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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
Development of Acetobacter sp. consortia for acetous fermentation of banana (Musa paradisiaca) fruit waste
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-08) Joshi, Nisha; Tewari, Lakshmi
The present study was carried out for identification and characterization of potential ethanol and acetic acid producing bacteria. Native bacterial and yeast cultures were isolated from diverse fruit sources. Based on qualitative and quantitative screening for ethanol/ acetic acid production four ethanogenic and acetic acid bacteria were selected. All the cultures were also evaluated for their osmo-,thermo-, and ethano-tolerance limits. Finally yeast isolate YBP1 and S. cerevisae were selected for alcoholic fermentation and three potential acetic acid strains were selected for acetous fermentation. Then, these potential microbial strains in mono/co-cultures were screened for their alcohol and acetic acid producing efficacies using banana cellulosic and non-cellulosic waste. Bioconversion of banana fruit pulp and peel employing potential strains into bio-ethanol was carried out in batch cultures through SHF and SSF process and subsequent conversion of bio-ethanol into bio-acetic acid was carried out through acetous fermentation in batch cultures. Influence of fortification of substrate with carbon, nitrogen and phosphorous source on ethanol/ acetic acid production through both the fermentation was also studied and was found to significantly enhance ethanol and acetic acid production from banana fruit waste. Alcoholic fermentation of banana pulp using yeast YBP1 was found superior over S. cerevisae. Higher ethanol/ acetic acid production from cellulosic banana fruit waste (peel) was recorded during SHF fermentation process. Maximum acetic acid yield (9.63 g/100g) could be recorded with banana pulp using YBP1+ AAB-Consortium-II. The acid producing fermentation medium was identified through TLC technique.
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
Biocontrol potential of indigenous fluorescent pseudomonads against banded leaf and sheath blight of maize incited by Rhizoctonia solani f.sp. sasakii
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-07) Brindhaa, N.T.; Sahgal, Manvika
Maize, the third most important cereal crop in the world is exposed to various abiotic and biotic stresses resulting in huge yield loss. The yield loss associated with fungal pathogens is estimated to be more than 10%. Of which, banded leaf and sheath blight (BL&SB) caused by Rhizoctonia solani f.sp.sasakii alone leads to 11-40 % yield loss. The aim of the study was to evaluate biocontrol potential of 22 indigenous (AS1-AS22) fluorescent pseudomonads (FLPs) (18 from Almora & 4 from Pithoragarh districts of Uttarakhand) against BL&SB. All the bacterial isolates were screened for in vitro antagonism against R.solani f.sp.sasakii coupled with hydrolytic enzyme production and plant growth promoting (PGP) traits. Out of these, two strains AS19 and AS21 showed the highest percent mycelial inhibition. The % mycelial inhibition with whole cell culture of AS19 and AS21 was 57.04 % & 54.07 respectively, whereas with cell free supernatant it was 67.41% & 65.74% respectively. Further, scanning electron micrographs revealed that hyphal cell wall at interaction zone of fungal mycelium with AS19 and AS21 was damaged. Both the FLP strains also inhibited the formation and germination of sclerotia in R.solani f.sp.sasakii and exhibited PGP traits like production of rhamnolipid, HCN, siderophore, IAA, ammonia and zinc and phosphate solubilization. They produced protease, chitinase, catalase, oxidase, gelatinase and H2S. Bacterial strains AS19 and AS21 were identified as Pseudomonas spp. through 16S rDNA sequence analysis with % similarity of 89.27 % and 86.62% respectively. The biocontrol efficacy of strains was evaluated in vivo through pot trial. The strains AS19 and AS21 significantly reduced the BL&SB disease incidence (DI). The reduction in DI ranged between 25-38.33%. Combined results of detached leaf assay and pot trial revealed that induced systemic resistance (ISR) was the mechanism of disease control. Foliar spray of two antagonistic isolates 48 hr before pathogen inoculation induced ISR and reduced the DI by half. The two strains AS19 and AS21 exhibited broad spectrum in vitro antifungal activity against Alternaria triticina, Bipolaris sorokiniana, Rhizoctonia maydis and Fusarium oxysporum f.sp.lentis. Future studies should focus on identification of the bacterial metabolite responsible for induced systemic resistance.
Open Access
Combined effect of nanochitosan and potential bioinoculant on maize plant and its rhizosphere
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-07) Agri, Upasana; Sharma, Anita
Present study focuses on the application of agriusable nanochitosan along with potential plant growth promoting rhizobacteria on maize plants under pot experiment. PC1 and PC4 were selected on the basis of their plant growth promoting properties and identified as Pseudomonas taiwanensis and Pantoea agglomerans with accession number MK106029 and MK106024 respectively. They were found compatible with each other and along with nanochitosan (40ppm) which was further supported by SEM results showing no inhibitory effect of Nch on bacterial cultures. Percent seed germination was significantly higher upto 98% in the combined treatment of nanochitosan and PGPR under pot experiment. Application of Nch and bioinoculants enhanced plant growth parameters. A significant increase (>1.5 fold) in carotenoid, total sugar, total phenol was observed in seedlings treated with nanochitosan and bacterial cultures after 30 days in comparison to control. Total catalase and peroxidase activity was found maximum in PC1+PC4+Nch treatment which accounted for 16.25 and 43.67 μmol/min/mg protein after 30 days of pot trial. Enhancement in total fluorescein diacetate and alkaline phosphatases enzyme activity was observed in PC1+PC4+Nch treated soil with (>1.6 fold increase) in comparison to control. Low level of dehydrogenase activity was found in Nch treated soils. physiochemical properties like oxidizable organic carbon, available phosphorus, and potassium and ammonical nitrogen were higher in Nch treated soil as compared to control. Improvement in soil health was observed in all treatments after 30 days in comparison to control. 1.5- 2 fold increase in total microbial population was observed in soil treated with Nanochitosan and bacterial cultures. Population based metagenomics analysis revealed no negative impact of nanochitosan on microbial population in total. It can be concluded from our findings that application of nanocompound along with bacterial treatments improved plantbacterial interaction, promoted plant parameters, increased microbial diversity and increased the level of soil health indicator enzymes.
