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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.