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Open Access
Implications of plant growth promoting rhizobacteria and their consortia for biofortification of Fe and Zn content in wheat under saline and non saline soils
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-06) Singh, Jyoti; Singh, Ajay Veer
Present study was an attempt to characterize plant growth promoting halotolerant rhizobacteria isolated from paddy rhizosphere of different region’s saline soils bearing massive competence for zinc solubilization and siderophore production. Furthermore, explorations were led to determine in situ effect of potential isolates and their consortia on growth and biofortification benefits through enhanced zinc and iron assimilation in wheat under saline and non saline soils. Total 146 bacteria were isolated from rice rhizosphere of both saline and non saline soils habitats and among these, 44 bacterial isolates were screened on the basis of zinc solubilizing potential, siderophore production and higher tolerance ability under NaCl and PEG stress. Finally, 4 bacterial isolates bearing multiple plant growth promoting potential were selected and characterized on the basis of morphological and biochemical tests. On the basis of bio compatibility, two bacterial consortia (C1 and C2) were developed from potential 4 isolates having zinc solubilizing and siderophore production ability under high NaCl concentration and used for the further study with selected four isolates. Selected bacterial isolates were characterized on the basis of 16S rDNA found to be phylogenetically related to four genera, namely Providencia vermicola, Pseudomonas protegens, Klebsiella pneumoniae and Pseudomonas sp. and were also found to have stress responsive acdS gene. UPLC-MS analysis of cell free supernatant obtained from zinc amended mineral medium revealed major organic acids (salicylic acid, carboxylic acid, acetic acid, succinic acid and lactic acids) produced by four bacteria depicts a key mechanism of zinc soubilization. All four isolates and two consortia were evaluated for their effect on seedling germination efficiency under different NaCl concentration, and among them consortium C2 showed prominent influence on germination efficiency over other treatments. Afterwards, isolates and consortia also showed positive and significant influence on agronomical as well as physiological and growth parameters through in situ green house pot experiment in Triticum aestivum var. PBW373 under both saline and non-saline soil systems. Moreover, the soil enzymatic activities were found at utmost level in soils (saline and non saline) of both consortium (C1 and C2). After harvesting, soil and plant samples were analyzed for zinc and iron assimilation through Atomic Absorption Spectroscopy (AAS). Both consortia and isolates (CP20 and CP17) assimilated enhanced level of Zn and Fe in wheat significantly over negative control. Proteomic studies revealed 33 differentially expressed proteins (23 up-regulated and 10 down-regulated) in 25th days seedling inoculated with C2 were related to stress tolerance, transporter and nutrient reservoir proteins. Present study provide an overview of employing microorganisms and their consortia as effective bioinoculants to drive biofortification of food crops in more eco-friendly environment to improve sustainable agriculture even in salinity stress.
Open Access
Differential proteomic studies of cold adapted diazotrophs and their impact on Cicer arietinum yield, nitrogen status and soil microbial community
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-05) Joshi, Divya; Goel, Reeta
Two psychrotolerant diazotrophs, Pseudomonas jesenii MP1 and Rhodococcus qingshengii S10107, were retrieved from department culture collection. They were tested for their impact on germination efficacy of seeds of three crops viz. chickpea, lentil and pea. Based on it, field trial experimentation was conducted on Cicer arietinum L. var. PG-186 at Norman E. Borlaug Crop Research Centre (NEBCRC), GBPUA&T to check its growth and development as well as soil health. Plant growth promotory activity of these strains were documented through increased plant growth in terms of shoot and root length, fresh and dry weight, nodule number, nodule fresh and dry weight (agronomical parameters), chlorophyll content and leaf nitrate reductase activity (biochemical parameters), nitrogen uptake, seed protein content and yield of the plants over control. Soil health was measured in terms of increased macro- and micronutrient content and soil enzyme activities unlikely to control. The results have shown that Pseudomonas jesenii MP1 with recommended dose of fertilizer (20 kg N ha-1) gave highest agronomical parameters, chlorophyll content, nitrogen content in plant, seed and straw, crop yield and harvest index over control treatment. However, MP1 alone gave highest nodule parameters, leaf nitrate reductase activity as well as bacterial (16S rRNA) and diazotrophic (nifH) copy number in soil. DGGE analysis showed that both diazotrophs remained persistent till 75 DAS. High throughput sequencing revealed soil with S10107 was characterized by the highest biodiversity indexes than control. Further, differential proteomics studies conducted on S10107 exhibit expression of proteins related to nitrogen fixation and stress response under N2-deficient conditions. Protein-protein interaction (PPI) showed interaction of major proteins as inevitable multifunctional nodes, maintaining the integrity of PPI’s network as their deletion could collapse the complete network into small clusters.
