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Chaudhary Charan Singh Haryana Agricultural University, Hisar
Chaudhary Charan Singh Haryana Agricultural University popularly known as HAU, is one of Asia's biggest agricultural universities, located at Hisar in the Indian state of Haryana. It is named after India's seventh Prime Minister, Chaudhary Charan Singh. It is a leader in agricultural research in India and contributed significantly to Green Revolution and White Revolution in India in the 1960s and 70s. It has a very large campus and has several research centres throughout the state. It won the Indian Council of Agricultural Research's Award for the Best Institute in 1997. HAU was initially a campus of Punjab Agricultural University, Ludhiana. After the formation of Haryana in 1966, it became an autonomous institution on February 2, 1970 through a Presidential Ordinance, later ratified as Haryana and Punjab Agricultural Universities Act, 1970, passed by the Lok Sabha on March 29, 1970. A. L. Fletcher, the first Vice-Chancellor of the university, was instrumental in its initial growth.
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ThesisItem Open Access Effect of mineral solubilizing bacteria and fly ash application on wheat (Triticum aestivum L.) crop(2022-08) Savita RaniCoal is a predominant source of global energy and contributes about 38% of the total energy production worldwide. Fly ash is one of the by-products generated in thermal power plants. Fly ash has been shown to have the potential to improve soil as well as crop health. Efforts are being made to study its compatibility with soil microflora by incorporating in soil in certain fixed proportions. Fly ash application along with mineral solubilizing bacteria can be explored to cut down the use of chemical fertilizers for achieving high crop productivity. Fly ash used in present investigation had grey color, sandy texture, alkaline pH, with trace amount of nitrogen, phosphorous, potassium, zinc, copper, manganese and iron. A total of 75 bacterial isolates were retrieved from fly ash contaminated soil by dilution and plating, out of which 21 bacterial isolates (MSB1-MSB21) exhibited zone of clearance on Pikovskaya’s, modified Aleksandrov and Zinc minimal medium. Maximum solubilization index for isolate MSB1 and MSB2 was 4.60 and 4.94 for phosphorous, 3.23 and 4.13 for potassium and 4.32 and 4.71 for zinc respectively. All twenty one MSB isolates were found to be IAA producers in the range of 0.12-9.82 μg/ml. Thirteen MSB isolates showed HCN production and five isolates were observed as siderophore producers. On the basis of biochemical characterization and partial 16S rDNA sequencing, the isolate MSB1 exhibited 98.28% similarity with Bacillus safensis strain FO-36b and isolate MSB2 was showing 99.68% similarity with Brevundimonas vesicularis strain Busing. The viable count of bacterial isolate MSB2 in nutrient broth amended with fly ash increased with increase in fly ash from 0.5 to 3.0% and decreased with further increase in fly ash concentration upto five percent. Maximum viable count of MSB2 was observed as 8.68 log cfu/ml at 3% fly ash concentration after 72h of incubation. Similarly, the bacterial isolate MSB2 survived in soil amended with 3% fly ash concentration with viable count 7.83 log cfu/g after 72h of incubation that decreased with increase in fly ash concentration and incubation time. Under pot house conditions, fly ash amendment in soil at 3% concentration and wheat seeds treated with culture of MSB2 resulted improved plant growth in terms of shoot weight, root weight and yield in terms of no. of seeds, no. of spikes, no. of tillers and seed weight in comparison with control. According to the findings of this study, fly ash (3%) combined with mineral solubilizing bacterium can be used to reduce the use of chemical fertilizer for achieving high crop yieldThesisItem Open Access Amelioration of salt stress in rice (Oryza sativa) by using cellulose immobilized ACC utilizing bacteria(CCS HAU, Hisar, 2022-09) Bharti; Pathak, D. V.Salt stress is one of the major abiotic stresses responsible for reducing plant growth and crop productivity. Plants subjected to excess salt initiate ionic imbalance, which leads to metabolism imbalances induced by ion toxicity and water