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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
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
Evaluation of endophytic bacteria for growth and stress tolerance of Trigonella Foenum-Graecum (Fenugreek) under saline stress conditions
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Naveen, K.; Tiwari, Lakshmi
Salt- affected soils are becoming greatest hindrance to crop production worldwide; the rate at which the arable land is lost every year due to these problems is imposing a greatest challenge to agricultural production. Hence in order to ameliorate the stress imposed by these soils on the crop plants, microbial mediated approach is selected and the present study is conducted to address this issue. Twenty seven indigenous endophytic bacteria were isolated from underutilized medicinal weed plant Phyllanthus sp and screened for in vitro pH (7.0-11.0) stress tolerance. Eleven alkalo-tolerant isolates were selected and further screened for in vitro salt tolerance(using NaCl concentration 0-12%) and plant growth promoting traits. The four superior isolates(PSBeP-8,PSBeP-11) showing higher stress tolerance and PGP properties were selected. The isolate PRBeP-10 was found to be most akalo-tolerant showing growth up to pH 10.0 while the isolate PSBeP-8 most salt tolerant with growth up to 10% NaCl concentration. Under normal conditions (pH 7.0;NaCl 0%) all the isolates showed P- solublization and siderophore production which was adversely affected with increasing salt and pH conditions. The isolate PRBeP-10 showed higher phosphate solublization under pH/salt stress conditions as compared to other cultures showing phosphate concentration of 61.18 μg/mL at pH 10.0 and 38.4 μg/mL at 10% NaCl concentration. Siderophore production at highly alkaline pH(pH 10.0) was shown by the isolate PRBeP-10(46.58% sid units in 72 h while at highly saline condition(10% NaCl) 28.3% sid units were recorded for PSBeP-8 in the same time. Thus the isolates PSBeP-8 and PRBeP-10 were found most halo-alkalo-tolerant retaining their p-solubilizing and siderophore producing abilities under pH and salt stress conditions. Both the isolates were evaluated for their in vitro and in vivo growth enhancing capabilities under normal and saline/alkaline conditions. Germination of fenugreek seeds was increased under salt and pH stress conditions due to inoculation with bacterial endophytes. During pot culture experiment, the mono-cultures of PRBeP-10 and PSBeP-8 and their co- culture inoculation enhanced the leafy biomass yield of fenugreek plants over their corresponding un treated controls grown in saline soil(of pH 9.0).Based on various morphological, biochemical traits and 16S rRNA gene sequence analysis, the isolate PRBeP-10 and PSBeP-8 were identified as Sphingobacterium sp PRBeP-10 and Pseudomonas aeruginosa PSBeP-8.
Open Access
Plant growth promotory effect of PGPR and their consortia on wheat under drought stress conditions
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Lohani, Swati; Singh, Ajay Veer
Plants under natural environmental conditions experience various kinds of abiotic and biotic stresses, results as severe deterioration in plant growth and development. According to this perspective, the present study was accomplished with the implementation of drought tolerant plant growth promoting rhizobacteria (PGPR) in wheat. For this study, total twenty bacterial isolates were retrieved from departmental culture collection. Drought tolerant twelve bacterial isolates were selected on the basis of growth present on tryptic soy broth medium supplemented with 8% poly ethylene glycol. These drought tolerant isolates were further analyzed for plant growth promoting activities (siderophore production, zinc solubilization, phosphate solubilization IAA production etc.) and five potent bacterial isolates were screened and further used for consortia development. These consortia along with single bacterial isolates were analyzed for plant growth promoting traits such as siderophore production, zinc solubilization, phosphate solubilization and ammonia production. Furthermore, an in situ greenhouse pot experiment was designed under drought stress to evaluate the potential of drought tolerant plant growth promoting bacteria along with their consortia isolates on wheat (Triticum aestivum var. PBW373). Results of greenhouse pot experiment confirmed that all bacterial isolates and their consortia were significantly able to improve plant growth and productivity in terms of agronomical parameters such as root length and plant height. Among all treatments, two bacterial isolates i.e. WRPa4 and SRK14 and two consortia CIII and CV proved as more potent for drought tolerance and plant growth. To determine the shelf life of consortia under different temperature conditions such as room temperature (25 °C), 4°C and outdoor conditions, a talc based bioformulation was prepared. Result of shelf life determination demonstrated that all consortia were able to survive for three months in all defined condition. The bioformulation these consortia CIII and CVand bacterial isolate SRK14 and WRPa4 could be used as bio protective and biofertilizer agent after further examination, at commercial scale in field under stressed conditions.
