Mitra et al., 2020, Biologica Nyssana 11(1) 11 (1) September 2020: 35-44 DOI: 10.5281/zenodo.4060292 Isolation and impacts of rhizobacteria from Saussurea obvallata (DC.) Edgew. (Brahma Kamal) Original Article Debasis Mitra Department of Biotechnology, Graphic Era (Deemed to be University), Dehradun, Uttarakhand, India debasismitra3@gmail.com (corresponding author) Navendra Uniyal Department of Biotechnology, Graphic Era (Deemed to be University), Dehradun, Uttarakhand, India navendraunniyal121@gmail.com Komal Sharma Department of Biotechnology, Graphic Era (Deemed to be University), Dehradun, Uttarakhand, India komal.sharma638@gmail.com Anju Rani Department of Life Sciences, Graphic Era (Deemed to be University), Dehradun, Uttarakhand, India teotia_anju29@rediffmail.com Lok Man Singh Palni Vice Chancellor, Graphic Era (Deemed to be Univer- sity), Dehradun, Uttarakhand, India lmspalni@rediffmail.com Akansha Chauhan Department of Biotechnology, Graphic Era (Deemed to be University), Dehradun, Uttarakhand, India akansha0911chauhan@gmail.com Prabhakar Semwal Uttarakhand State Council for Science and Technol- ogy (UCOST), Vigyan Dhaam, Dehradun, Uttara- khand, India. semwal.prabhakar@gmail.com Poonam Arya Department of Biotechnology, Graphic Era (Deemed to be University), Dehradun, Uttarakhand, India aryapoonam73@gmail.com Received: November 06, 2019 Revised: January 21, 2020 Accepted: January 26, 2020 Abstract: The rhizospheric association of bacteria in terrestrial plants across the hilly region has tremendous potential for plant growth-promotion (PGP) and protection. This is the first study which aims to isolate some rhizo-bacteria from the different rhizospheric soil samples of S. obvallata in The Himalayan region. Genotypic, biochemical and PGP traits shows genetically similar bacteria species to have diverse PGP potential as shown by IAA production of 87.567 μg ml-1 by Bacillus sp. D5 and phosphate solubilization potential 30 mm halo zones are shown by Bacillus sp. D9. Growth of isolates was in the ranges of temperature (0-60 °C), salt (0-20%), which shows their tendencies towards surviving harsh environmental conditions. The isolates were further evaluated on Amaranthus cruentus, which shows a significant improvement in growth compared to control. The novel study finds the bacterial community at high altitudes to survive extreme climatic variability through association with plants by developing a commensalistic relationship with host. Key words: S. obvallata, indigenous rhizobacteria, plant growth promotion, amaranth Apstract: Izolacija i uticaj rizobakterija iz Saussurea obvallata (DC.) Edgew (Brahma Kamal) Bakterijska rizosferna asocijacija na terestričnim biljkama širom brdskog re- giona ima ogroman potencijal za stimulaciju biljnog rasta (PGP) i zaštitu. Ovo je prva studija koja ima za cilj izolovanje nekih rizobakterija iz različitih uzoraka zemljišta rizosfere vrste Saussurea obvallata u regionu Himalaja. Genotipska, biohemijska i PGP svojstva pokazuju da genetski slične vrste bakterija imaju raznovrstan PGP potencijal, kao što pokazuje IAA produk- cija od 87.567 µg/ml od strane Bacillus sp. D5 i potencijali za solubilizaciju fosfata od 30 mm halo zone od strane Bacillus sp. D9. Rast izolata je bio u rasponu temperature (0-60°C) i soli (0-20%), što pokazuje njihove tenden- cije prema preživljavanju surovih uslova životne sredine. Izolati su dalje pro- cenjeni na vrsti Amaranthus cruentus, što je pokazalo značajno poboljšanje rasta u odnosu na kontrolu. Nova studija otkriva da bakterijska zajednica na velikim nadmorskim visinama preživljava ekstremne klimatske varijacije povezivanjem sa biljkama i razvijanjem komensalističke veze sa domaćinom. Ključne reči: S. obvallata, autohtone rizobakterije, stimulacija biljnog rasta, Amaranthus Introduction Saussurea obvallata (DC.) Edgew. (Brahma Kamal), the state flower of Uttarakhand, India is an endemic herb of the Himalayan region (encompassing the Indian Himalayan Region, Northern Burma and Southwest China). The plant is distributed at an al- titudinal range of 3000–4800 m (Semwal and Pant, 2013). Brahma Kamal is used for the preparation of traditional medicines by the local peoples in Tibet and other places including Garhwal Himalayas. It is well known that indigenous rhizobacteria exert ben- eficial effects on plants productivity and sustenance of soil health through their capacity for phosphate solubilization (Sarikhani et al., 