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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 Interaction of Sesbania rhizobia with different species of Sesbania and kharif legumes(CCSHAU, Hisar, 2020-11) Jeniffer Christeena Masih; Gera, RajeshSesbania is an important wild legume as it can grow in wide range of soils like alkaline, waterlogged and acidic soils. It has high nitrogen fixation properties due to its association with rhizobia in both root and stem nodules. Rhizobia from wild legume like Sesbania may function as excellent plant growth promoting bacteria.Therefore, the present research was carried out to study the interaction of Sesbania rhizobia with different species of Sesbania and kharif legumes. A total of 25 Sesbania rhizobial isolates, which includes five isolates each from Sesbania aculeata, S. sesban, S. grandiflora, S. rostrata (root nodulating and stem nodulating), were included in the present investigation. Out of which 21 rhizobial isolates were obtained from departmental culture collection and 4 rhizobial isolates were isolated from soils collected from different locations of India using trap plant method. All the rhizobial isolates were able to produce IAA and ammonia, however, 92, 48 and 48 % rhizobia had the ability for phosphate solubilization, bacteriocin and siderophore production, whereas, 60% of rhizobia were able to utilize ACC. All the rhizobial isolates showed the presence of nifH and nodC genes. Five rhizobial isolates namely SSKr(ii), SGMg, SAUd(i), SRKr(iv)/r and SRTn/s from each Sesbania species were selected on the basis of different plant growth promoting traits, nodulation efficiency and amplification of nodC and nifH gene, to study their effect on different Sesbania species, mungbean and pigeonpea under sterilized conditions. The rhizobial isolates; SRKr(iv)/r (root nodulation) and SRTn/s (stem nodulation) were found to be most efficient isolates on the basis of nodule number and fresh nodule weight in cross nodulation within Sesbania species and other legumes. These isolates were also tagged with gfp gene to study their colonization on different parts of Sesbania rostrata. Recovery of GFP marked strains from root, root nodules, stem and stem nodules was 95 to100%. However, recovery of gfp marked strains from the surface of root and root nodules varied from 92 to100% while on the surface of stem and stem nodules, it ranged between 25 to 33%. Rhizobial isolate; SRKr(iv)/r showed good nodulation efficiency in all four Sesbania species and pigeonpea as compared to other rhizobial isolates under unsterilized conditions. Nodule occupancy of GFP marked strains; SRKr(iv)/rGFP+ and SRTn/sGFP+ under unsterilized condition was found to be 33-54 and 92% in case of root and stem nodules, respectively of Sesbania rostrata.ThesisItem Open Access Development of biocontrol agent against phytopathogenic fungi of tomato (Lycopersicon esculentum Mill.) crop.(CCSHAU, Hisar, 2020-07) Kapoor, Divya; Rakesh KumarIn the present study, seventy-eight bacterial isolates were retrieved from the tomato rhizosphere soil and screened for their antagonistic activity against Fusarium oxysporum f. sp. lycopersici, Alternaria solani and Rhizoctonia solani under in vitro conditions. Thirteen isolates, NAU5, NAU10, NAU13, NAU20, NAU21, NAU23, NAU29, NAU32, NAU36, NAA59, NAA60, KBA43 and KBA44 showed broad inhibition zones against A. solani measuring more than 20 mm. Six isolates, NAU19, NAU20, NAU23, NAU24, NAU27 and NAU30 resulted in broad inhibition zones against F. oxysporum f. sp. lycopersici measuring 10.1-20 mm. Six isolates, NAU20, NAU30, NAU35, LBV58, NAV66 and NAV73 resulted in broad inhibition zones against R. solani measuring 15.1-25 mm. Among the retrieved 78 isolates, 28% of rhizobacteria produced high amount of IAA and seven isolates, NAU4, NAU5, NAU10, NAU20, NAU28, KBA43 andKBV47 produced maximum IAA. The amount of IAA production by different isolates varied from 3.62μg/ml (NAV69) to 26.96μg/ml (NAU5). Among 78 isolates tested for chitinase assay, two isolates, NAU13 and NAV68 showed maximum activity. Out of 78 isolates, siderophore production was observed in 42% of retrieved bacteria and five isolates, NAU19, KBV45, KBV46, LBV56 and NAV75 produced maximum orange halo zone. HCN was produced by 10% of isolates and highest HCN was observed by 6 isolates, NAU4, NAU6, NAU20, NAU29, NAU41 and NAA59, qualitatively. Seven isolates, NAU5, NAU20, NAU30, NAU35, NAA59, NAV68 and NAV75 were selected and total of 27 treatments were prepared on the basis of antagonistic and plant growth promoting attributes tested in vitro for further evaluating under pot house conditions. Isolate NAU5 showed maximum shoot length, shoot weight, root weight, fruit yield and disease control (100%) against F. oxysporum f. sp. lycopesici. Isolate NAV75 showed maximum shoot length, shoot weight, root weight, fruit yield and disease control (87%) against A. solani. Isolate NAU35 showed maximum shoot length, shoot weight, root weight, fruit yield and disease control (100%) against R. solani. Irrespective of the fungi, isolate NAU5 showed biocontrol activity against all the three phytopathogens. The seven promising isolates showed the PGPR activities in the order NAV75> NAV68> NAA59> NAU5> NAU35> NAU20> NAU30 irrespective of the fungi in pot house studies. The isolates NAU5 against all the three fungi, NAU35 against R. solani and NAV75 against A. solani can be further tested on the tomato fields as potent biocontrol agents. On the basis of biochemical characterization isolate NAU5 was found to belong to Alcaligenes, NAU35 to Bacillus and NAV75 to Burkholderia genus.ThesisItem Open Access Impact of co-inoculation of plant growth promoting actinomycetes and biofertilizers on mustard (Brassica juncea L.) crop(CCSHAU, Hisar, 2020) Umang; Leela WatiMustard (Brassica juncea L.) a major oilseed crop and the second largest agriculture commodity after cereals in India, plays a significant role in Indian economy. Due to inconsistent climatic changes, occurrence of diseases and implementation of unsuitable dosage of chemical fertilizers and pesticides, overall production of mustard has been comparatively static for last two decades regardless of the use of numerous traditional and breeding approaches. Actinomycetes, a class of actinobacteria are Gram-positive bacteria found in soil and possessing various plant growth traits can play significant role in improving growth and yield of mustard crop. In the present study, two biofertilizer cultures viz., Azotobacter chroococcum and phosphate solubilizing bacterium (PSB) along with seven available actinomycetes isolates AK1, AK3, AK6, AK11, AK17, AK20 and AK37 were screened for different plant growth promoting traits including IAA production, phosphate solubilization, potassium solubilization, zinc solubilization, siderophore production, HCN production, cellulose and chitin degradation under in vitro conditions. The actinomycetes isolates exhibited three or more plant growth promoting traits and were compatible with standard biofertilizer cultures. Actinomycete isolates exhibiting multiple plant growth promoting traits along with biofertilizer cultures were evaluated for growth promotion of mustard under pot culture conditions during 2019-2020. Actinomycete isolate AK17 was adjudged best in improving growth andseed yield of mustard crop when coinoculated with Azotobacter chroococcum and PSB. The isolates AK20 & AK37 were found promising in improving vegetative growth only. The actinomycete isolate AK17 identified by 16S rDNA sequencing showing 99% similiarity with Streptomyces pseudgriseolus, could further be explored as bioinoculant in agricultural practices.ThesisItem Open Access Impact of chitinase producing bacteria on thrips infestation in citrus plantation(CCSHAU,HiSAR, 2020) Sharma, Nidhi; Pathak, D.V.The studies on chitinase activity of bacterial isolates against thrips on Kinnow were carried out at citrus orchard with the objective Impact of chitinase producing bacteria on thrips infestation in citrus plantation. Plant growth promoting rhizobacteria (PGPR) are root associated bacteria and benefit plants by providing growth promotion through a variety of mechanism. The public concern over the harmful effects of chemical pesticides on the environment and human health has enhanced the search for safer, environmentally friendly control alternatives. Due to the importance of chitinolytic enzymes in insect, nematode, and fungal growth and development, they are receiving attention in regard to their use as biopesticides and microbial biocontrol agents. Therefore, the present study was aimed to isolate and characterize the chitinolytic bacterial isolates and also evaluate their efficacy. A total of eighty morphologically distinct bacterial isolates were retrieved from soil samples of different microbiomes on King‘s B media and these bacterial isolates were screened for chitinase production out of these twenty isolates was showed zone of clearance on the collodial chitin agar plates where zone of clearance and enzyme activity index were used for initial screening of chitinolytic bacteria. Twenty out of eighty bacterial isolates were selected as chitinase producers on the basis of zone size ranging from 12.40 mm to 38.50 mm and were further screened for quantitative chitinase production. Quantitatively it was screened for chitinase enzyme activity on the basis of release of N-acetylglucosamine units (U) by action of chitinase, maximum enzyme activity was