Indian Phytopath. 67 (2) : 126-133 (2014) RESEARCH ARTICLE Characterization of bacteria from the rhizosphere of Persea bombycina with multiple growth promoting traits JINTU RABHA1, DHRUVA KR. JHA1*, HRIDIP KR. SARMA2, AMRITA ACHARYA3, USHA CHAKRABORTY3 and BISHWANATH CHAKRABORTY3 1 Microbial Ecology Laboratory, Department of Botany, 2Department of Biotechnology, Gauhati University, Guwahati 78 1014, Assam, India 3 Immuno-Phytopathology Laboratory, Department of Botany, North Bengal University, Siliguri 734 013, West Bengal, India ABSTRACT: The present study was carried out to screen such beneficial rhizobacterial isolates that could be utilised for improvement of growth of som plant (Persea bombycina Kost.), a primary food source of the muga silkworm. Rhizobacteria was isolated from the rhizospheric soil collected from the som germplasm conservation site located at Regional Muga Research Station, Central Silk Board, Boko, Assam (GPS location 25.990 N, 91.250 E). Sixty-one percent of the total 18 isolates showed phosphate solubilisation as well as NH3 production. On the contrary, fewer isolates (33%) produced indole acetic acid in vitro. In addition, some of the isolates depicted antagonistic activity against Pestalotiopsis disseminata and Phyllosticta persea, the two phytopathogens responsible for causing grey blight and leaf spot diseases respectively in P. bombycina. Molecular identification of two potent plant growth promoting rhizobacteria (PGPR) isolates was carried out by amplification of their partial 16s rRNA and sequencing. Based on in vitro results, one PGPR isolate Pseudomonas sp. GUDBPKA301 was screened for in vivo study. A significant improvement in growth measured in terms of increase in shoot length, number of leaves and branches was observed when one year old som saplings were bacterized under pot conditions. Results clearly suggest that Pseudomonas sp. GUDBPKA301 is a potential PGPR which can be used as an efficient microorganism for enhancement of plant growth in som cultivation. Key words: Persea bombycina, Pestalotiopsis disseminata, Phyllosticta persea, plant growth promoting rhizobacteria Plant rhizosphere microflora, particularly plant growth promoting rhizobacteria (PGPR) are defined as beneficial bacterial communities that inhabit the rhizosphere of plants and stimulate the growth of the host plant either directly or indirectly (Kloepper and Scroth, 1978). They induce direct effects by producing plant growth regulators such as indole acetic acid (Bharucha et al., 2013; Patten and Glick, 2002) or facilitate nutrient uptake by the plants by solubilizing essential minerals such as phosphate (Rodriguez and Fraga, 1999) in the rhizosphere. Some bacteria can fix atmospheric nitrogen (Zhang et al., 1996) and also produce siderophores (Kloepper et al., 1980; Katiyar and Goel, 2004). Indirect mechanisms include induction of systemic resistance through the synthesis of antimicrobial compounds (Kloepper et al., 1981; Raajimakers et al., 2002) or lytic enzymes having negative impact on the growth and sustenance of deleterious microorganisms thriving in the vicinity of plant rhizosphere (Kloepper et al., 2004). Kloepper et al. (2004) established that the application of plant growth promoting rhizobacteria (PGPR) that colonize the rhizosphere of plants result in improvement of plant health. Stimulation and protection of different crops by PGPR have been demonstrated many times under controlled conditions and field trials (Lucy et al., 2004). Persea bombycina Kost (som tree) is a medium sized evergreen tree belonging to family Lauraceae. It is an economically important plant of North-eastern India *Corresponding author: dkjhabot07@gmail.com and is widespread in Assam because of its importance as the primary host plant of the golden silk producing silkworm, Antherea assama Helfer. From studies reported earlier, it has been found that the growth, development and economic characters of the