spiced paste was formulated by adding 0.5% onion, 0.1% cumin, 0.1% red chilli and 0.01% black pepper to the salted paste. To develop fermented paste formulations, salted and spiced paste was fermented using Lactobacillus rhamnosus GG for 2 days at 35±2°C. The unfermented mushroom spiced and salted paste was preserved with sodium benzoate @ 0.065% while fermented formulations were kept devoid of the any chemical preservative. The paste formulations were packed in glass jars and retort bags, and thereafter treated in boiling water bath for one hour. In organoleptic evaluation, the spiced fermented mushroom paste scored (8.3) significantly higher from the salted fermented formulation (7.7). Nutritional analysis of all the formulations revealed that spiced fermented mushroom paste contained highest content of crude protein (23.67%) and moisture content (36.72%) while lowest content of ash (3.01%), fat (0.51%), crude fiber (7.92%) and carbohydrates (28.17%). Highest fat (0.92%) was recorded in salted formulation. The overall energy was recorded highest (233.23J) in salted mushroom paste which was 10.04% higher than fermented spiced paste. The shelf life studies revealed that unfermented and fermented paste formulations showed spoilage on 3rd and 28th day of storage in glass jars at room temperature, as a indicated by total bacterial count of more than permissible limit of FSSAI, India. In contrast, longer shelf life of 56 days was observed for fermented mushroom paste stored in glass jars in refrigerated conditions. Variation of pH was indicator of spoilage, it was found that mushroom pastes stored in retort bags varied significantly in pH and were more prone to bacterial as well as fungal contamination irrespective of the temperature conditions. However, when stored in glass jars, the fermented (salted and spiced) mushroom pastes showed numeric variation in pH at 4ºC. The antioxidant analysis revealed that DPPH radical scavenging activity of fermented paste formulations increased significantly upto 21 days followed by gradual decrease till 56th day. While in case of unfermented paste steep decrease in DPPH activity was observed during storage. Total phenolic content of all the paste formulations was found to be decreased with storage. Above results showed that fermented spiced mushroom paste was best formulation, thus was selected for incorporation to culinary recipes. Incorporation of this mushroom paste at 25%, 10% and 45% was found best in noodles, spread and aloo gobhi sabji, respectively. It was concluded that fermentation of Volvariella volvacea spiced mushroom paste with L. rhamnosus GG culture for two days significantly enhanced its nutritional properties, antioxidants activity and shelf life upto 56 days; hence can be a good value addition to most perishable mushroom.ThesisItem Restricted Production and physicochemical analysis of black tea based functional beverage(Punjab Agricultural University, 2022) Tanushree; Katyal, PriyaKombucha is a functional beverage which is usually prepared by fermenting sweetened black tea with Tea fungus (Symbiotic consortium of acetic acid bacteria and different yeast known as SCOBY). Aim of the research was to prepare a fermented kombucha beverage having higher antioxidant activity and sensory acceptance. To produce a sensory acceptable beverage, standardization of inoculum concentration was done, in which 5% inoculum concentration was considered to be appropriate. For production of a fermented beverage with higher amount of antioxidant activity, tea concentration (5-15 g/L) and sugar concentration (40-80 g/L) were statistically optimized by Central Composite Rotable Design in ―Response surface methodology‖ (RSM) using Design Expert 13. Kombucha beverages prepared with different concentration of tea and sugar were fermented for 7 days at 25º C. Kombucha with tea concentration of 15 g/L and sugar concentration of 80g/L was found to be the beverage having highest antioxidant activity. Physicochemical and microbiological properties of Kombucha beverages were analyzed during fermentation and results demonstrated that chemical characteristics such as pH, TSS (ºBrix), total sugars and reducing sugars decreased while titrable acidity, total phenolic content, antioxidant activity, vitamin C and alcohol content increased with fermentation time. Developed Kombucha beverages were sensory evaluated by semi-trained panel of judges on a nine point ‗Hedonic scale‘. Fermented Kombucha containing tea concentration of 5 g/L and sugar concentration of 80 g/L received the highest overall acceptability sensory score of 8.15. Kombucha beverages selected by RSM and the one having highest sensory score were stored at refrigerated conditions for 7 week. The parameters viz., pH, TSS (ºBrix), total sugars, reducing sugars, titrable acidity, total phenolic content, antioxidant activity, vitamin C content, alcohol content were analyzed at every 7 day interval. Beverage was also checked for contaminants such as coliforms and mold growth. It was found that overall acceptability of Kombucha beverage remained high during the period of 7 weeks.ThesisItem Restricted Antioxidant and antimicrobial properties of Pleurotus djamor (Rumph. ex Fr.) Boedijn enriched with selenium (Se) and zinc (Zn) nanoparticles(Punjab Agricultural University, 2023) Samson, Debbarma; Sharma, ShivaniThe biofortification