Growth enhancement in vegetable crops by multifunctional resident plant growth promoting rhizobacteria under tropical Island Ecosystem

Plant growth promoting rhizobacteria have been used to improve crop production. A total of 114 bacterial isolates were recovered from the rhizosphere soils of healthy plantation and vegetable crops grown in Andaman Islands. These isolates were evaluated in vitro for plant growth promoting traits, hydrolytic enzyme production and antagonistic activity. Among the isolates, NPB6 and MNB1 were positive to 8 out of 10 properties tested followed by NFB3, MKP3, NNB4 and NTB2. In the dual culture assay, NFB3 showed highest inhibition against the plant pathogen Macrophomina spp (33.3%) and S. rolfsii (23%). Six most promising isolates were selected and identified as Bacillus and Pseudomonas spp. on the basis of Microbial Identification System and 16S rDNA. These isolates significantly increased the seed germination, vigor index, radical and plumule length however, the individual isolates effect varies with crop. All the six isolates enhanced the root and shoot length of brinjal, chilli and okra seedlings while Bacillus cereus (NPB6) in brinjal, B. stratosphericus (NFB3) in chilli and Pseudomonas fluorescens (NNB4) in okra were most effective. The promising isolates can be used in combination with other beneficial native microbes for plant growth promotion even under abiotic stresses and promotion of organic agriculture.


INTRODUCTION
Soil micro-organisms belonging to different groups are known to play prominent role in plant-soil interactions at the rhizosphere (Mantelin and Touraine, 2004).Among them bacteria that colonize the rhizosphere (rhizobacteria) are very important and beneficial for plant health.They are referred to as plant growth promoting bacteria (PGPB) or plant-growth promoting rhizobacteria (PGPR) (Kloepper, 1993).The principal mechanisms of growth promotion include production of growth stimulating phytohormones, solubilization and mobilization of phosphate and siderophore production whereas antibiosis, inhibition of plant ethylene synthesis and induction of plant systemic resistance against pathogens are favourable health effects (Gutierrez-Manero et al., 2001;Whipps, 2001;Idris et al., 2007;(Gutierrez-Manero et al., 2001;Whipps, 2001;Idris et al., 2007;Richardson et al., 2009).However, only 1-2% of rhizobacteria are known to promote plant growth, which are associated with many plant species and commonly present in varied environments (Antoun and Kloepper, 2001).Some of the most promising genera reported to exhibit plant growth promotion are Bacillus, Pseudomonas, Azospirillum, Azotobacter, Enterobacter and Serratia (Hurek and Reinhold-Hurek, 2003).
The interactions of rhizobacteria in the rhizosphere soil play a pivotal role in transformation, mobilization, solubilization of nutrient, from the nutrient pool in the soil and subsequent uptake by the crops.As a result, PGPR increases germination rate, root length, leaf area, chlorophyll content, protein content, nitrogen content, tolerance to drought, shoot and root weight and delayed leaf senescence (Cakmakci, 2005;Babalola et al., 2006).Therefore, soil microorganisms including PGPR play prominent role in the maintenance of soil health (Lucy et al., 2004).
A significant increase in growth and yield of cowpea and other important crops in response to seed bacterization with PGPRs have been reported by many workers (Amara and Dahdoh, 1997;Asghar et al., 2002;Minaxi et al., 2012).This considerably reduces the input cost on fertilizers and pesticides.In the tropical Islands of Andaman and Nicobar brinjal, tomato, chilli and okra are the major vegetable crops cultivated and consumed.Out of the total consumption of fertilizers and pesticides, a significant amount is used for these crops (DES, 2011).Keeping this in view, field survey was conducted to collect rhizosphere soil samples from different locations followed by in vitro and green house experiment was conducted to find out the promising native PGPR strains with multifunctional properties, which can be used for increasing the productivity of vegetable crops in these islands, in particular and other parts of the world, in general.

Sampling site
The tropical island of Andaman and Nicobar is located in the Bay of Bengal 1200 km off the eastern coast of main land India.It experiences annual mean temperature of 32°C, annual rainfall of 3180 mm and high evaporation during the summer months.The rhizosphere soil samples from vegetable and plantation crops were collected from across North  and their cropping history were recorded.The soil samples were brought to the laboratory in an icebox and processed further for bacterial isolation.

Isolation of bacteria
Bacteria from the rhizosphere soil samples were isolated using serial dilution method on King's B agar and Nutrient agar medium.This was incubated at 28C for 48 h.A total of 114 colonies were recovered on King's B agar and nutrient agar, which were purified with repeated culturing and maintained in 20% glycerol at -20C.These isolates were used to study its plant growth promoting properties and antagonistic activities.