silkworm are influenced to a great extent by nutritional content of their food plants (Neog et al., 2011). Besides, quality of cocoons and quantity of raw silk produced is also dependent on the quality of leaves supplied to the growing larvae (Khanikar and Unni, 2006). It is, therefore, evident from the earlier reports that an understanding of physiological and biochemical potential of som plants that provide good quality leaves are essential towards proper growth of muga silkworm larvae for better silk production. The plants also suffer from a number of foliar diseases that affect the quality and quantity of leaves thereby affecting adequate silk production (Thangavellu et al., 1988; Das and Benchamin, 2003). The chemicals used for controlling plant diseases cannot be used at random, as they might negatively affect the beneficial insects (Gamo and Hirobe, 1977). Biological control through the application of beneficial rhizospheric microorganisms par ticular ly those that exhibit antimicrobial activity against foliar fungal pathogens could be useful for controlling the diseases. The present investigation therefore, was carried out to screen beneficial rhizobacteria (PGPR) that could be utilised to promote growth traits of som leaves and reduce the severity of foliar diseases caused by fungal pathogens. Indian Phytopathology 67 (2) : 14-21 (2014) MATERIALS AND METHODS 127 was observed by adopting the method of Lorck et al. (1948). Sampling sites Antagonistic activity against foliar fungal pathogens The sampling site for the present study was located at 25.99° N and 91.25° E latitudes close to the som germplasm conservation site, Central Silk Board, Boko, Assam. The recorded annual rainfall of the area ranges between 1500 mm to 2600 mm with an average humidity of 75 percent and maximum and minimum temperature of 38.5 °C and 7°C. Soils were collected from the rhizosphere of Persea bombycina Kost (som plants) at depths (0-5cm top soil) and were packed in polythene bags using sterile scalpel. The collected soil samples were carried to the laboratory in cold sealed ice boxes to ensure that microorganisms remained active and alive. Isolation and characterization of rhizospheric bacteria Processed soil was serially diluted and spread plated on Pikovskaya agar with following composition (in g/l) Yeast extract 0.5, Dextrose 10.0, Calcium phosphate 5.0, Ammonium sulphate 0.5, Potassium chloride 0.2, Magnesium sulphate 0.100, Manganese sulphate 0.0001, Ferrous sulphate 0.0001 and Agar 15.0. The plates were incubated at 28°C for 48 hours in a BOD incubator (Caltan, India). For intended isolation of Bacillus species of soil bacteria, soil pre-treatment method as described by Knight and Proom (1950) was followed. Briefly, soil suspension in broth was heated at 70°C for 10 min. It was followed by plating on nutrient agar (Himedia) and incubated at 37°C. Purification of bacteria was done by repeated streaking on nutrient agar plates; pure cultures of single colony isolates were maintained in nutrient agar slants. The biochemical characterization and identification was done according to standard manuals (Cappuccino and Sherman, 2008) and Bergey’s Manual of Systematic Bacteriology (Garrity et al., 2005). In vitro screening of PGPR for plant growth promoting traits The rhizobacterial isolates were screened based on halo zone formation (phosphate solubilization) in Pikovskaya agar. Production of indole acetic acid (IAA) in vitro was detected by the method of Brick et al. (1991). Quantitative estimation of phosphate solubilization was done in Pikovskaya broth supplemented with tricalcium phosphate according to Chlorostannous reduced molybdophosphoric blue color method as was described by Jackson (1958). Quantitative estimation of IAA was done following the method of Loper and Schroth (1986) using media supplemented with tryptophan (100µg/mL) and recording the absorbance at 535 nm. The isolates were tested for production of