Open Access
Development of immobilized microbial consortia for decolorization of synthetic industrial dyes
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Shalu Priya; Tewari, Lakshmi
Industrial application of different types of synthetic toxic dyes in huge amount is of major concern with regard to environment and human health. Due to their carcinogenic and mutagenic properties these dyes are damaging the ecosystem and their natural flora and fauna. Therefore, the present investigation was carried out for the efficient bioremediation of seven toxic synthetic industrial dyes (direct brown, diazo black, congo red, direct black, turquoise blue, malachite green and brilliant green), which are widely used in different industries, using fourteen microbial (bacteria, fungi, yeast, microalgae and cyanobacteria) cultures. All the microbial cultures were initially screened for their in vitro dye decolorization efficiencies. Then, the selected isolates were further evaluated spectrophotometrically for the quantification of dye decolorization for all the dyes. Four fungal cultures, (PMF2, WFPN1, WDP2 and PPS1) showed maximum decolorization for most of the azo dyes, where as the microalgae (PASM, PHSM and PRSM) and the cyanobacteria (Synechococcus sp.) showed maximum decolorization for the dyes malachite green and brilliant green only in 4-7d. The microbial cocultures (consortia) were developed using the potential microbial monocultures to increase the extent of dye removal for the four selected dyes (congo red, direct black, malachite green and brilliant green). The decolorizing efficiencies shown by the selected fungal consortia for the azo dyes (congo red, direct black) and triaryl methane dyes (malachite green, brilliant green) were significantly higher than that for their monocultures. The consortia showed complete decolorization of the liquid cultures in lesser time (3-4d and upto 2d, respectively). The effect of certain factors such as concentration of dye and pH of the medium were also found to have a great impact on microbial dye decolorization. The developed microbial consortia (PHSM + Synechococcus) was able to grow and decolorize the synthetic dye (malachite green) upto a conc. of 1000 mgL-1 and could also tolerate high alkaline pH range decolorizing the dye upto pH 11. Reduction in the values of BOD, COD, electrical conductivity and pH of the treated samples as compared to their uninoculated control counterparts was also recorded. The immobilization of the co-cultures of microalgae and cyanobacteria was done on corn cob. The immobilized co-cultures showed rapid decolorization of the dye malachite green with better dye decolorizing efficacy. The extent of dye decolorization during microbial decolorization process was monitored by UV-visible spectroscopy, FTIR and SEM analysis.
Open Access
Comparative microbial diversity under conventional and organic farming practices for chickpea cultivation
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-07) Singh, Pranjali; Goel, Reeta
An experiment was conducted during the year 2017-18 on two fields of chickpea (variety Pant Kabuli chana 1) which were subjected to conventional and organic farming practices. The aim of the study was to analyse the effects of these two entirely different farming systems on the soil quality, microbial population, agronomical parameters and productivity of the crop and quality of the food grain produced. The study used a holistic approach to study the interplay between these factors. It was found that the soil physico-chemical properties of the organic plot were far better than the conventional plot. DGGE analysis did not show any remarkable difference between the two systems in terms of bacterial diversity. Nevertheless, the relative abundances of the Operational Taxonomic Units showed temporal variations. 16S rDNA quantification revealed more microbial load in the conventional soil at all the stages of cultivation. The diazotrophic abundance estimated using nifH copy number showed that the number of diazotrophs increased in the conventional soil while in the organic soil there was a decrease in the number of diazotrophs during the course of cultivation. The agronomical parameters of chickpea plant increased under organic cultivation. Moreover, the grain yield of the crop was more under organic cultivation (1345.5 kg ha-1) as compared to conventional cultivation (896.5 kg ha-1). The Fe and Zn content of organically produced chickpea grain was almost double of the conventionally produced grains. Hence, organic cultivation led to better quality of soil, chickpea grain and increased yield of the crop. Therefore, organic agriculture is recommended due to its low cost input and less intensive nature.
Open Access
Microbial consortia development and their evaluation for the growth of wheat under drought stress conditions
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-07) Panwar, Manisha; Singh, Ajay Veer
Drought considered as one of the major abiotic stress that severely affects overall plant growth and productivity worldwide compare to any other abiotic stress. Therefore, sustainable approaches are needed to assist plants growth under drought stress in order to mitigate crop losses. The present study was designed to develop microbial consortia of plant growth-promoting rhizobacteria and their bio-protective effects on wheat and determined shelf life of microbial consortia under different bioformulation preparations. In the current investigation nine bacterial isolates were selected as prominent drought tolerant bacteria on the basis of better growth potential in nutrient broth supplemented with 10% poly ethylene glycol (PEG). Drought tolerant isolates showed remarkable plant growth promoting properties such as production of siderophore, ACC deaminase, exopolysaccharide and efficient solubilization of zinc and phosphate and selected for consortium development on the basis of their biocompatibility assay. These bacterial consortia and their individual bacterial isolates were further tested comparatively for different plant growth promotory functional traits. The biochemical study of the bacterial isolates also confirmed their ability to produce various extracellular enzymes and utilization of diverse substrates as their carbon source. Furthermore, plant growth promotory potential of microbial consortia were assessed through in situ pot experiment in greenhouse with wheat (Triticum aestivum var. PBW 373) plants inoculated with or without consortia under drought stress conditions. The results of in situ pot experiment confirmed the efficiency of consortia to confer drought stress tolerance and retained higher biomass as compared to non-inoculated plants. Inoculated plants showed high content of chlorophyll and carotenoids, higher relative water content and proline content, significantly lower MDA content in the vegetal tissues hence confirmed the PGPR mediated amelioration of drought tolerance in wheat. Furthermore, bioformulation of bacterial consortia were prepared by using talc and sugarcane bagasse as carrier and evaluated for shelf life on different conditions for 90 days. The survival rate of bacterial consortia was higher in talc-based bioformulation at 90th day at 4 ºC. Above results demonstrated that these developed bacterial consortia could be used as bio-inoculant after further evaluation under field conditions to enhance crop productivity under drought stress for sustainable agriculture.

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