Assessment of plant growth promoting traits
Indole 3-Acetic Acid (IAA) production by the isolates was estimated by qualitative method (Naik et al., 2008) and it was further confirmed by quantitative estimation (Benizri et al., 1998).Bacteria were cultured overnight in Luria-Bertani broth in the dark at 30C.The bacterial cells were removed from the culture medium by centrifugation at 8,000 g for 10 min. 1 ml of supernatant was mixed vigorously with 2 ml of Salkowski's reagent and incubated at room temperature in the dark for 30 min.The absorbance at 535 nm was measured and the concentration of IAA produced was estimated from a standard IAA graph and expressed as micrograms per milliliter (Patten and Glick, 1996).
Two days old pure bacterial culture grown in nutrient agar was streaked on King's B medium amended with an indicator dye.Change of blue color of the medium to yellow halo surrounding the bacterial growth indicated the production of siderophore.The reaction of each bacterial strain was scored either positive or negative to the assay (Schwyn and Neilands, 1987).
All bacterial isolates were screened for inorganic phosphate solubilization according to the method suggested by Verma et al. (2001).A loopful of fresh bacterial culture was streaked onto Pikovaskaya's medium amended with inorganic phosphate and the plates were incubated at 28±2C for 4 days.A clear halo around the bacterial colony indicated solubilization of mineral phosphate.

Analysis of extracellular enzyme activity
Bacterial isolates were analyzed for production of six enzymes viz.amylase, cellulase, chitinase, lipase, protease and pectinase by plate method.Proteolytic activities of the cultures were screened qualitatively in a medium containing skimmed milk (HiMedia, Mumbai).Zones of precipitation of paracasein around the colonies appearing over the next 48 h were taken as evidence of proteolytic activity.For cellulase activity, mineral-salt agar plate containing 0.4% (NH 4 ) 2 SO 4 , 0.6% NaCl, 0.1% K 2 HPO 4 , 0.01% MgSO 4 , 0.01% CaCl 2 with 0.5% carboxy methyl cellulose (CMC) and 2% agar (Hi-Media) were surface inoculated.Iodine solution was used to detect cellulase activity as described by Kasama et al. (2008).The clear zone formation around the growing colony was considered as positive.The lipase activity of bacterial isolates was determined according to the diffusion agar methods in which nutrient agar medium was supplemented with CaCl 2 .H 2 O 0.01%.Tween 80 was sterilized for 20 min at 121C and added to the molten agar medium at 45C to give a final concentration of 1%.The medium was shaken until the Tween 80 had dissolved completely and then poured onto Petri dishes.For positive test, an opaque halo that occurred around the colonies was considered as positive.

Antagonistic activities against plant pathogenic fungi
The antagonistic effects of bacterial isolates were tested against two fungal plant pathogens namely: Scelerotium rolfsii and Macrophomina sp.For this, the bacterial isolates were streaked at a distance of 3.5 cm from rim of individual Petri plate containing potato dextrose agar (PDA) medium.6 mm mycelial disc from a 7day old PDA culture of fungal pathogens were then placed on the other side of the Petri dish and the plates were incubated at 28C for 4 days.The percent inhibition was calculated by using the formula: Where, I is percent inhibition of mycelial growth over the control, C is mycelial growth of fungal pathogen in control plate and T is mycelial growth of fungus in bacteria inoculated plate.The experiment was carried out in three independent replicates.

Identification of the selected isolates with Biolog system
Six potential bacterial isolates were selected and tested for preliminary biochemical characterization as per standard methodologies (Collins and Lyne, 1980).The identities of the bacterial isolates were revealed on the basis of Biolog carbon source utilization.Bacterial suspensions were inoculated into Biolog GENIII Micro plates and incubated at 30C for 24 h.The results were interpreted with Biolog Micro Log TM , Release 4 software (Biolog, Hayward, CA).

16S rDNA amplification and phylogenetic analysis
Genomic DNA was extracted using the method described by Chen and Kuo (1993).The extracted DNA was dissolved in 20 µl TE buffer and used as a template for the Polymerase Chain Reaction (PCR).The PCR mix included 3 U of Taq DNA polymerase, 10X Taq buffer A, 2.5 mM concentrations of each dNTP (Bangalore GeNeI, Bangalore India), 1 µl each of pA 5'-AGAGTTTGATCCTGGCTCAG-3' and pH 5'-AAGGAGGTGATCCAGCCGCA-3' primers (Edwards et al., 1989) and 20 ng of the template DNA in a total volume of 50 µl.Amplification of 16S rDNA was performed in a Gene Amp-PCR system 9700 (Applied Biosystems) under the following conditions: 130 s of denaturation at 92C followed by 35 cycles of amplification with a 60 s denaturation at 92C, 30 s of annealing at 48C and 130 s of extension at 72C.An extra extension step of 360 s at 72C was added after completion of the 35 cycles.Amplified PCR products were sequenced and were aligned with 16S rDNA gene sequences available by the BLASTN search in the NCBI, GenBank database (http://www.ncbi.nlm.nih.gov).Phylogenetic tree was generated after performing multiple alignments (CLUSTAL W version).The method of Jukes and Cantor (1969) was used to calculate evolutionary distances, phylogenetic dendrogram was constructed by the neighbour-joining method and the tree topologies were evaluated by performing bootstrap analysis of 1,000 dataset using MEGA 3.1 software (Kumar et al., 2004).The sequence obtained in this study was deposited in the GenBank nucleotide sequence database under the accession number JX885487, JX960423-26 and JX9604230 (6 sequences).