2019), indole ace- tic acid (IAA) (Selvakumar et al., 2008), ammonia, HCN and cell wall degrading enzyme production (Tsegaye et al., 2019) etc. PGPR are heterogeneous groups of microbes associated with the plant’s root in diverse ways (Nath et al., 2015). In the rhizobac- © 2020 Mitra et al. This is an open-access article distributed under the terms of the Creative Commons At- tribution License, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 35 terial population, 2-5% of total the beneficial rhizo- bacteria are involved in plant growth promotion (Bakker and Schippers, 1987; Antoun and Kloepper 2001; Ahemad and Kibret, 2014). Over 95% of the rhizobacteria existing in the roots and plants obtain many nutrients through the soil bacteria (Ji et al., 2014). Different PGPR, including associative and symbiotic bacteria such as Pseudomonas sp., Azos- pirillum sp., Azotobacter sp., Rhizobium sp., Kleb- siella sp., Enterobacter sp., Alcaligenes sp., Arthro- bacter sp., Burkholderia sp., Bacillus sp., and Ser- ratia sp. groups have been used for their beneficial effects on plant growth and improvement (Kloep- per and Beauchamp, 1992; Höflich et al., 1994). Rhizospheric microbial population mainly promote plant health, they stimulate plant growth directly by producing or changing the concentration of plant growth regulators (Kloepper et al., 1980; Ramette et al., 2003; Sparzak-Stefanowska et al., 2019) like gibberellic acid, IAA, cytokinins (Glick, 1995; Ves- sey, 2003), asymbiotic N2 fixation (Ardakani et al., 2010; Bhattacharyya and Jha, 2012). These popula- tions also achieve antagonism against phytopatho- genic microorganisms by producing antibiotics, fluorescent pigment, enzyme, cyanide, phenolics, signal compounds (Niranjan et al., 2005), as well as solubilization of mineral phosphates and other nu- trients (Johri et al., 1999; Sharma and Johri 2003). Inoculums have been used to increase plant yields in several countries, which is reported in research arti- cles (Dursun et al., 2019) and commercial products are currently available (Backer et al., 2018). We are addressing for the first time isolation of high altitude rhizobacteria from S. obvallata (Brah- ma Kamal) rhizospheric soil in the Himalayan re- gion and their beneficial effects, harsh environment survival strategy and plant growth promotion in Am- aranthus cruentus. We proposed that these isolates may also have the ability to express activity towards promoting sustainability in harsh environments. Material and methods The four rhizospheric soil samples of S. obvallata were collected from different place of Kedarnath val- ley, Uttarakhand, India and samples collection sites were described by Semwal et al. (2018) and Mitra et al. (2019). The soil samples were serially diluted and 100 μL of dilution sample was plated onto nutri- ent agar medium (NAM) (HiMedia, MV002, India) for the isolation of potential PGP. All isolated strains were stored at -20 °C in nutrient broth containing 15% (v/v) glycerol. Morphological and biochemical characteriza- tion tests viz. IMViC test, citrate utilization, starch hydrolysis, phenylalanine deaminase, urease test, casein hydrolysis, H2S production, peroxidase test, nitrate utilization, and growth in different media - MacConkey’s agar, esculine hydrolysis, mannitol salt agar tests were carried out followed by MacFad- din, 1985; Harley and Prescott, 2002; Anija, 2003; Dubey and Maheshwari, 2007; Cappuccino and Sherman, 2008; Brown, 2009 respectively. To iden- tify the morphological characteristic of all isolates, Gram staining, negative staining, shape, margin, el- evation, form, motility and pigment were recorded. A loopful of bacteria was inoculated and incu- bated into pre-sterilized peptone broth containing 1% of tryptone for 48 h at 28 °C. After 48 h, 1 mL of Kovac’s reagent (HiMedia, India) was added to all tubes including control, gently shaked for few sec- ond and left to react for 5-10 minutes. The appear- ance of red/ pink ring at the top is the clear indication of IAA production (Loper and Schroth, 1987; Dubey and Maheshwari, 2007). As described in Gordon and Weber (1951), the standard graph of IAA was pre- pared for the quantitative estimate of IAA produc- tion. Different IAA concentrations were prepared as aqueous solution of IAA ranging from 5-150 μg ml-1. To each 1 ml of the standard, 2 mL of 2% FeCl3 (0.5 M) in 35% perchloric acid i.e. Salkowaski reagent was added and readings were