recorded for isolate B1 (3.905) followed by B5 (3.52) and D2 (1.35). A total of twenty bacterial isolates, were studied on the basis of their morphological and microscopic characterstics which showed a variation in margins, elevation, shape, pigmentation, Gram‘s reaction and cell shape. These bacterial isolates was biochemically characterized, isolate B5 was found positive for various biochemical tests such as citrate utilization test, Voges Proskauer (VP) test and negative for methyl red test. All the twenty isolates was screened for multifarious plant growth promoting traits viz. IAA production and HCN production. Three bacterial isolates which exhibited maximum chitinase activity were further evaluated for their biocontrol potential against thrips of citrus under field conditions. Kinnow were sprayed with three different bacterial isolates B1, B5, D2 (106 cfu/ml), dimethoate phosphamide spray and nimbicide at different concentrations (1.5ml/L, 2ml/L respectively). One isolate B1 showed maximum biocontrol efficacy in reduction of thrips population -79.66 percent against thrips in kinnow in comparison to control where -37.70 percent of reduction of thrips population was recorded. The percent infestation recorded for B1 isolate was 20 percent as compared to control where infestation was 36 percent. The mean population density recorded for B1 was 0.4 as compared to control where density of thrips was 0.96. The identification of B1 was tentatively confirmed by biochemical characterization and it was identified as strain belonging to Bacillus sp.ThesisItem Open Access Studies on molecular mechanisms underlying rhizobia promiscuity in different Khariflegumes(CCSHAU, Hisar, 2020) Rana, Barkha; Gera, RajeshMungbean, mothbean, cowpea, clusterbean, urdbean and pigeonpea are economically important kharif legumes commonly grown in rain-fed areas particularly South-West Haryana and Rajasthan state. It is possible to improve its symbiotic property for different kharif legumes through identification and manipulation of genetic factors that controls the promiscuity of Rhizobium. So the present investigation was undertaken to study molecular mechanism underlying rhizobia promiscuity in different kharif legumes. A total of 10 rhizobial isolates along with three rhizobial mutants were obtained from Molecular Microbiology laboratory of Microbiology Department that were retrieved from arid and hyper-arid zones of South-West Haryana and Rajasthan state. All selected rhizobial isolates were checked for their authenticity in six different kharif legumes after 45 days of growth under sterilized conditions. Promiscuous rhizobial mutants (PKR177m30, PKR188m85 and PKR194m93) have the ability to nodulate all six kharif legumes with high nodulation efficiency. Evaluation of these isolates for cellulase activity both on CMC agar plates and in liquid broth revealed that promiscuous rhizobial mutants showed highest cellulase activity both on CMC agar plates and in liquid broth as compared to other rhizobia. Molecular mechanism of rhizobial promiscuity was studied by amplifying and detecting relevant nodulation genes such as nodD, nodT, nodXandnolA in different rhizobia. PCR conditions were optimized to amplify a particular gene. All the 13 selected rhizobial isolates showed the presence of nodD and nodX genes, whereas amplification of nodT was observed in the isolates capable of nodulating 6, 5 and 4 different legumes. However, the expression of nolA gene was fairly good with single amplified PCR product in the rhizobia capable of nodulating 6, 5 and 4 different kharif legumes, amplification of nolA with single PCR amplified product of 672 bp was observed in isolates nodulating 6, 5 and 4 different while rest of the isolates showed poor expression of nolA gene with multiple bands 672 & 323bp. The mutant PKR188m85 performed better in terms of nodulation efficiency and plant growth parameters in pigeonpea, mungbean, clusterbean and mothbean whereas PKR177m30 performed better in cowpea and urdbean followed by PKR188m85 than their respective parent and reference strains under sterilized conditions.ThesisItem Open Access Characterization and mass production of chitosan from fungi(CCSHAU, Hisar, 2020-06) Aathira S. Kumar; Malik, KamlaChitosan is a linear cationic biopolymer consisting of β (1-4) bonds between 2-amino-2- deoxy-D glucopyranose and 2-acetamido-2- deoxy-D-glucopyranose. It is non-toxic, biocompatible, biodegradable, antimicrobial agent and has high charge density which paves way for its numerous applications especially in the field of agriculture, food and pharmaceuticals. Chitosan, besides chitin, occurs in fungal cell walls particularly of Ascomycetes, Basidiomycetes and Zygomycetes. The enzymatic deacetylation of chitin