deficit generated by hyperosmotic stress. Plant growth-promoting microbes enhance plant growth under salt stress by different mechanisms. In this context 32 bacterial strains were isolated from soil collected from salt-affected regions. Out of these14 isolates were found to tolerate NaCl concentration up to 15%(w\v). All bacterial isolates were quantitatively screened for IAA production, Ammonia production, and qualitative screening was done for zinc solubilization, potassium solubilization, phosphorous solubilization, and salt tolerance. Out of all bacterial isolates, 23 were found to utilize ACC. On the basis of ACC utilization activity, and PGPR traits, two potential isolates, STB11 and STB15 were selected for further studies. Most bio-fertilizers are unable to establish in the rhizosphere due to lack of nutrients and stress caused due to excess salt and water deficiency. In this study, for the first-time effect of bacterial cellulose in agriculture field was explored. Isolated ACC utilizing bacteria were immobilized on bacterial cellulose and evaluated for ameliorating salt stress in rice crop. A total of 26 cellulose-producing bacteria were isolated from different rotten fruits and kombucha tea. Only one (CPB26) among them was selected as a potential cellulose producer, based on the amount of bacterial cellulose produced. Central Composite Design based Response Surface Methodology (CCD-RSM) was employed to design the optimization experiments. Temp. 35C, pH 6, and incubation days 5 were found to be optimum for maximum cellulose production. Selected isolate CBP26 preferred peptone as nitrogen source and glucose as carbon source. Among metal ions, Mg2+ and Fe2+ were found to support bacterial cellulose production, highest at 0.15%(w/v) concentration, whereas Zinc did not support bacterial cellulose production. Out of four different enhancers, 1%(v\v) ethanol enhanced cellulose production. For confirmation of the bacterial cellulose, pellicle of bacterial cellulose was treated with cellulase enzyme, which digested it completely in 24h. Ultrafine Bacterial cellulose nanofibrils of size 20nm were visualized under scanning electron microscopy. FTIR spectra of bacterial cellulose obtained from analysis was found similar to those reported previously and typical bacterial cellulose thermal behavior was observed upon thermogravimetric analysis. X-RD analysis revealed that the bacterial cellulose was ordered crystalline with a crystallinity index of 77.9% and 11nm crystal size. The water holding capacity of bacterial cellulose was found to be 360%. The water evaporation rate of soil mixed with bacterial cellulose was less than the soil without bacterial cellulose. Dried bacterial cellulose was converted to powder form to carry out further studies. Selected bacterial isolates STB11 and STB15 were immobilized on powdered bacterial cellulose by absorption and incubation method. After the incubation step, immobilized bacterial cells were coated on rice seeds (Pusa 1121). Sowing of these coated seeds was done in pots (4 seeds/pot) under pot house conditions. Different RSC water i.e.,4, 8, 10 was used for irrigation of crops. The selected bacterial isolates were found to be effective in terms of rice plant growth such as plant height varied from 13cm -78.5cm from 15 to 90 days after sowing (DAS), maximum in T20 (RSC8+BCI STB15) 78.5cm at 90 DAS, root length (7.99-29.9cm), maximum in T21 (RSC 10+BCI STB15) 29.9cm, fresh weight of shoot and root varied between 1.49- 3.43 and 0.3-1.34g/plant respectively, dry shoot and root weight varied between 0.15-0.49 and 0.15-0.18g/plant respectively. Selected bacterial isolates also enhanced N (2.31- 13.66mg/plant-1) and P (0.10-0.27mg/plant-1) and increased superoxide dismutase activity in plants (1.07-3.54Unit/g FW), maximum in T20 (RSC8+BCI STB15) 3.5 unit/g FW. Rhizospheric bacterial viable count of rice plants was found maximum at 60 days after sowing in T20 followed by T21 i.e., 8.09 and 8.06 log Cfu/g of soil, respectively. On the basis of partial 16s rDNA sequencing bacterial isolate STB11 was identified as Bacillus pumilus, STB15 as Enterobacter spp. Bacterial cellulose producer CPB26 was identified as Gluconacetobacter liquefaciens. All the three identified bacterial gene 16s rDNA sequences were submitted to NCBI and