ammonia using tryptone water as mentioned by Cappuccino and Sherman (2006). Antibiotic susceptibility profile was observed using disc diffusion method described by Kirby and Baur (1966). Screening for protease activity was done by observing the formation of halo zone in skim-milk agar (Cappuccino and Sherman, 2006). HCN Production of the isolates Test fungi for the experiments were obtained from CMETRI, Central Silk Board, Lahdoigarh, Jorhat, Assam. Both the fungi viz. Pestalotiopsis disseminata and Phyllosticta persea were identified and selected as causative agents of grey blight and leaf spot diseases of som. The dual culture method was used to observe antagonistic activity of PGPR against test pathogens. Molecular characterisation of isolates Two of the Pseudomonas isolates namely PKA-105/3 and PKA-105/5 were grown in nutrient broth at 30°C. Cells were harvested after 24h and processed for DNA extraction using Himedia Bacterial Genomic DNA extraction kit (HiMedia Laboratories, Mumbai, India). Amplification of 16S rRNA gene sequence was performed by polymerase chain reaction (PCR) with the primers pairs 1406F (5’-TGYACACACCGCCCGT-3’) and 155R (5’-GGGTTBCCCCATTCGG-3’) to yield a product of 780 bp as described by Ikeda et al. (2004). The PCR reactions were carried out in 25 µl volumes containing 2.5µl of 10X PCR buffer, 1.5 µl of 50 mM MgCl2, 0.5µl of a mixture containing each of the dNTPs at a concentration of 2.5 mol/l, each primer at a ûnal concentration of 5.0 pM, 2 µl of DNA containing 100 ng and 1 U of Taq DNA polymerase. The amplification reaction was carried out for 35 cycles. After a single cycle comprising of initial denaturation for 3 min at 95°C was followed by 34 cycles with 40s of denaturation at 94°C, 60s of annealing at 50°C and 1 min of extension at 72°C. After 34 cycles, there was a final extension for 10 min at 72°C. PCR amplifications were performed using C1000 thermal cycler (Bio-Rad, USA, thermal cycler). The resulting PCR products (amplicons) were separated electrophoretically in 1.2% agarose gel, stained with EtBr (10 mg/ml) in 1X TAE buffer and photographed in Gel documentation system (Gel Logic 212 PRO Carestream, USA). Sequencing of partial 16s rRNA gene and sequence analysis The amplified PCR product was purified and sequenced at Xceleris laboratories, Ahmadabad, India using the same eubacterial primers as described above. The chromatograms obtained were edited in BioEdit Sequence Editor Software (Version 7.2.0) (Hall et al., 1998). The sequence data so obtained was aligned and analysed for identification, confirmation as well as to find the closest homolog by a BLAST (Altschul et al., 1997) search. Known sequences were selected from BLAST results and downloaded. Multiple sequence alignment was performed along with the downloaded sequences from GenBank using CLUSTAL X2.0 (Larkin et al., 2007). Manual adjustments of the sequences were made wherever necessar y. Phylogenetic analysis was performed using the dnaml module of the MEGA5 128 (Tamura et al., 2011) package. Maximum composite likelihood method (Tamura et al., 2004) was employed to generate the phylogenetic relationship among the sequences. The robustness of the inferred trees was evaluated by bootstrap resampling. The two sequences were submitted to the NCBI GenBank nucleotide database. Evaluation of in vivo plant growth promoting potential Based on in vitro studies conducted one potent PGPRPseudomonas sp. GUDBPKA301 was selected for evaluation of plant growth promotion in som under pot conditions. The bacterium was cultured in 150mL Nutrient broth media and grown in orbital shaking incubator (Remi Model. RIS-24BL, India) at 34ºC for 48 hours and cells were harvested by centrifugation at 12000 g. The cell pellet so obtained was diluted/ suspended in sterilized double distilled water to yield a cell count 107 cfu ml–1 (~ 0.5 O.D). The bacterial suspension was applied as a soil drench, 200 ml per plant, to the rhizosphere