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Open Access
Biodegradation of cypermethrin and profenofos using indigenous bacteria and their subsequent influence on microbial dynamics of rhizospheric soil of wheat and maize
(G.B. Pant University of Agriculture and Technology, Pantnagar, District Udham Singh Nagar, Uttarakhand. PIN - 263145, 2022-07) Singh, Harshita; Sharma, Anita
Cypermethrin and Profenofos degrading bacterial isolates were recovered from the pesticide contaminated site of agricultural fields of U.S. Nagar, Uttarakhand. Recovered bacterial isolates were enriched to grow in Minimal Salt Medium in the presence of Cypermethrin and profenofos (20 to 100 ppm). Two bacterial isolates (HS2A and HS2B) were selected for the study on the basis of their ability to degrade and utilize Cypermethrin and Profenofos as a source of Carbon and phosphorus. Both the isolates tolerated 100ppm of Cypermethrin and Profenofos. Bacterial isolates were characterized on the basis of morphological, biochemical and molecular tests. HS2A was characterized as Bacillus amyloliquifaciens and HS2B with Staphylococcus gallinarum showed more than 95% s e q u e n c e homology. HS2A and HS2B biodegraded Cypermethrin and Profenofos (100ppm) in Minimal Salt Medium. After 36 h, the pattern of Cypermethrin and Profenofos degradation was 88.9% > 76.31% and 84.62% > 79.03% by HS2A and HS2B respectively. Degradation of both the pesticides in soil slurry was 61.54% > 61.21% and 54.38%, 67.01% > 66.80% and 63.86% by mixture of HS2A+HS2B and HS2A, HS2B respectively after 15 days. HS2A also decolorized toluidine blue in MSM. Pot experiment conducted on maize and wheat using unsterilized soil spiked with Cypermethrin (0.806 ml/100ml water) and Profenofos (0.714 ml/100ml water) showed best performance in consortium treatment. GC-MS analysis of intermediates of biodegraded Cypermethrin and Profenofos with bacterial isolates did not show the presence of toxic metabolites after 36 hours in Minimal Salt Medium. Bacterial isolates degraded Cypermethrin and Profenofos into Benzene, 1,3-bis(1,1-dimethylethyl)-, Dodecane, 4,6-dimethyl-, Dodecane, n-Hexadecanoic acid, Eicosane, 2,4-dimethyl- and 13-Hexyloxacyclotridec-10-en-2-one Hexane, 3,3,4- trimethyl. Soil health and microbial diversity were better in treated soil than control.
Open Access
Exploration of zinc solubilizing bacteria and their prolific contribution in zinc biofortification of rice grains
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-12) Upadhayay, Viabhav Kumar; Singh, Ajay Veer
The current study was an endeavor to characterize zinc-solubilizing bacteria, bearing massive plant growth-promoting traits, from the rhizosphere of millets (barnyard millet and finger millet). In addition, the study was conducted to evaluate the effect of potential bacterial strains and their consortia on growth and the biofortification benefits by assessing improved level of zinc assimilation in rice under pot and field conditions. A total of 130 bacteria were isolated and tested for zinc solubility characteristics, of which only 10 bacterial isolates were selected based on their zinc solubilization potential. Finally, 3 bacterial isolates (BMAR64, FMAR105 and BMRR126) were selected based on the quantitative pattern of zinc solubility potential and their compatibility with one another, and were used for the development of bacterial consortia. These bacteria solubilized Zn profoundly determined quantitatively in liquid broth by AAS and SEM-EDX. These zinc-solubilizing bacterial strains were characterized on the basis of morphological, biochemical tests and 16S rDNA sequencing. These isolates BMAR64, FMAR105 and BMRR126 were recognized as Pantoea rodasii, Serratia marcescens and Burkholderia cepacia, respectively, and they were also found to have genes for organic acid production (gcd and gdh). All three bacterial isolates and their consortia were evaluated for their effect on rice plants (variety- Pusa Basmati -1) in a pot trial in which the treatment with bacteria and their consortia with ZnO supplementation established narrative of Zn-biofortification and improvement in the growth of rice plant. The zinc solubilizing bacterial consortia (Consortium 1 and Consortium 2) with ZnO supplementation exhibited the maximum Zn content in grains of rice (variety- Pusa Basmati-1) in both the Terai and Katchar region of Northern part of India. The treatments containing single bacterial inoculants (BMRR126) with 60% RDF and Consortium (BMAR64+FMAR105+BMRR126) with 100% RDF with ZnO amendment exhibited promising level of Zn content in grain of rice plant (variety- Pant Dhan 18) grown in Terai region. All treatments containing zinc-solubilizing bacterial strains increased rice crop productivity in terms of several aspects such as plant height, dry matter accumulation, number of tillers and effective tiller, grain yield, straw yield, biological yield, panicle length, number of grains per ear, and thousand grain weight. The soil parameters (pH, EC, organic carbon, NPK, available Zn, and soil dehydrogenase activity) also positively influenced under all bacterial treatments compared to uninoculated control. The present study gives an impression of the use of zinc solubilizing bacteria and their consortia as effective bioinoculants (supplementation with economic feasible source of zinc, i.e. ZnO) to harness zinc biofortification of rice crops in more ecofriendly way for sustainable agriculture. Our study clearly highlights the need for zinc-solubilizing bacteria to achieve the benefits of zinc biofortification in different regions.