potential of biogenic selenium (Se) and zinc (Zn) NPs derived from P. djamor and P. sajor-caju were evaluated in terms of yield, protein content, antimicrobial activity, antioxidant and phenolic content. The fruit body extracts of P. djamor and P. sajorcaju were used for the synthesis of Se and ZnO NPs using the precursor salts (Na2SeO4 and ZnC4H6O4) at different concentrations (0.4 to 1.0 mM). The visual colour change in the extracellular extracts of Pleurotus spp. treated with salts were observed (deep orange red and white for Na2SeO4 and ZnC4H6O4, respectively). This visual color change indicated the formation of biogenic NPs for both Se and Zn salts. Characterization of the NPs through UVVis spectroscopy revealed the optimized precursor salt concentration (0.4 mM for P. djamor and 0.8 mM for P. sajor-caju) and incubation time spans (72 hours for BioSeNPs-PD, BioZnONPs-PD, BioSeNPs-PSC and 96 hours for BioZnONPs-PSC). TEM imaging revealed spherical and roughly spherical shape for Se and ZnONPs, respectively with average particle size of 124 and 68 nm and 84 and 91 nm for PD and PSC BioSeNPs and BioZnONPs respectively. FTIR analysis revealed the presence of specific functional groups that denote the formation of characteristic nanoparticles. The maximum yield was found in BioZnNPs-PSC treated P. sajor-caju with an average fruit body yield of 56.16 ± 4.6 kg/100 kg dry straw. The BioZnNPs-PD treated P. djamor fruit body showed the highest total soluble protein content with a value of 2.98±0.07 mg g-1. The highest Zn level was observed in BioZnONPs (P. djamor) enriched wheat straw (133.1 ± 3.54 ppm), while Se content was highest (95.67 ± 2.98 ppm) in BioSeNPs fortified P. sajor-caju. BioZnONPs-PD exhibited the highest antibacterial activity against S. aureus, while BioSeNPs-PD and BioSeNPs-PSC were equally effective against E. coli. The biofortification of P. djamor with BioZnONPs exhibited the highest H2O2 and FRAP activities, while the fortification of P. sajor-caju with BioZnONPs showed the highest DPPH radical scavenging activity. The phenolic content of biofortified fruit bodies was significantly higher than the non-supplemented control, with biogenic Se/ZnO NPs was found to be more effective in enhancing phenolic content than inorganic supplementation. Thus, this study revealed that bio-fortification of P. djamor and P. sajor-caju with Zn and Se BioNPs played an important role in increasing the yield, protein content, elemental composition, antioxidant, and phenolic content, as well as exhibited antimicrobial activity which could be helpful in enhancing the nutraceutical value of mushrooms which may be used as a future food supplement.ThesisItem Embargo Biological control of Sclerotinia sclerotiorum causing stem rot disease of sunflower using bacterial endophytes(Punjab Agricultural University, Ludhiana, 2023) Rajwinder Kaur; Vyas, Pratibha; VyasThe aim of present study was to screen antagonistic bacterial endophytes against Sclerotinia sclerotiorum causing stem rot disease in sunflower. A total of 42 bacterial endophytes were acquired from the Department of Microbiology and Sclerotinia sclerotiorum from the Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana. Out of 42 bacterial isolates, four isolates exhibited antagonistic activity varying from 20.0 to 36.6% in dual plate assay and 35.4 to 92.0% in liquid media against S. sclerotiorum. The effect of bacterial endophytes on the ultrastructure of S. sclerotiorum was observed by scanning electron microscopy (SEM) which showed thinning, rupturing, breakage and shrinkage of the fungal hyphae in micrographs. In addition to antagonistic activity, bacterial endophytes also exhibited phosphate solubilization, and production of siderophores, HCN, auxins, ammonia, lipase, cellulose and amylase, while tested negative for protease, pectinase and xylanase production. Based on morphological features, biochemical test and MALDI-TOF-MS analysis, the isolates were identified as Sphingobacterium spiritivorum EB1, Bacillus sp. EB6, Pseudomonas aeruginosa EB21 and Serratia liquefaciens EB23. All the four bacterial strains exhibited ability to produce different volatile organic compounds with the highest number of VOCs produced by Bacillus sp. EB6 and the least by Pseudomonas aeruginosa EB21. The SEM analysis showed the colonization of the plant tissues by all the four bacterial strainsvalidating their endophytic nature. The bacterial treatments singly or in combinations also significantly enhanced the vigour index of sunflower ranging from 250 to 630.5. A significant increase in growth parameters, chlorophyll content, and nutrient content of shoot and soil was also observed with the bacterial treatments singly or in combinations over the uninoculated control in sunflower grown in pots in glasshouse conditions. The disease control by the bacterial treatments varied from 66.7 to 88.9%. The results indicated that the consortium of four strains was statistically not different from the consortium of SsEB1+ BaEB6 +SlEB23, indicating thereby the potential of these strains as potential biocontrol agent against Sclerotinia sclerotiorum. As the future line of recommendation, the formulation containing the consortium of these strains may be developed and tested in sunflower to control stem rot disease caused by Sclerotinia sclerotiorum in field conditions.