taken after 30 min- utes at 535 nm using UV-Visible spectrophotometer (Thermo Scientific™ GENESYS-10S-UV). The standard graph was finally prepared by plotting con- centration of IAA in ppm vs optical density at 535 nm. Bacterial cultures were grown for 48 h in nu- trient broth. Fully grown cultures were centrifuged at 4000 rpm for 20 minutes at 4 °C. The superna- tant (1 mL) was mixed with 4 mL of Salkowaski reagent. Samples were left at 28 °C for 30 minutes. Development of pink color indicates the produc- tion of IAA. Optical density was measured at 535 nm with a spectrophotometer. The concentration of IAA produced by bacterial cultures was measured with the help of standard graph of IAA (Loper and Schroth, 1987). Phosphate solubilization ability of the isolates was evaluated in Pikovskaya’s (PKV) agar medium (HiMedia, India) incorporated with tri-calcium phosphate (TCP) [Ca3 (PO4)5] as insolu- ble phosphate. PKV media plate was prepared and point inoculation was transferred in each plate at 28 °C for 5 days. After incubation, positive phosphate solubilizing bacteria which give clear zone around the colony were measured (Fiske and Subbarow, 1925). Isolated strains were grown overnight in 10% trypone soy agar supplemented with glycine (4.4 gly L-1) (HiMedia, India) (Bakker et al., 2003). A What- man no. 1 filter paper soaked in 2% sodium carbon- ate and 0.4% picric acid solution was placed to the underside of the petri dish lids (Macfaddin, 1980; results explained by Cook, 1993) and kept at 28 °C for 4 days. For the ammonia production, all isolated 36 BIOLOGICA NYSSANA ● 11 (1) September 2020: 35-44 Mitra et al. ● Isolation and impacts of rhizobacteria from Saussurea obvallata (DC.) Edgew. (Brahma Kamal) bacterial strain was grown in 4% peptone broth for 48 h at 28 °C. Detection of ammonia production was done by adding dropwise of 1.5 mL Nessler’s reagent (HiMedia, India) (Bakker et al., 2003). Cel- lulase enzyme production was determined from clear zone test in CMC (Carboxyl methyl cellulose) agar (CMC - 100.00 g, peptone - 5.0, agar: 15.00 g, distilled water - 1000 mL, pH: 7.2) and Czapek- mineral salt medium (NaNO3 - 2.2 g, K2HPO4 - 1.0 g, MgSO4.7H2O - 0.7 g, KCl - 0.5 g, CMC - 5.0 g, pep- tone - 2.5 g, agar - 18.0 g, distilled water - 1000 mL, pH - 6.0) (Bakthavatchalu et al., 2012). Two days after incubation, 2 mL of Congo red solution (0.1% Congo red in 1 M NaCl) was added in each plate. All the plates were then gently shaken. A positive result was indicated when a clear zone and light develops (Qadri et al., 1988). Protease activity (casein degra- dation) was determined from the clear zone in skim milk agar (skim milk powder - 100.0 g, peptone - 5.0 g, agar - 15.0 g, distilled water - 1000 ml and pH - 7.2) (Vijayaraghavan and Vincent, 2013). The biofilm experiment was performed in 10 mL glass bottles. Three milliliters of nutrient broth (without NaCl) was dispensed and inoculated with an overnight experiment culture. The bottles were incubated under the static condition for 5 days, the wells were washed thrice with distilled water and the biofilm was stained with 1% crystal violet dye solution for 15 min, and again the wells were rinsed thrice with distilled water to wash off the unbound dye. Quantification of the biofilm biomass was done by absolute alcohol and recording the absorbance at 600 nm according to Srinandan et al. (2010). Antibi- otic resistance testing was determined by disc diffu- sion method (penicillin: SD028-1VL, erythromycin: SD083-1VL, streptomycin: SD091-1VL, chloram- phenicol: SD153-1VL and tetracycline: SD133- 1VL, HiMedia, India) using nutrient agar plates. The bacterial suspension was inoculated on the nutrient agar plate by swabbing to give a smooth lawn and antibiotic discs were properly placed on the plate for overnight. The plates were observed and recorded the zone of inhibition around the antibiotic disc. All isolates antibiotic profile diversity was analyzed by NTSYS 2.02e software, where 0 denotes suscepti- ble and 1 denotes resistance to a particular antibiotic source. The data were analyzed using the Similar- ity for Qualitative data subroutine of NTSYS-PC2 package. The isolates were grouped as per Jaccard’s similarity index by the unweighted paired group method using arithmetic means (UPGMA) and de- picted as dendrogram (Ansari et al., 2014). Antibi- otics test result were graphically determined by the R-Heatmap plot analysis where 0 denotes suscepti- ble and 1 denotes resistance to a particular antibiotic source. 