of one year old saplings, twice at an interval of 15 days. The growth promotion was studied in terms of increase in height, number of leaves and number of lateral branches in comparison to water control. Each treatment was carried out in six replications under same physical and environmental condition (temperature 28–32°C; R.H. 70–80%). Observations were recorded after three months of final application of bacteria to soil. RESULTS A total of eighteen isolates (eleven isolated in PKA medium and seven isolated in TSA medium by heat treatment method) were characterized on the basis of their cultural conditions, morphological features and biochemical characteristics using standard methods as described by Cappuccino and Sherman (2008) and the Bergey’s Manual of Systematic Bacteriology (2005) (Table 1). Of the total eighteen isolates, twelve isolates were selected based on their phosphate solubilizing potential in Pikovskaya agar (Pikovskaya, 1948) and production of indole acetic acid which were further subjected to assessment of in vitro plant growth promotion traits. Phosphate solubilization The phosphate solubilizing potentiality of isolated rhizobacteria in liquid PKA medium supplemented with tricalcium phosphate is presented in table 2. All the 12 isolates showed positive results. Five isolates viz. TSA2A5, TSA-1A1, TSA-1A2 and PKA-103/3 and PKA-103/ 7 solubilized more than 300 µg/mL of phosphate in supplemented media. IAA production Four of the total of twelve bacterial isolates under study produced indole acetic acid when media was supplemented with 100µgmL-1 DL- tryptophan (Table 2). The Bacillus isolate TSA-1A5 produced maximum Indian Phytopathology 67 (2) : 14-21 (2014) concentration of IAA (75µgmL -1 ) followed by Pseudomonas isolates PKA-105/3 > PKA-105/5 > PKA103/3 respectively. Ammonia production, protease activity and HCN production The potentiality of the bacterial isolates for ammonia production, protease activity and HCN production is presented in table 2. None of the isolates however produced HCN in vitro. Antagonistic activity of the test isolates Five of the twelve isolates were found to show antagonistic activity against test foliar fungal pathogens (Pestalotiopsis disseminata and Phyllosticta persea) of som plant when tested in vitro. All the five isolates were able to inhibit the growth of P. disseminata (Table 2). The Bacillus isolate TSA-2A1 produced largest inhibition zones against P. disseminata. On the contrary, only two Pseudomonas isolates viz. PKA-105/3 and PKA-105/5 were able to inhibit the growth of P. persea. Molecular characterization of PGPR and phylogenetic analysis with standard sequences Molecular characterization of two PGPR isolates namely PKA-105/3 and PKA-105/3 was done by partial 16S rRNA sequence (800 bp) analysis, and these sequences were deposited in GenBank Bank nucleotide database, KF571703 (Pseudomonas sp. PBR1) and KF571704 (Pseudomonas sp. PBR2) respectively. Dendrogram analysis revealed that the two bacteria are closely related to each other genetically, together forming a separate cluster and also bears very high homology (ranging from 98-99%) to some ex type Pseudomonas spp., among which two Pseudomonas stutzeri (Fig. 2) isolates appear closest relatives. In vivo plant growth promotion Significant increase in growth was observed in all the som morphotypes treated with PGPR ((Table 3). Increase in height ranged from 21.48% (S1-morphotype) to 44.75% (S-8 morphotype). Increase in number of leaves from 34.78% (S-4) to 56.98% (S-2) was observed. Number of branches showed increase of 43.10% (S1 treated plants) to as high as 82.05% in som S-5 morphotype (Table 3). DISCUSSION Significant increase in growth and yield of agronomical important crops in response to inoculation with PGPR have been reported. The positive effect of many soil bacteria on plants is mediated by a range of mechanisms including improvement of mineral nutrition, enhancement of plant