allotted the accession numbers.ThesisItem Open Access Studies on antagonistic activity of bacteria against root knot nematode(s)(CCSHAU Hisar, 2022-09) Antil, Sonam; Pathak, D.V.Root-knot nematodes under the genus Meloidogyne are world widely distributed plant parasites that cause serious damages to many important agricultural crops such as potato, cotton, tomato, brinjal, etc. Bacteria associated with roots and rhizosphere of many plant species have been extensively tested for the control of various soil borne pathogens including plant parasitic nematodes. Four bacterial isolates- KMT-4, KMT-5, KMT-8 and KMS-6 were originally isolated from nematode infested rhizospheric soil belonging to research field of Department of Nematology, CCS HAU, Hisar and have demonstrated potential nematicidal activity against root-knot nematode Meloidogyne javanica. All the isolates showed HCN and siderophore production but chitinase activity was exhibited by KMT-5 only. The bacteria were identified based on 16S rRNA gene sequence analysis as: Bacillus aryabhattai (KMT-4), B. cereus (KMT-5), B. megaterium (KMT-8), B. altitudinis (KMS-6). For in vitro bioassays, M. javanica eggs were isolated from infected roots and juveniles (J2) were obtained through MBFT. Effect of bacterial strains on egg hatching and J2 mortality was checked by preparing the extracellular and intracellular extracts of all four bacterial strains and tested their activity on M. javanica eggs and J2. The larval mortality and egg-hatching inhibition rates of M. javanica were increased with the rising concentration of extracellular extracts of all the bacterial strains and the duration of exposure. A pot experiment was also planned in spring 2021 on tomato to check the genetic stability of the bacterial strains. All four bacteria reduced nematode infection and promoted plant growth far better than control and chemical treatment as well. The nematicidal bacterial extracts were subjected to crude extraction from extracellular and intracellular extracts for proteinaceous as well as non-proteinaceous nematicidal compound(s). Crude extraction done for extracellular non-proteinaceous was found potent of all the bacterial strains when tested against M. javanica eggs and J2 with B. cereus KMT-5 showing maximum activity. Thus, this study aimed to separate, purify, and identify nematicidal compound from B. cereus KMT-5 and to validate its anti-M. javanica activities. Compound was purified through silica gel column chromatography using ethyl acetate: methanol as mobile phase with increasing polarity gradient and fractions obtained were checked via thin-layer chromatography. Similar fractions were combined and tested against M. javanica eggs and J2. Out of five subfractions, subfraction III (FIII) showed maximum activity. The nematicidal compound from FIII was extracted via prepTLC and subjected to HPLC. Structural identification was conducted through H1-nuclear magnetic resonance (NMR) spectroscopy. The nematicidal compound was identified as phytosphingosine based on H1-NMR shifts and structure was in silico verified using MestReNova 14.2 software. Purified nematicidal compound phytosphingosine from B. cereus KMT-5 was then tested against M. javanica eggs and J2. Only 5.6 J2 were obtained after 96h of treatment with phytosphingosine whereas in control there were 98.0 J2 showing a significant difference in hatching rate. The mortality rate of M. javanica J2 reached to 94.6% after 72h of exposure to phytosphingosine.ThesisItem Open Access Production, extraction and characterization of astaxanthin from microalgae(Chaudhary Charan Singh Haryana Agricultural University hisar, 2022-09) Satish Kumar; Satish Kumar; Rakesh Kumar; Rakesh KumarIn the present investigation, five microalgal isolates were retrieved from different regions of Himachal Pradesh and Haryana. Out of five, two isolates, PLM1 and PLM2, produced astaxanthin. Astaxanthin production potential of PLM1 was low; therefore, only PLM2 was selected for further studies. Based on molecular analysis, PLM1 and PLM2 were identified as novel species named Desmodesmus sp. nov. PLM1 and Desmodesmus sp. nov. PLM2. Optimized culture conditions include sodium nitrate (20 mM), glucose (3mM), potassium chloride (32mM), pH (7), incubation temperature (27°C), and 15 days of