is the major mechanism for synthesis of chitosan in fungi. Hence, the biological alternatives (fungi) have been used for chitosan production through fermentation technologies. Chitosan has broad antimicrobial spectrum to which gram-negative, gram-positive bacteria and fungi are highly susceptible. In the present study, characterization and mass production of chitosan from fungi was standardized fermentation conditions and determined its antimicrobial properties against pathogenic microorganisms. A total of 18 morphologically different fungal isolates, 17 bacteria and 3 actinomycetes were isolated on chitin agar medium. Out of which, only 6 fungal isolates (FC1, FC3, FC7, FC8, FC9 and FC 16), 2 bacterial isolates (BC 1 & BC 12) and 1 actinomycete (AC 1) showed positive results by production of yellow colour on the chitin agar media supplemented with p-nitroacetanilide as indicator. FC 3 was the most efficient fungal isolate with highest yield of chitosan (0.096 g/100ml). Maximum chitosan production (0.265 g/100ml) was observed at temperature (35˚C) and pH 5 after 96 h of incubation. Glucose (0.309 g/100ml) and yeast extract (0.332 g/100ml) severed as the best carbon and nitrogen source for highest production of chitosan from FC3. When the growth media supplemented with agro-industrial waste @1% paddy straw +1% glucose (w/v) the yield of chitosan (0.315 g/100ml) was increased. The fungal isolates (FC3) showed maximum chitosan production in submerged fermentation (0.533 g/10g paddy straw) as compared to solid state fermentation (0.182 g/10g paddy straw). Therefore, mass production of chitosan from fungal isolate FC 3 was carried out by submerged fermentation in a bioreactor (BioFloR 120) and the yield of chitosan was found to be 5.37 g/l. Further, the chitosan extracted from isolate FC 3 was estimated for degree of deacetylation and it was observed 88.5%. On the basis of morphological and molecular characterisation, the fungal isolate FC 3 was identified as Aspergillus flavus. The antibacterial activities of chitosan at different concentrations were examined against Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus and Xanthomonas. The maximum inhibition zone (8. 0 mm) was observed at 1000 ppm against Escherichia coli. Whereas, the highest percentage of inhibition was observed at 3000 ppm for Rhizoctonia solani (90.73 %) and Fusarium oxysporum (76.27%).ThesisItem Open Access Evaluation of plant growth promoting actinomycetes on chickpea (Cicer arietinum L.)(CCSHAU, 2019) Kavita Rani; Leela WatiChickpea is the second most important cold season food legume grown globally on a very wide area. However, global yields of chickpea have been relatively stagnant for the last two decades due to unpredictable climatic changes, incidence of diseases and extensive application of chemical fertilizers and pesticides. Actinomycetes are one of the major components of rhizosphere microbial population and are useful in soil nutrient cycling as well as plant growth-promotion. Since last few years, studies on beneficial traits of actinomycetes regarding plant growth promotion and biocontrol activities had opened new avenues for their applications in sustainable agriculture. In the present investigation, total 40 (AK1-AK40) actinomycete isolates were retrieved from different soil samples and chickpea nodules collected from CCS Haryana Agricultural University, Hisar farms. All the isolates were assessed for different plant growth promoting traits including IAA production, P solubilization (P-SI), Zn solubilization (Zn-SI), siderophore production, HCN production, salt tolerance, biocontrol potential against F. oxysporum, ACC utilization and ammonia excretion. Based on the results of various PGP traits, isolate AK3 with high P-SI (2.78) and Zn-SI (3.92); AK6 with high HCN production; AK11 with good P-SI (2.70) and Zn-SI (3.27) and AK34 with high IAA production (20.95μg ml-1) were evaluated for growth promotion of chickpea under pot culture conditions. Isolate AK3 found promising in terms of nodulation, growth and yield parameters under pot culture conditions was evaluated on chickpea variety HC-5 under field conditions (during 2017-2018). Coinoculation of AK3 along with Mesorhizobium 1233 was beneficial for plant growth and yield of chickpea. The promising actinomycete isolate AK3, identified as Streptomyces griseoruber on the basis of partial 16S rDNA sequencing, can be further tested on different locations for use as biofertilizer.ThesisItem Open Access Biochemical characterization and toxicity Studies of biosurfactants produced by yeast(CCSHAU, 2019) Dolly Rani; Sangwan, SeemaThe biosurfactant production potential of two yeasts, Meyerozyma guilliermondii YK 32 and M. guilliermondii YK 22 was investigated using total