tolerance to biotic and abiotic stress, modification of root development, as well as suppression of soil-borne diseases (Glick, 1995; Kloepper et al., 1989). The bacterial traits involved in these activities, include Isolate name Gram reaction Cell shape Starch Hydrolysis Indole test Methyl red Citrate utilization Glucose Mannitol Lactose genus/species Esculin Nitrate Oxidase Possible Bacterial species PKA-103/3 +ve rod - - - - - - - - + - Unknown PKA-103/4 -ve rod - - + - + - - + - - Proteus mirabilis PKA-103/5 -ve rod + - - + - - - - + - Pseudomonas sp. PKA-103/7 -ve rod - - - - + - + - - - Unknown PKA-105/1 -ve rod + - - + - + - - + + Pseudomonas. Sp. PKA-105/3 -ve rod - - - + - - - - + + Pseudomonas sp.GUDBPKA301 PKA-105/4 -ve rod + - - + - - - - + - Pseudomonas sp. PKA-105/5 -ve rod - - - + - - - - + + Pseudomonas sp.GUDBPKA501 PKA-107/2 -ve rod + - - + - - - - - - Pseudomonas mallei PKA-107/3 -ve rod + - - - - - - - + - Unknown PKA-107/4 -ve rod - - - + + - - + + - Unknown TSA-1A1 +ve rod + + + - + + - + + - Bacillus sp. TSA-1A2 +ve rod - - - + + - - + + - Unknown TSA-1A4 +ve rod + + + + - + - + + - Bacillus sp. TSA-1A5 +ve rod + - + + + - - + + - Bacillus sp. TSA-2A1 +ve rod + + - - + + - + + - Bacillus sp. TSA-2A4 +ve rod + + + + - + - + + + Bacillus sp. TSA-2A5 +ve rod + + + + + - - + + - Bacillus sp. Indian Phytopathology 67 (2) : 14-21 (2014) Table 1. Biochemical characterization of rhizobacteria 129 Indian Phytopathology 67 (2) : 14-21 (2014) 130 Table 2. Plant growth promoting traits of the rhizobacterial isolates In vitro growth inhibition Name of Phosphate IAA isolate Solubilization Production Pestalotiopsis Phyllosticta NH3 Protease HCN production activity production (in µg/ml) (in µg/ml) disseminata persea PKA-103/3 337.18 ±2.39 20.50 ± 0.84 - PKA-103/7 308.57 ±1.67 - - - + + - - + - PKA-105/3 56.51 ± 3.00 53.66± 0.98 25.26±1.04 - 18.43±1.26 + - - PKA-105/5 60.83 ± 2.84 47.86 ± 0.63 PKA-107/4 230.58±1.80 - 32.36±1.67 21.56±1.21 + - - 30.33 ±1.10 - + + TSA-1A1 324.32±2.37 - - - - + + - TSA -1A2 306.14±1.97 - - - + - - TSA-1A4 124.47±1.72 - - - + + - TSA-1A5 196.85±1.93 74.78 ± 1.60 - - + + - TSA-2A1 151.98±2.09 - 37.56±1.03 - + + - TSA-2A4 248.89±0.87 - - - + + - TSA-2A5 351.87(±2.07) - 17.46 ±1.29 - + + - Table 3. Growth improvement in pot grown som plants following application of PGPR S1 S2 S3 S4 S5 S6 S7 S8 Morphotype Height (in cm) No. of leaves No. of branches 6.60 ± 0.50 Control 45.96 ± 0.57 18.80 ± 0.86 Treated 58.54 ± 0. 98 (16.80)* 35.20 ± 0.58 (46.59) 11.60 ± 0.92 (43.10) Control 38.06 ± 0.87 16.00 ± 0.70 3.60 ± 0.50 Treated 53.70 ± 0.71 (29.12) 37.20 ± 0.66 (56.98) 10.60 ± 0.50 (66.03) Control 34.02 ± 0.90 11.80 ± 0.86 2.20 ± 0.37 Treated 50.94 ± 0.54 (33) 20.60 ± 0.92 (42.71) 10.60 ± 0.50 (79.24) Control 28.42 ± 0.97 15.00 ± 0.70 2.20 ± 0.37 Treated 47.82 ± 0.98 (41) 23.00 ± 0.70 (34.78) 8.80 ± 0.37 (34.09) Control 41.66 ± 0.85 19.20 ± 0.58 1.40 ± 0.24 Treated 59.94 ± 0.54(30.49) 30.20 ± 0.73 (36.42) 7.80 ± 0.37 (82.05) Control 33.74 ± 0.99 15.00 ± 0.54 1.60 ± 0.24 Treated 46.84 ± 0.88 (27.96) 28.40 ± 0.50 (47.18) 5.20 ± 0.37 (69.23) Control 23.88 ± 0.89 12.40 ± 0.50 1.20 ± 0.37 Treated 34.16 ± 0.61 (30.09) 21.40 ± 0.67 (42.05) 6.20 ± 0.37 (80.64) Control 23.48 ± 0.98 12.80 ± 0.58 2.00 ± 0.31 Treated 42.50 ± 0.82 (44.75) 26.20 ± 0.48 (51.14) 6.40 ± 0.50 (68.75) Average of 3 replicates per set with 5 plants each. ± = Standard error; Difference between control and treated significant at P = 0.01 in all varieties as determined by Paired t-test using SPSS 19 statistical software. * - Percentage increase with reference to control. nitrogen fixation, phosphate solubilization, iron sequestration, synthesis of phytohormones, modulation of plant ethylene levels, and control of phytopathogenic microorganisms. By virtue of their rapid rhizosphere colonization and stimulation of plant growth, there is currently considerable interest in exploiting these rhizosphere bacteria to improve