Open Access
Effect of nanosilicon dioxide on maize and its rhizosphere under the influence of indigenous bioinoculant
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-07) Bharti; Sharma, Anita
Nanotechnology is a science that has wide application in almost all the developing fields including chemical, manufacturing, medical and agriculture sector. Nanoparticles are atomic or molecular aggregates of 1 to 100 nm in size. Two bacterial isolates (PC1 and PC4) were selected on the basis of their plant growth promoting properties and used as bioinoculant along with/without nanosilicon dioxide in a pot experiment on maize. Based on molecular analysis PC1 and PC4 showed homology with Pseudomonas taiwanensis and Pantoea agglomerans respectively and were allotted with accession number MK106029 and MK106024 respectively. Amplification of acdS (ACC deaminase) gene was observed in PC1 and PC4 isolates with amplicon size of 500bp and 600bp respectively. Scanning electron microscopy and biocompatibility assay on nutrient agar plate revealed the compatibility of both the isolates with each other. PC1 and PC4 produced 44% and 25% siderophore unit, solubilised 3.49mg/l and 3.63mg/l zinc and 382.2μg/ml and 361.9μg/ml phosphate respectively while consortium of the two (PC1+PC4) produced 31% siderophore unit, solubilised 3.70 mg/l zinc and 478.6μg/ml phosphate. Combined application of bacterial consortium (PC1+PC4) and nSiO2 showed enhancement in agronomic parameters of maize plants. Average plant height and number of leaves were increased by 1.38 and 1.28 folds over control respectively after 30 days. Values of total chlorophyll (3.32mg/g), carotenoid (0.326mg/g), total sugar (50.47mg/g) and total soluble protein (18.42 mg/g) in maize plant were also high in PC1+PC4+nSiO2 treatment. Total phenol and flavonoid content was 2.75 mg/g and 0.544mg/g respectively on 30th day when treated with PC1+PC4+nSiO2. Level of antioxidant enzymes (catalase and peroxidise) were found to be maximum in treatment (PC1+PC4+nSiO2) which accounts for 15.09μmol/min/mg protein and 60.92μmol/min/mg protein respectively on 30th day. Total bacterial count and population of K and P solublizers was maximum in of the soil treated with PC1+PC4+ nSiO2. Enhancement of 70.6% in total bacterial count in PC1+PC4+ nSiO2 treatment over control was reported. An increase of 2.14, 1.57 and 1.43 times over control in the activity of FDA, dehydrogenase and alkaline phosphatase respectively was observed in PC1+PC4+nSiO2 treatment. Population based metagenomics analysis revealed no negative impact of nanosilicon dioxide on microbial population in total. Thus, our study with PGPR and nanosilicon dioxide treated maize plants revealed that inoculation of beneficial microorganism in combination with nanocompounds is an effective method for enhancing the growth of maize and maintaining the soil health.
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
Effect of plant growth promoting rhizobacterial consortia with nanoparticle and plasma treated seeds under abiotic stress conditions in wheat
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-06) Kaviya, N.; Singh, Ajay Veer
The plants existing in natural environmental conditions are being continuously exposed to different biotic and abiotic stresses that results in reduced plant growth and development. In this aspects, the present study was accomplished with the implementation of drought tolerant plant growth promoting rhizobacteria (PGPR) along with two different nanoparticles namely nano zinc-oxide and nanochitosan under drought stress conditions and with plasma treated seeds under heat stress conditions. For this study six drought tolerant bacterial isolates were retrieved from departmental culture collection. Those cultures were tested for their drought tolerant ability and further analyzed for several PGP traits (siderophore production, zinc solubilization, phosphate solubilization, IAA production, etc.) and based on the compatibility test the cultures were choosen to make consortia. This developed consortium was also tested for different PGP traits. Furthermore, two microbial consortia was choosen based on PGP traits and used for seed treatment of wheat (Triticum aestivum L.) along with nano zinc-oxide and nano chitosan treated seeds to be evaluated under drought stress conditions and with plasma treated seeds to be evaluated under heat stress conditions in field conditions. The results of this experiment showed that the treatment of Nano zinc-oxide, Nano chitosan and Microbial Consortia 1 (Zn+C+MC1) from drought stress conditions and the treatment of Plasma and Microbial Consortia 1 (P+MC1) from heat stress conditions showed significant improvement in several physiological and biochemical characters of leaf like chlorophyll and carotenoid content, antioxidant enzymes like catalase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD), protein content, proline content, malondialdehyde content and relative water content when compared with their respective control. The agronomical and yield parameters as well as the zinc and iron micronutrient content in soil and grain; Bacterial dynamics in soil was also significantly higher in Nano zinc-oxide, Nano chitosan and Microbial Consortia 1 (Zn+C+MC1) treatment under drought stress conditions and Plasma and Microbial Consortia 1 (P+MC1) treatment under heat stress conditions. The shelf life of consortia was also evaluated and the results demonstrated that the shelf life life of Microbial Consortia (MC1) at 4°C was significantly better compared to Microbial Consortia 2 (MC2) and storage at room temperature. Therefore, the application of Microbial Consortia 1 (MC1) along with nanoparticles and plasma could be a novel strategy to improve crop production under abiotic stress conditions to maintain the agricultural sustainability.

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