Open Access
Rhizospheric bacterial diversity in different Dalbergia sissoo Roxb. provenances
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Joshi, Samiksha; Sahgal, Manvika
Dalbergia sissoo Roxb., common name shisham is a leguminous tree known internationally for its priced timber. Therefore, there is an urgent need to understand the cause and ways to control large scale mortality of mature trees in natural forests. Soil microbes play an important role in tree growth and health. Hence, a comparative study of bacterial communities in shisham rhizosphere at three provenances Lachhiwala, Tanakpur and Pantnagar was carried out through illumina-MiSeq sequencing. The bacterial community composition and diversity based on V3-V4 16S rDNA region was variable and distinct for each provenance. The bacterial diversity index values indicated by species richness (Chao metric), species richness and evenness (Shannon metric) and count of unique OTUs (observed species metric) were significantly higher at Pantnagar than at Lachhiwala and Tanakpur with highest coverage for Lachhiwala (88.86%) and least for Tanakpur (67.19%). diversity analysis depicted closer relationship between rhizospheric bacterial diversity at Tanakpur and Lachhiwala (both healthy forests) than at Pantnagar (forest with mortality). Phylum Proteobacteria was abundant at Lachhiwala and Tanakpur whereas Acidobacteria predominated at Pantnagar. Amongst genera, Pseudomonas, Flavobacterium, Bacillus, Paenibacillus, Sphingomonas, Nitospirae and Massilia were abundant at Lacchiwala and Tanakpur. However, Williamsia, Blastocatella, Methylobacterium, and Brevibacterium at Pantnagar. Metagenomic bacterial diversity was linked to several soil biotic (population and enzyme activities) and abiotic properties (pH, organic carbon, EC and available N, P, K and micronutrients). Highest bacterial count was recorded at Tanakpur followed by Lachhiwala and Pantnagar. Soil enzyme activities such as acid and alkaline phosphatases and urease were highest at Lachhiwala. In contrast FDA and Dehydrogenase were highest at Tanakpur. A strong positive correlation was observed between all enzyme activities and soil nutrients (macro and micro). Since phosphatase activities and available phosphate in soil was higher so phosphate solubilizing bacteria (PSB) were recovered and characterized. Inorganic phosphorous solubilizing ability of bacteria ranged between 285.78 μg ml-1 to 891.38 μg ml-1 These PSB’s exhibited multiple plant growth promoting traits also. In all 18 PSBs were distinguished into seven ARDRA groups. These were identified as Pseudomonas sp., Klebsiella sp., Streptomyces sp., Pantoea sp., Kitasatospora sp., Micrococcus sp. and Staphylococcus sp. through 16SrDNA sequence analysis. Out of eighteen, sixteen bacterial isolates were positive for 82 bp pqq C gene whereas six for 72 bp pqq A gene.
Open Access
Optimization of thermo-alkalotolerant cellulase enzyme production from Aspergillus terreus PPCF for conversion of delignified bamboo wood biomass into fermentable sugars
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-01) Heena Parveen; Tewari, Lakshmi
Renewable biofuels are currently considered as more sustainable, cost effective and eco-friendly alternative to fossil fuels. Present study explores the possibility of utilizing bamboo biomass as sustainable feedstock for alcohol industry. Efforts were also made to identify potential cellulase producing native microbial cultures that play an important role in the hydrolysis of lignocellulosic biomass. Seventy two bacterial and fungal cultures were isolated from diverse sources, screened for cellulase enzyme activities both qualitatively and quantitatively and potential fungal isolate PPCF exhibiting maximum β-glucosidase (0.517UmL-1) and FPase (0.166 UmL-1) activity was selected for the bioconversion process. The cellulase enzyme production from PPCF was optimized using OVAT and statistical approach which enhanced the FPase activity to 10.78 U mL-1 and β-glucosidase to 6.62 U mL-1 . The dry powdery biomass of Bambusa balcooa was first subjected to chemical and biological pre-treatment to remove the lignin content for enhancing saccharification efficiency. The chemical pretreatment followed by biological treatment with lignin degrading fungus (Lenzites elegans WDP2) was optimized and alkaline-oxidative pre-treatment followed by treatment with Lenzites elegans decreased the lignin content from 28% to 8%. The pre-treated substrate was than hydrolysed into fermentable sugars by utilizing cellulase enzyme from the fungal isolate PPCF. Further statistical optimization of bamboo biomass was done of delignified biomass. Maximum saccharification (56.32%) was observed with enzyme loading of 20.09 FPU g-1 of substrate at 55oC in 24 h. Liquid chromatography mass spectrometry of saccharified fluid of the treated samples revealed the presence of glucose along with other compounds. The pretreated saccharified biomass was monitored through FTIR and SEM analysis to reveal the chemical changes and occurrence of porosity in the biomass. Based on phenotypic and 5.8 S ITS rRNA gene sequencing, the isolate PPCF was identified as Aspergillus terreus PPCF.