37 To find out the optimal conditions for the growth of bacterial isolate, the effect of different tempera- ture levels on the development of isolates was exam- ined. 0 °C, 4 °C, 28 °C, 37 °C and 60 °C were mainly used and bacterial suspension (100 μL) was placed in a tube containing 10 mL nutrient broth. The tube was incubated at the predetermined temperatures for 48 hours. The effect of different salt concentra- tion on bacterial growth was studied with nutrient broth containing salt concentrations ranging from 0-20%. The effect on different salt concentration (0% and 8% NaCl) for indole-3-acetic acid produc- tion in tryptone broth. This was investigated by us- ing tryptone broth containing 0% and 8% NaCl and following 48 h of incubation, the effects on growth rate were measured by spectrophotometric measure- ments at 660 nm. The pot experiment was conducted (two times) in the net-house, Graphic Era (Deemed to be Universi- ty), Dehradun (30°19’N, 78°04’E, 650 m) with two times sterile soil. Amaranthus cruentus seeds were surface sterilized, the seeds were dried for 2 hours in sterile conditions and subsequently planted into the pots prepared for treatments. There were five replicates per treatment with 15 seeds planted per pot. Two bacterial isolates viz. Bacillus sp. D5 and Bacillus sp. D9 were selected based on PGP activity. Soil treatments consisted of 0.5 kg Lignite (carrier material) and 100 mL of inoculum (D5 and D9; sep- arately) solution and sterile distilled water was the control. After 30 days, the length and mass of shoots and roots were taken to determine the effect of treat- ments on the growth of A. cruentus plants. Diversity and statistical analysis were done by using NTSYS 2.02e software. Experimental data were analyzed using standard analysis of variance (ANOVA) and HAU, Hisar online statistical analysis software Results Ten PGPR isolates (Pseudomonas sp. D1, Bacillus sp. D2, Bacillus sp. D3, Bacillus sp. D4, Bacillus sp. D5, Bacillus sp. D6, Bacillus sp. D7, Bacillus sp. D8, Bacillus sp. D9 and Bacillus sp. D10) were cho- sen from a total of eighty-five isolates using the seri- al dilution technique of rhizospheric soil samples of S. obvallata. All isolates were screened and selected on the basis of growth rate, morphological (Fig. 1) and biochemical characteristics. This is the first re- search report that the D1, D2 and D3 isolated from Hathi Parwat (HP), D4 and D5 from Maha Panth (MP), D6 and D7 from Madhu Ganga (MG) and D8, D9 and D10 from Cheer Ganga (CG) of Kedarnath valley. Similarly, Mitra et al. (2019) reported that some dominant fungi viz. Phanerochaete chrysospo- rium, Aspergillus fumigatus and Trichoderma longi- brachiatum was found and identified through 16S rDNA sequencing (ITS4 and ITS5) from the same BIOLOGICA NYSSANA ● 11 (1) September 2020: 35-44 Mitra et al. ● Isolation and impacts of rhizobacteria from Saussurea obvallata (DC.) Edgew. (Brahma Kamal) 38 rhizospheric soil samples of S. obvallata. Biological communities in the Indian Himala- yas include one of the biggest altitudinal encompass in the world and a portion of the more extravagant gatherings of wild and medicinal plants are found in this area (Chauhan et al., 2015). Presentation and exploitation of PGPR in agro-biological systems im- prove plant-microbes interactions that may influence biological communities supportability, farming effi- ciency, and ecological quality (Zahid et al., 2015). The rhizosphere is a special specialty for different kinds of microorganisms in the soil (Agrawal and Johri, 2014). Semwal et al. (2018) reported that S. obvallata rhizospheric and non-rhizospheric soils macro - micro nutrients analysis and range of differ- ent nutrients availability viz. nitrogen, phosphorus, potassium, zinc, copper, iron, manganese in normal and suggested that they are helpful for develop- ment and growth for this endangered plant. So, in our present study, ten PGPR bacterial isolates were isolated and screened for PGP abilities (Kloepper et al., 1988). This study was conducted to isolate the native low temperature growing potential PGPR from the Himalayan region rhizospheric soil of S. obvallata plant and showed that some common PGPRs like Bacillus sp. and Pseudomonas sp. were present in this rhizospheric area. Similarly, Majeed et al. (2015) reported that they are isolated some PGPR for the root endosphere