crop production. Fig. 1. PCR amplification of PGPR 16S rRNA gene profiles. Lane 1: mol mass marker (100 bp ladder), Lane 2 and 3 represent partial 16s rRNA of Pseudomonas sp. GUDBPKA301 and Lane 3 and 4 represent PCR product for Pseudomonas sp. GUDBPKA501 The present study revealed that rhizosphere of som plant harbours numerous beneficial bacteria with multiple plant growth promoting attributes. Significant variation was observed in magnitude of the functional traits determined in vitro. These isolates were screened for their ability to produce IAA, solubilization of phosphate and other PGPR activity. Solubilization and mineralization of phosphate (P) by phosphate-solubilizing rhizobacteria is one of the most important physiological traits in plant growth promotion by the PGPR (Rodriguez and Fraga, Indian Phytopathology 67 (2) : 14-21 (2014) 131 Fig. 2. Phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic relationship between Pseudomonas sp (GUDBPKA301 and GUDBPKA501) and other closely related species of Pseudomonas selected based on similiraty (e”97%) by NCBI blast search. Tree was constructed based on maximum-likelihood method using MEGA5 software 1999). In the present study out of the total eighteen isolates studied, eleven isolates solubilized phosphate in solid media while twelve of them showed positive results in liquid media. Bacillus isolate TSA 2A5, showed best phosphate solubilizing potential. However, an important factor observed was the contrasting results obtained for the ability of the PGPR regarding phosphate solubilization whereby differences in solubilizing potential was evident and distinct in both solid and liquid media formulations. Solubilization of phosphate was more enhanced in solid PKA medium compared to PKA liquid medium. This was true for the Pseudomonas isolates PKA-105/3 and PKA-105/5 when compared to other isolates. These unusual observations may be attributed to entrapment of phosphate in agar thereby inducing the isolates to more stressful conditions of nutrient/mineral acquisition when compared to liquid medium where the probability of media acclimatization and availability of nutrients/minerals or even metals is perhaps a nonstressful. Liquid media screenings illustrate an isolate’s ability to solubilize Calcium phosphate (CaHPO4) under non-stressful conditions, but solid media screenings demonstrate that P solubilization results as a matter of stressful conditions (Murumkar et al. , 2012). This limitation for P solubilization on the solid media could have been caused by the unavailability of water in the solid media and subsequently limited nutrient supply (Mitchell and Wimpenny, 1997). The effectiveness of Bacillus isolates in solubilizing phosphate is comparable to results reported earlier (Murumkar et al., 2012). Production of indole acetic acid (IAA) is widespread among plant-associated bacteria (Patten and Glick, 1996). Only four isolates produced the plant growth regulator IAA among which highest producer was the Bacillus isolate TSA-1A5. The production of Ammonia, however, was a common trait encountered in all the isolates. None of the isolates was found to produce Hydrogen cyanide (HCN) in media amended with glycine though this trait has also been linked to inhibition of PGPR activity (Alstrom and Burns, 1989). More than 65 percent of the isolates produced halo zone in skim milk agar depicting protease activity. Fate of the beneficial PGPRplant interaction depends on the exercise of plant beneficial traits in the rhizosphere which would depend primarily on the survival of the microbe in the rhizospheric zone. Three isolates of Pseudomonas (PKA-105/3, PKA105/5 and PKA-107/4) and two of Bacillus (TSA-2A1 and TSA-2A5) were found to be positive in the inhibition of the two test fungal pathogens viz. Pestalotiopsis disseminata and Phyllosticta persea. Such characteristic might be taken advantage of in direct application of antagonists or their bio-formulation