Open Access
Application of conventional and metagenomic approaches to analyse the effect of nanocompounds and indigenous bioinoculants on the health of soil and Zea mays
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-01) Parul; Sharma, Anita
Present study puts forward the application of nanocompounds (nanogypsum, nanozeolite, nanochitosan and nanophos) in agriculture practices for the sustenance of agriculture system under pot and field conditions. Impact of nanocompounds was studied on soil and maize health using microbiological, biochemical and metagenomic approaches. Out of 20 bacterial isolates recovered from nanogypsum treated soil, 2 best plant growth promotory rhizobacteria (PC1 and PC4) were selected which showed best growth in the presence of nanogypsum and identified as Pseudomonas taiwanensis and Pantoea agglomerans with assession number MK106029 and MK106024 respectively. Treatment of nanogypsum with PC1 and PC4 showed 97.22% and 98.61% germination compared to control which had only 69% and 70.27% germination under sterilized and unsterilized condition in the pot experiment. Percent seed germination was also significantly higher in the combined treatments of nanocompounds, PGPRs under field condition. In the pot trial conducted in sterilized and unsterilized soil on maize, application of nanogypsum and bioinoculants enhanced the plant health parameters by 1.5 fold. A significant increase (>2 fold) in leaf area, chlorophyll, carotenoid, total sugar, total phenolic content and antioxidant enzymes was observed in the seedlings treated with different combinations of nanocompounds and bioinoculants under pot and field trials. GC-MS analysis of 60 days old maize leaves revealed a clear cut difference in the concentration of phenolics, acid esters and sugars in treated plants than the control. Combined treatment of nanocompounds and PS2 and PS10 enhanced maize yield significantly in field condition. Improved in soil health was observed. Physicochemical parameters like oxidizable organic carbon, available phosphorous, potassium, ammonical nitrogen and nitrate nitrogen were higher in treated soil as compared to control. Application of nanocompounds enhanced total and NPK dissolving bacterial population in the experimental soil. Soil health indicator enzymes showed higher activities in all treated soil. Two to three fold increase in the activity of FDA hydrolysis, dehydrogenase, alkaline phosphatase, β-glucosidase, amylase and arylesterase was observed in nanocompound and PGPRs treated soil than the control. Copy number of bacterial population enhanced in treated soil as compared to control. T-RFLP analysis showed that nanocompounds treated soil had more number of bacteria as compared to control. Metagenomics study of soil under the treatment of nanogypsum with PC1, nanozeolite and nanochitosan with PS10 showed overall increase in bacterial population compared to control in pot and field trial. Rarefaction curve exhibited a steeper slope for nanogypsum along with PC1, nanozeolite and nanochitosan along with Bacillus sp. treated soil than the control and demonstrated greater genetic diversity. Proteomic study of bacterial isolates showed that the nanocompounds used in the study were not toxic for the bacterial isolates hence can be used as bioinoculants in the presence of nanocompounds.