and rhizosphere of wheat from the Himalayan region and isolates showed PGP ability like IAA production, N2 Fixation and P - solubiliza- tion. Agrawal and Johri (2014) reported that fifty- five rhizobacteria were isolated, characterized and screened PGP attribute from the central Himalayan region, forest sites; altitude 3500, 12000 and 3100 ft. Biochemical test results of all bacterial isolates were analyzed by Heatmap plot and represented the results where black colour denotes negative and gray colour denotes positive activity (Fig. 2). Heat- map plot colour analysis showed that most (approx. <50%) isolates has positive biochemical activity. IAA production is a natural property of rhizo- bacteria that enhance and facilitate plant growth and development. Quantitative assay of IAA production of rhizobacteria was done by the spectrophotomet- Fig 1. The figure shows the percentage abundances of bacteria as determined under microscope Fig 2. Biochemical test results represented by Heatmap plot (MR and VP: MR/VP test, C: Citrate utilization, SH: Starch hydrolysis, PD: Phenylalanine deaminase, UT: Urease test, CH: Casein hydrolysis, H2S: H2S produc- tion, PT: Peroxidase test, NR: Nitrate utilization, MA: MacConkey’s agar, EH: Esculine hydrolysis and MSA: Mannitol salt agar) BIOLOGICA NYSSANA ● 11 (1) September 2020: 35-44 Mitra et al. ● Isolation and impacts of rhizobacteria from Saussurea obvallata (DC.) Edgew. (Brahma Kamal) 39 ric analysis. The concentration of IAA produced by bacterial cultures was measured with the help of the standard graph of IAA. In this study, six qualitative screened positive bacterial isolates were taken for the quantitative assay and D5 isolate IAA produc- tion is 87.567 μg ml-1. Phosphate solubilizing bac- teria used as inoculants simultaneously increases phosphate uptake by the plant and crop yield (Ro- dríguez and Fraga, 1999). Phosphate solubilization activity was determined by the development of the clear zone around the bacterial colony. Isolate D9 is a high phosphate solubilizer bacterial strain which is used for the treatments. D9 showed the 30 mm zone (diameter) on PKV agar media (Fig. 3A) after 5 days of incubation. Four phosphate solubilizer bacteria strains were grown in PKV medium (pH 6.35) for 3 and 5 days at 28 °C. After the given time, pH of the culture medium was checked by pH meter and decreased pH rate can be observed in Fig. 3B. In the similar study, Panday et al. (2002) reported and isolated P. corrugata from the low-temperature lo- cation of Sikkim and reported that all isolates could grow and solubilize phosphate in the range of 4 to 35 °C. All isolates were grown in 10% tryptone soy agar with glycine (4.4 gly L-1) on the plates sealed with parafilm®. The production of HCN was determined by the change in colour of filter paper from yellow to red-brown. After the incubation of all bacterial culture in medium, the Nessler’s reagent was added. Ammonia produced by bacterial isolates formed the yellowish-brown colour in the medium. Cell wall degrading enzyme production was determined by congo red method and protease activity was de- termined by clear zone formation on the test plate. By this, the production of the most important plant growth hormones was recorded (Fig. 4). Fig 3. A - Bacillus sp. D9 isolate phosphate solubiliza- tion zone on PKV media after 5 days; B - pH level in PKV broth Fig 4. Isolates plant growth-promoting test results are evaluated graphically by Heatmap plot where gray color: (+) PGPR and black colour: (-) PGPR. (IAA: IAA production, PS: Phosphate solubilization, HCN: HCN Production, AP: Ammonia Production, C: Cellulase Test and P: Protease test) Fig 5. Biofilm formation by isolates BIOLOGICA NYSSANA ● 11 (1) September 2020: 35-44 Mitra et al. ● Isolation and impacts of rhizobacteria from Saussurea obvallata (DC.) Edgew. (Brahma Kamal) 40 Quantification of the biofilm biomass results by the bacterial isolates are presented in Fig. 5. Isolates D2, D5, D7 and D9 showed high biofilm formation after 5 days of incubation. The antibiotic sensitivity was determined by disc diffusion method, where five antibiotics were used and the results of resistance and susceptibility of the isolates are presented in Fig. 6A. Antibiotic diversity of the isolates was analyzed by NTSYS and dendrogram, where D2 and D5 showed sensitivity to all tested antibiotics (Fig. 6B). Heatmap plot showed that D6, D7, D8, D9 and D10 are resistant to