incubation time for the green and red stages. The media with optimized composition were re-named as Modified BG-11 or M-BG11 medium. After achieving maximum biomass yield, cells were harvested and inoculated in nitrogen, phosphorous limiting media supplemented with 0.2% NaCl and 4.4mM sodium acetate and incubated at 27℃ for 15 days under the illumination of the white light of intensity 3000 lux. After 15 days, microalgal red cyst cells from BG-11 and M-BG11 medium were harvested, lyophilized, stored, and directed for astaxanthin extraction using different solvents. Maximum astaxanthin extraction resulted from cells grown in M-BG-11 medium was 21.13 ppm using 4 M HCl. The extracted astaxanthin was confirmed for its presence using UHPLC, FTIR, and Raman spectroscopy. Astaxanthin extracted from Desmodesmus cells grown in two different media was evaluated for DPPH free radical scavenging activity. Maximum DPPH free radical scavenging activity (78.69%) and free hydroxyl radical activity (37.81%) were observed for astaxanthin extracted using 4M HCl. The lyophilized dried biomass was stored and evaluated for degradation at different temperatures, including -20°C, 4°C, 25°C, and room temperature. Maximum degradation (88%) was observed at room temperature after 20 weeks of storage for the BG11 medium. The red-light illumination enhanced the vegetative growth of Desmodesmus sp. PLM2, whereas blue-light illumination caused stress in Desmodesmus sp. PLM2, thereby triggering astaxanthin biosynthesis. The extracted astaxanthin was inhibiting the growth of Bacillus subtilis, Staphylococcus aureus, and E. coli.ThesisItem Open Access Evaluation of silicate solubilizing bacteria and fly ash application on rice (Oryza sativa L.) crop(Chaudhary Charan Singh Haryana Agricultural University hisar, 2022-09) Chopra, Gourav; Leela WatiFly ash has been reported beneficial for soil and plant health as it can act as soil conditioner and has varying amount of different nutrients required for plant growth. Use of chemical fertilizers for agriculture in present time has become a serious threat for environment and sustainability, therefore use of fly ash in agricultural system can reduce the demand of chemical fertilizers. Fly ash used in present study was of grey colour, sandy texture and alkaline pH. Different plant nutrients i.e. nitrogen phosphorus, potassium were detected in trace amount whereas silicon was present in high concentration. Total 52 bacterial isolates were screened for silicate solubilization on Bunt and Rovira medium out of which 40 bacterial isolates (SSB1-SSB40) showed clear zone and highest silicate solubilization index (2.67) was observed for isolate SSB2. Six bacterial isolates (SSB2, SSB6, SSB17, SSB26, SSB30 and SSB32) exhibiting high silicate solubilization were tested for different plant growth promoting traits. All bacterial isolates were able to produce indole acetic acid and excrete ammonia in the range of 5.72-12.40μg/mL and 1.75- 4.60μg/mL, respectively. Three bacterial isolates (SSB2, SSB6 and SSB30) were found positive for siderophore production. Bacterial isolates SSB2 and SSB32 have shown growth in nutrient medium having 10% NaCl concentration. Highest silicate solubilization (36.69 mg/L and 23.98 mg/L) was exhibited by SSB2 after 21 days in liquid medium having 0.25% magnesium trisilicate and 0.25% fly ash, respectively. Optimum concentration of fly ash to be amended in liquid medium was found 0.35% as amendment of 0.45% fly ash resulted decreased silicate solubilization by bacterial isolates. On the basis of biochemical characterization and partial 16S rDNA sequencing the isolate SSB2 found promising under in vitro studies was identified as Pseudomonas baetica. The viable count of bacterial culture SSB2 in nutrient broth amended with different concentrations of fly ash increased upto 3% fly ash and hampered with further increase in fly ash concentration and increased incubation period. Survival of bacterial culture enhanced on fly ash amended in soil from 0.5-3% whereas it was impeded by further increase in concentrations. Under pot house conditions, amendment of fly ash (3%) in soil along with RDF and treatment of rice seedlings roots with bacterial culture SSB2 resulted in improved plant growth and grain yield in comparison with control.