Open Access
Functional and molecular characterization of rhizobacterial antagonists against foliar blight pathogens of wheat, Bipolaris sorokiniana and Alternaria triticina
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-02) Jaggi, Vandana; Sahgal, Manvika
Foliar blight pathogens affect the wheat production and growth worldwide. The application of chemical fertilizers to control the disease has harmful effects to human health and environment. Fortunately, rhizobacteria can be used as an alternative strategy for the sustainable disease management. In present study, a total of forty five rhizobacteria isolated from rhizosphere soil of wheat cultivated in five agroclimatic regions across four India states were tested for in vitro antagonistic activity against two foliar blight pathogens; Bipolaris sorokiniana and Alternaria triticina causing wheat spot blotch and leaf blight respectively. In addition, all 45 rhizobacteria were tested for production of hydrolytic enzymes (chitinase, protease, cellulase, amylase, and lipase) and PGP traits (solubilization of phosphate & zinc, production of ammonia and siderophore). Six isolates scoring (Ʃ=>12) on bonitur scale were selected for further studies. The isolates P10, UP11, MH13, MP17, MH12, and MP14 were subsequently identified as Bacillus methylotrophicus (MN099430.1), Bacillus subtilis (MN099431.1), Bacillus sp. (MN099432.1), Streptomyces sp. (MN099435.1), Lysinibacillus sp. (MN099433.1), and Staphylococcus epidermidis (MN099434.1) respectively. In vivo pot trial revealed that seed bacterization plus booster dose of bacterial treatments resulted in lowest disease incidence (DI %) than foliar spray 24 hrs after pathogen infection against both the foliar blight pathogens. Reduction in spot blotch incidence in wheat plants treated with the isolates P10 (T2) UP11 (T3) was significant with48.66% and 50.33 % DI respectively. Likewise, percrent reduction in the incidence of leaf blight by P10 and UP11 was 49.26% and 50.33% respectively. The pot experiments also revealed that P10 and UP11 considerably increased plant biometric parameters. Therefore, B. methylotrophicus strain P10 and B. subtilis strain UP11 were equally effective to control both the foliar blight pathogens individually as well as in a complex. Antifungal metabolites produced by P10 and UP11 were analyzed in ethyl acetate extract by GC-MS and 29 and 30 compounds were identified respectively. A compound, isobutylhexahydropyrrolo [1, 2-a] pyrazine-1, 4-dione was dominant and abundant in both the strains. In silico studies revealed a strong interaction (ΔG-6.76) between the dominant compound and, ceratoplatinin, a phytotoxic protein of fungus B. sorokiniana. Hence, we suggest that strain P10 and UP11 has the potential to be used in sustainable agriculture.