penicillin and D1 showed resistance to erythromycin and tetracycline (Fig. 6C). Bacterial growth under different temperature and NaCl concentration were determined by measuring the optical density (OD) at 660 nm by spectropho- tometer (Fig. 7A, B). In this study D2, D5 and D9 bacterial isolates demonstrated the highest growth rate at 0 C, while 28 and 37 were optimal growth temperatures for most other isolated strains. All iso- lates showed low growth rate at 60 °C. Our study suggested that the most suitable salt concentrations differed among the strains. The result showed that optimal growth and survival were at 0% for D8, 0.5% for most of the strains (D1, D2, D3, D7, D9 and D10), while three strains (D4 D5 D6) had the best growth at 5% NaCl concentration (Fig. 7B). IAA is a common product of L-tryptophan me- tabolism by numerous microorganisms but in the medium salt concentrations affects the growth rate of microorganism. All isolates were grown in tryptone broth with different salt concentration. D6 and D7 growth rate were higher at 8% NaCl and D9 growth rate was constant at 0% and 8% NaCl (Fig. 8). The results of the soil treatments with potential PGPR isolates viz. D5 and D9 on the growth of A. cruentus showed significant effect in enhancing the plant’s shoot - root height and weight of inoculated plants as compared to uninoculated control (Fig. 9). In experiment 1, D5 and D9 showed a positive result in the development of shoot but in experiment 2, D5 showed high effect in enhancing shoots and roots as compared to control. Discussion PGPR are originally free-living soil bacteria that colonize the rhizosphere or the tissues of living plants. Once the bacteria are associated with the plant by colonizing the rhizosphere around the roots, the rhizoplane (root surface) or the root it- self (Rodriguez-Navarro et al., 2007), the bacteria can increase the development of the plant through indirect or direct mechanisms. In our study, PGPR isolates viz. D4, D5, D6 and D9 showed positive for phosphate solubilization and pH ranged from 4.00 – 5.00. D9 bacterial isolates showed the highest zone in PKV plate. Highest biofilm formation showed by rhizobacterial isolates D1, D2, D5, D7 and D9 was very evident at 600 nm readings. From the compat- ibility results, D1, D5 and D9 isolates were selected for the pot experiment to assess its performance in terms of growth, nutrient uptake and stress condi- tions survival ability. Pot experiment results showed Fig 6. A - Effect of antibiotics to different isolates; B - Antibiotics diversity of isolates analyzed by NTSYS; C - Antibiotics test results analyzed by Heatmap plot where gray color: Resistance and black color: Suscep- tibility (P10: Penicillin, E10: Erythromycin, S25: Strep- tomycin, C25: Chloramphenicol, T10: Tetracycline BIOLOGICA NYSSANA ● 11 (1) September 2020: 35-44 Mitra et al. ● Isolation and impacts of rhizobacteria from Saussurea obvallata (DC.) Edgew. (Brahma Kamal) 41 Fig 7. A - Isolates growth under the different temperature; B - Isolates growth under the different salt concentration Fig 8. Growth rate in tryptone broth with different salt concentration BIOLOGICA NYSSANA ● 11 (1) September 2020: 35-44 Mitra et al. ● Isolation and impacts of rhizobacteria from Saussurea obvallata (DC.) Edgew. (Brahma Kamal) that soil treatments with these isolates significantly enhanced the plant growth of inoculated amaranth plants as compared to uninoculated control. Conclusion In our study, we used Bacillus sp. D5 and Bacil- lus sp. D9 as a bio-inoculum and phosphate solu- bilizer from the rhizospheric soil samples of S. ob- vallata (Brahma Kamal) in The Himalayan region. The present study has clearly demonstrated the PGP trails and application on amaranths with Bacillus sp. D5 and Bacillus sp. D9 having a positive influence in growth parameter in comparison with the control. The study suggested that low-temperature bacterial community growth has multifunctional characteris- tics in growth promotion and develops a green bio- fertilizer for hill farming. Acknowledgements. We are grateful to the Founder and President, Prof. (Dr.) Kamal Ghanshala, Graphic Era (Deemed to be University), Dehradun and giving stu- dent research grants for this research work. We are also grateful to Prof. Ashish Thapliyal, Professor and Head, Department of Biotechnology, Graphic Era (Deemed to be University), Dehradun. 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