via foliar application. From the investigations conducted, it may be concluded that most of the above-tested isolates could exhibit more than two or three PGPR traits, which may promote plant growth directly or indirectly or synergistically. Overall, it was a convincing set of experiments whereby isolates PKA-103/3, PKA-105/3, PKA 105/5, TSA-1A5 and TSA-2A5 depicted many important characteristics necessary for productive PGPR traits in plant pathogen interactions. Molecular identity of two potential PGPR was confirmed by 16s rRNA sequencing and NCBI BLAST search. Results have confirmed the identity of two potent PGPR isolates to be belonging to Genus Pseudomonas. However, as more than one standard NCBI GenBank sequences have shown similarity of 99% with our sequences, we do not ascertain a particular species name to our isolates and consequently we have deposited our sequences as Pseudomonas sp. GUDBPKA301 (isolate PKA-105/3) under Accession no. KF571703 and Pseudomonas sp. GUDBPKA501 (Isolate PKA-105/5) under Accession no. KF571704. Application of the bacteria ( Pseudomonas sp. GUDBPKA301) as aqueous suspension resulted in significant increase in growth measured in terms of height, leaf numbers and number of branches. In a recent 132 study conducted on rice (Yadav et al., 2014), combination of different PGPR including Pseudomonas spp. have proven to be more effective combination for rice production. Chakraborty et al. (2013) have reported use of native Bacillus pumilus isolated from rhizosphere of tea for growth promotion in tea, showing positive PGPR traits in vitro . In this case Pseudomonas sp. GUDBPKA301 also depicted multiple PGP traits which might have acted synergistically in promoting growth under pot conditions. These results were obtained despite existence of other soil microflora as unsterilized soil was used in potting the saplings. This is a good indication of its rhizosphere competence in som. Similar results have been obtained on plant growth promoting effects of two Bacillus strains OSU-142 (N2-fixing) and M3 (N2-fixing and phosphate solubilizing) when these were tested alone or in combinations on organically grown primo cane fruiting raspberry (cv. Heritage) plants in terms of yield, growth, nutrient composition of leaves and variation of soil nutrient element composition (Orhan et al., 2006). In another study, application of Pseudomonas fluorescens isolate Pf1 resulted in increased shoot and tuber length of Coleus in addition to biocontrol activity (Vanitha and Ramjegathesh, 2014). In all, the present study throws light into the possibility of using the isolates in enhancement of plant growth promotion in Som. Conclusive from in vitro experiments and effective in vivo results, PGPR isolate Pseudomonas sp. GUDBPKA301 stands out to be a potential bioinoculant for its use as bio-fertilizing agent in integrated practices for improving the health status of som plants which was otherwise only restricted to traditional practices such as application of cow-dung and NPK fertilizers (Unni et al., 2009). Considering that the growth stimulating effect was evidence of only 12 weeks of seedling growth, it is envisaged that further studies on plant growth promoting efficacy of Pseudomonas sp. GUDBPKA301 under field conditions, in addition to optimization of parameters related to delivery system of PGPR for successful mediation of growth promoting potential are some inevitable criterions. In addition, molecular based insight on underlying mechanisms involved in PGPR associated growth regulation in som might pave path for future research. ACKNOWLEDGEMENTS The authors are grateful to DBT, Govt. of India for funding the DBT Twinning Research Project for North East. 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Studies on the influence of host plants and effect of chemical stimulants Received for publication: December 31, 2013 Accepted for publication: April 22, 2014 View publication stats