Open Access
Bioconversion of photooxidatively delignified woody biomass of Salix alba into bioethanol using C6/C5 co- sugar fermenting yeast Pichia kudriavzevii BGY1
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-03) Bisht, Neha; Tewari, Lakshmi
Presently bioethanol is considered as the most sustainable and eco- friendly fuel in contrast to non- renewable fuels. The present study was carried out for identification and characterization of potential ethanogenic yeast. Native yeast cultures were isolated from diverse fruit sources. Based on qualitative and quantitative screening for ethanol production two ethanogenic yeast were selected and subjected to UV and gamma mutagenesis with aim to enhance stress tolerance limit and sugar fermenting ability. Based on phenotypic and 5.8 S ITS rRNA gene sequencing, the isolate BGY1 and its mutant BGY1- γ m were identified as Pichia kudriavzevii BGY1 and Pichia kudriavzevii BGY1- γ m. The woody biomass of Salix alba was first subjected to chemical, biological, chemi- biological and photonanocatalyst mediated pretreatments for removing lignin and improving saccharification efficiency. Combined photonanocatalyst induced and cellulase enzyme aided alkaline oxidative pretreatment decreased the lignin content from 18.7 to 4.0 %. The pretreated substrate was then saccharified into fermentable sugars using cellulase enzyme from the fungal isolate (Aspergillus terrus PPCF). Optimization of saccharification resulted in 57.2 % of saccharification efficiency in 24 h. Then, the potential microbial strains in mono/co-cultures were screened for their alcohol producing efficacies using saccharified Salix alba wood biomass. Bio-conversion of saccharified wood employing potential strains into bio-ethanol was carried out in batch cultures through SHF and SSF process. Finally the co- culture of P. Kudriavzevii BGY1 and P. Kudriavzevii BGY1-γ m were selected for alcoholic fermentation. Alcoholic fermentation of saccharified Salix alba wood powder using co- culture of yeast P. kudriavzevii BGY1 and P. kudriavzevii BGY1- γ m at 32º C under SSF batch fermentation process was found superior over batch fermentation under SHF process. Under optimized conditions maximum ethanol yield 13.0 % was achieved with saccharified Salix alba wood.
Open Access
Identification and characterization of siderophore producing bacteria from Dalbergia sissoo Roxb. forest ecosystem
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2020-02) Srivastava, Pragati; Sahgal, Manvika
Dalbergia sissoo Roxb. (family Fabaceae) is a large deciduous multipurpose timber tree. This high value cash tree is getting depleted due to dieback and wilt induced mortality. Hence, there is an urgent need to find ways for solving this problem. Soil microbes play a major role in plant health. Hence a comparative study of bacterial communities in the shisham forest (from rhisophere and bulk soil) at AFRC, GBPUAT, Pantnagar was done in cold dry (Oct- Dec) and monsoon wet (Jan-March) season through metagenomic approach. The variation in bacterial community in rhizosphere and bulk soil was not significant at both phylum and genus level. However there was variation within the seasons. Amongst ten most abundant bacterial phyla eight were common in both seasons. Common reported phyla were Acidobacteria, Actinobacteria, Bacteriodetes, Gemmatimonadales, Nitrospirae, Proteobacteria, Planctomyces, and Verrucomicrobia. Two genera Chloroflexi and Firmicutes were reported only in monsoon wet season and Cyanobacteria only in cold dry season. At genus level the variation was more pronounced. During monsoon wet season, the most abundant genera were unclassified iii1_15 within Acidobacteria, unclassified genus WD21010, Kaistobacter and Flavobacterium and genera WD2101;f ,Nitrospira, Kaistobacter, Pseudomonas, Candidatus Solibacter were abundant in cold dry season. The soil was alkaline and iron content was sufficient. The bioavailability of iron is low in alkaline soils. Thus, a total of twenty siderophore positive bacterial isolates were recovered from shisham forests in both the seasons. Of which, on the basis of qualitative and quantitative CAS assays, ten siderophore positive isolates were further identified and characterized. Through 16SrDNA sequencing and analysis, isolates were identified within genus Pseudomonas, Burkholderia and Streptomyces. Single factor optimization experiment revealed that siderophore production in all ten strains was between 100-70% siderophore unit (SU) and was significantly affected by pH, ammonium nitrate (NaNO3), and 50 μM FeCl3.6H2O. Siderophore production in five best siderophore positive strains Burkholderia territorii, Streptomyces lavendulae, Pseudomonas monteilli, P. chlororaphis and P. benzenovorans was optimized using response surface methodology. It was 70-90% SU with each strain having specific requirement for optimum siderophore production. FTIR and LCMS studies identified a derivative of ornibactin and pyochelin as siderophore in Burkholderia territorii strain whereas pseudomonine and salicyclic acid as siderophores in Pseudomonas monteilii strain.
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.

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