Open Access
Documentation of the bacterial and diazotrophic diversity from Garhwal Himalaya through culturable and unculturable approaches
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-07) Saurabh Kumar; Goel, Reeta
To study the bacterial diversity and soil physiochemical properties of Garhwal Himalaya, total five soil samples were collected from different locations of Garhwal Himalaya namely Harsil, Gangotri, Uttarkashi, Dhanaulti and Kandakhal. Based on the primary screening through quantification of 16SrDNA and nifH genes and 16SrDNA DGGE analysis, Gangotri and Dhanaulti were selected for further study. Comparative soil physiochemical characterization of the both soil samples revealed that the Kandakhal soil was neutral in pH, poor in soil organic matter (SOM) and total Kjeldal nitrogen (TKN) and rich in micronutrient. However, Gangotri soil was found alkaline, rich in TKN and SOM and poor in mineral nutrients. Thus, it was found that the soil physicochemical properties vary significantly with the altitude. Metagenomics of Kandakhal soil revealed that the Proteobacteria (31%) was the major phylum followed by Bacteroidetes (20%), Fermicutes (9%), Acidobacteria (7%), Actinobacteria (6%) and Chloroflexi (5%). However, in phylum Proteobacteria, Alphaproteobacteria (8%), Betaproteobacteria (8%), Gammaproteobacteria (8%) and Deltaproteobacteria (7%) were uniformly distributed. On the other hand in Gangotri, total 31 bacterial phyla were observed where, Proteobacteria (38.49 %), Acidobacteria (17.88 %), Actinobacteria (14.48 %), Bacteroidetes (7.89 %), Gemmatimonadetes (7.87 %) and Chloroflexi (5.94 %) were dominant bacterial phyla. However, in phylum Proteobacteri, Alphaproteobacteria (16.88%) was the most abundant bacterial class followed by Betaproteobacteria (9.44%). Presence of large numbers of unidentified OTU in Gangotri soil suggests that it has unique bacteria diversity. Further, an indigenous diffusion chamber was designed and incubated in-situ for cultivation of readily not cultivable bacterial from Gangotri soil which revealed the potential of this device for isolation of bacteria which were previously difficult to isolate from conventional methods. Moreover, six psychrophilic diazotrophic bacteria were isolated form Gangotri soil and identified as Pseudomonas helmanticensis, Arthrobacter humicola, Brevibacillus invocatus and Pseudomonas mandelii etc. However, no psychrophilic diazotrophic were isolated from the Kandakhal soil. Further investigation was carried out to study the cold adaptation in Pseudomonas helmanticensis at molecular level. This was the first time when proteomics of psychrophilic diazotrophs Pseudomonas helmanticensis was reported under the cold stress. Differential proteomics of P. helmanticensis under cold stress through LC-MS analysis revealed that majority of upregulated and unique proteins expressed under cold stress were functionally involved in RNA metabolism, translation and energy production. Moreover, major upregulated proteins were molecular chaperones which assist in the proper protein folding and RNA stability at low temperature. Therefore, both culture dependent and independent studies revealed that the Kandakhal and Gangotri soil harbor rich bacterial diversity. However, diversified culturable psychrophilic nitrogen fixers were only present in the Gangotri soil. Thus, Garhwal region of the western Indian Himalaya is rich in cold adapted bacterial diversity which could be explored for basic understanding of cold adaptation besides its applied significance.
Open Access
Impact assessment of pesticides on soil health through conventional and metagenomic approaches and their biodegradation using indigenous microbes
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Gangola, Saurabh; Sharma, Anita
Application of agro-pesticide technologies, hybrid seeds and systematic irrigation in agriculture practices has contributed significantly in enhanced food production and achieving green revolution. On the other hand injudicious use of synthetic chemicals has developed environmental pollution which is posing serious health problem in human beings. In this study attempt has been made to reduce pesticide pollution with the help of isolated indigenous bacteria. To isolate pesticide degrading bacteria, pesticide contaminated soil samples were collected from the agricultural fields of Gularbhoj, Bhatbhoj and Dineshpur, Uttarakhand. Out of 12 bacterial isolates recovered from the contaminated soil, only four isolates (1B, 1D, 2D and 3C) were selected for bidegradation studies. Bacterial isolates tolerated up to 450 ppm pesticide(s) and utilized cypermethrin, imidacloprid, fipronil and sulfosulfuron as C source in minimal medium and showed maximum degradation of cypermethrin (95%). Consortium of four bacterial isolates degraded cypermethrin (99%), imidacloprid (99%), fipronil(95%) and sulfosulfuron(93%) in 15 days as compared to control (7%). Molecular characterization of 1B, 1D, 2D and 3C showed 99-100% homology with Bacillus brevis (MG948470), Bacillus subtillis (MG948470), Bacillus cereus (MH341691) and Bacillus sp. (MH341692) respectively. Individual bacterial isolate(s) and their consortium showed maximum