eight types of carbon sources followed by extraction and characterization of final product. Maximum biosurfactant production monitored in terms of oil displacement (9.0cm) was obtained using a mixture of olive oil and whey at 4%(v/v) concentration of each in case of M. guilliermondii YK 32. Its cell free supernatant yielded maximum 12.77g/L crude biosurfactant using acetone precipitation method. Total eight types of crude biosurfactants obtained using different carbon sources (BS1-BS8) were subjected to further characterization. The preliminary characterization using TLC revealed yellow spots in case of BS1, BS4, BS5 and BS8 having Rf 0.33, 0.41, 0.45 and 0.38, respectively indicating the presence of carbohydrate and lipids while a ninhydrin spray detected purple to reddish purple spots in BS2, BS3, BS6 and BS7 with Rf value varied from 0.23 to 0.93 depicting protein and lipid contents. The biosurfactant produced using a mixture of olive oil and whey (BS6) as substrate contained highest protein content (28.61 mg/g) followed by BS2 (20.90 mg/g) and BS1 (15.94 mg/g) while a mixture of molasses and whey supported the production of crude biosurfactant (BS8) containing highest carbohydrate (28.82 mg/g) as well as lipid content (A260nm, 16.85). All the crude biosurfactants exhibited neutral behavior during ionic charge determination. Contrary to chemical surfactant (1%SDS) which inhibited the germination of chickpea, the increasing concentration of biosurfactants supported the germination of chickpea giving a maximum germination index equaled to 423 % at 0.8% concentration of biosurfactants BS4.Further, all the biosurfactants were non toxic to yeast multiplication and did not show any antimicrobial activity against Rhizobium, a bioinoculant for nitrogen fixation. Being supportive to chickpea germination and nontoxic to yeast, the neutral biosurfactant produced by M. guilliermondii YK 32 may be tested for application in agricultural practices, cosmetics and pharmaceutical industries.ThesisItem Open Access Bioremediation of phenolic compounds in sewage water using bacterial consortia(CCSHAU, 2019) Diksha; Rakesh KumarSewage water is a rich source of organic waste and nutrients in the form of nitrates and phosphates, due to which it can be used in agriculture after proper treatment. Sewage water contains various harmful compounds like phenolics which need to be treated before discharge. The direct discharge of sewage water into natural water sources leads to the obstructive impacts on the water sources and aquatic life, sewage water becomes an open source of harmful microbes and pollutants. To demolish these problems the treatment of domestic waste water is must and its reuse for the domestic as well as agriculture purpose can become an eco-friendly solution for the conservation of water. A total of 29 bacterial isolates were retrieved from seven sewage water samples collected from Sewage Treatment Plant, CCS HAU, Hisar. All bacterial isolates were screened for phenol degradation in mineral salt medium supplemented with 250, 500 and 750 ppm concentration of phenol. Out of 29, only 8 bacterial isolates were able to grow in mineral salt medium having 750 ppm concentration of phenol. These eight bacterial isolates were further observed for growth upto 1500 ppm concentration of phenol, out of eight, only four bacterial isolates (NI6, NI5, NR1 and JC1) were able to grow upto 1250 ppm concentration of phenol. Cultural conditions, inoculum size and aeration were optimised in culture medium as well as sewage sludge. Fifteen percent inoculum and 2 LPM aeration rate were found to be optimum for efficient phenol degradation. Least residual phenol in culture medium and sewage sludge at 15% inoculum was left with bacterial isolate NI6 (265.2 ppm and 607.30 ppm respectively). Same was observed in culture medium and sewage sludge at 15% inoculum and 2 LPM aeration in lab scale fermenter for bacterial isolate NI6 (352.3 ppm and 761.27 ppm respectively). A consortium of four bacterial isolates (25% each of NI6, NI5, NR1 and JC1) was found to leave least 276.93 ppm residual phenol in sewage sludge as compared to individual isolates. Consortium was further tested for removal of phenolic compounds, COD, BOD and Heavy metal at STP, CCS HAU, Hisar. At 9th day of application, highest phenolic compounds degradation was observed in sewage sludge (120 ppm) and outlet of clarifier (172.9 ppm), thereafter phenolic compounds concentration started to increase. At 9th day COD reduced from 460 mg/L to 60 mg/L and 100 mg/L to 40 mg/L while BOD decreased from 153 mg/L to 18 mg/L and 28 mg/L to 17 mg/L in sewage sludge and outlet of clarifier respectively. Removal of Zinc and Chromium was observed in outlet of clarifier and sewage sludge.