degradation of imidacloprid (99%) after 15 days as compared to control (7%) in soil slurry. Common metabolites of cypermethrin biodegradation by 1D were Phenol, 3-(2,2-dichloroethenyl) 2,2-dimethyl cyclopropane carboxylate, chloroacetic acid, 1-decanol, cyclododecylamine, cyclopentane, palmitoleic acid, decanoic acid and undecyl ester. Based on GC-MS analysis of biodegraded products of cypermethrin by 1D, a new pathway is proposed. Under stress conditions production of enzymes under stress was higher than control. Km values of the enzymes were low in stress. Amplification of esterase (EST) gene from 1B, 1D, ALDH gene from 1B, 3C and laccase gene from 1D, 2D and 3C was observed. Proteomic analysis of ID and 2D isolates showed increase in stressed response proteins and catabolic/pesticide degrading proteins as compared to control. Metagenomic study of 2 soil samples by DGGE revealed that number of bands were high in unspiked but contaminated soil as compared to pesticide spiked contaminated soil. Maximum copy number of 16S rDNA was observed in Dineshpur soil (pesticide contaminated soil) followed by Gularbhoj (control and pesticide contaminated soil) and Dineshpur (Control) in qRTPCR study. Metagenomic analysis of the pesticide contaminated (Dineshpur 1D, Gularbhoj 2G) and uncontaminated soil samples (Dineshpur1DC and Gularbhoj 2GC) revealed that total reads for 1D (893,487) were high as compared to 1DC (99,265). Total reads for 2G (562,416) were low as compared to 2GC (873,083) soil sample. Results of metagenomic study showed the presence of Proteobacteria (Kaistobacter, Serratia,), Firmicutes (Clostridium, Bacillus), and Actinobacteria (Nocardioides,Arthrobacter) Dineshpur (ID). Gularbhoj (2G) soil showed the presence of Firmicutes (Clostridium), Actinobacteria (Nocardioides) and Chloroflexi (Bellilinea, Anaerolinea, Caldilinea). Repeated application of the pesticides in the agriculture fields forces to adopt new microbial population with simultaneous replacement of the older one. Present study recommends the application of indigenous microorganisms in biodegradation of common agriculture pesticides. Study on metabolomics and proteomics gives significant insight of three major genes responsible for biodegradation of pesticides.
Open Access
Biodegradation studies of electronic waste using indigenously developed microbial consortia
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Debbarma, Prasenjit; Goel, Reeta
The management of electronic waste (e-waste) becomes a global issue in this digital era. Existing conventional practices are harmful for dealing with e-waste, therefore, indigenous soil bacteria were explored for e-waste treatment through enrichment culture approach followed by screening, identification and their bioformulation used for in situ investigation. Soil bacteria were enriched in the presence of e-waste after 30 days of incubation under standard laboratory conditions. This finding was established through optical density and _-max values that were higher in the case of soil enrichment than the control. Later, the identification and characterization of isolates was done by polyphasic approach. The optimum tolerance level (OTL) concentration of solubilized e-waste was found to be 3 mg mL-1 which was determined through UV-vis spectrophotometer. The selection and biocompatibility testing of potential isolates were performed for the formation of bacterial consortia. Two potential bacterial consortia were developed, viz. consortium C-1 comprises Achromobacter insolitus strain PE2 (MF943156), Acinetobacter nosocomialis strain PE5 (MF943157), Pseudomonas resinovorans strain PE8 (MF943158), Pseudomonas aeruginosa strain PE10 (MF943159) and Stenotrophomonas pavanii strain PE15 (MF943160) and consortium C-2 which is composed of all above four bacterial strains except strain PE10. The consortium C-3 consist of Microbacterium sp. strain MK3 (DQ318884), Bacterium Te68R strain PN12 (DQ423487) and Pseudomonas putida strain MK4 (DQ318885) was also used which is previously defined for its ability to degrade variety of polymers. Talc based bioformulation was prepared by mixing the talc in the sterilized dish with an active consortium. E-waste granules were incubated with freshly prepared bioformulation in soil pits for 9 months. The biodegradation of e-waste by the selected strains during enrichment and in situ experiment was confirmed by FT-IR, TGDTG-DTA, SEM, EDX and elemental analysis, respectively. The in situ trial of e-waste biodegradation using formulated consortia has shown the significant degradation by C-1 and C-3 followed by C-2. Bacterial community analysis revealed that the used strains were persisting in the experimental pits throughout the trial period. Thus, this study besides providing direct and standardized protocol for screening and selection of efficient e-waste utilizing bacteria is also demonstrating potential consortia which are ready to be used.
Open Access
Impact assessment of nanozeolite and nanochitosan on plant-soil-microbe interaction using conventional and molecular methods
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-08) Khati, Priyanka; Sharma, Anita

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