Seed germination rate and seedling vigour index
Brinjal (CARI Brinjal-1), chilli (Cv.K2) and okra (Cv.Kashi Lalima) seeds were surface-sterilized with 70% ethanol for 2 min and in 2% sodium hypochlorite for 2 min followed by ten times washing in sterile distilled water.The surface sterilized seeds were inoculated by soaking in the respective rhizobacterial suspension (10 8 cfu/ml) for 45 min at 28 ± 2C.The coated seeds were incubated in petri dishes lined with moist filter paper and incubated at 28 ± 2C.Seeds immersed in sterilized distilled water served as control.The seed germination rate and vigor index were calculated after five days of incubation using the formula given below (Zucconi et al., 1981).

Number of germinated seed
Germination rate (%) = x100% Number of total seed tested Plume and radicle lengths were recorded for the calculation of vigor index Vigor index = (mean of plumule + radical lengths) x germination rate (%).

Soil analysis
Physico-chemical characterization of experimental soil collected from the vegetable block of Central Agricultural Research Institute, Port Blair was carried out before filling the pots.The soil pH, EC, available potassium, phosphorous and nitrogen were determined as per the standard procedure (Jackson, 1973).Briefly, pH and EC was measured both in supernatant and in the suspension using pH and Electrical Conductivity meter.The soil was medium in 0.51% organic carbon content, available N (base hydrolysable N), available phosphorus (P) (Bray 1-P, 0.03 mol L -1 NH 4 F + 0.025 mol l -1 HCl extractable), available potassium (K) (1 mol L -1 NH 4 OAc extractable) were determined.

Preparation of inoculum and seed bacterization
Pure colony of each multi trait plant growth promoting (PGP) bacteria was inoculated in 100 ml of Nutrient Broth and incubated at 30 ± 2C in an orbital incubator shaker for 3 days at 130 rpm.Seed bacterization was carried as per standard procedure of Silva et al. (2003).Three different varieties of vegetable crops viz., brinjal, chilli and okra were used in this study.Seeds of brinjal, chilli and okra were surface-sterilized as mentioned above.Approximately, 500 seeds from each vegetable crop were immersed in appropriate inoculum of PGP bacterial for 1 h at a concentration of 10 8 cfu/ml in sterile saline water (0.85%).Bacterized seeds were then air dried in laminar air flow hood and sown immediately.

Experimental design
The treatments were arranged in a Completely Randomized Block Design (CRBD) and were placed on a platform in the greenhouse.The treatments were as follows: T1, control (seeds coated with media); T2, seeds coated with NFB3; T3, NPB6; T4, MKP3; T5, MNB1; T6, NNB4 and T7-NTB2.Ten seeds of each brinjal, chilli and okra were sown in each plastic pots of 15 cm diameter containing 1 kg of sterile field soil.The seedlings were grown at a temperature of 28-32C and 85% relative humidity in a greenhouse under a day-night cycle of 11-13 h natural light.The soil was moistened to 60% water-holding capacity and was maintained at this moisture content by watering to weight every day.Uninoculated seeds sown in pots served as control.In each of the treatment, the plants were harvested 3 weeks after the emergence of the seedlings.The morphological characteristics of each plant were recorded viz.root length, shoot length and total number of secondary root of each plant.

Statistical analysis
Data of morphological characteristics of seedlings were statistically analyzed using the general linear model software Agres (3.01) and Agdata.The means were compared using the least significant difference (LSD) method at P ≤ 0.05.

Distribution of rhizobacteria
There was a significant variation in the distribution of rhizobacteria in the soils samples of different crops.The total bacterial count in Middle Andaman samples ranged from 0.6 to 77 x 10 3 cfu ml -1 and most of the samples belong to the rhizosphere of spices.In North Andaman samples, the value ranged from 1.6 to 52 x 10 3 cfu/ml wherein most of the samples were collected from the rhizosphere of vegetable crops.The number of cfu was more in coconut + vegetables with regular supply of organic manures whereas regular use of only inorganic fertilizers in rice-vegetable resulted in the reduction of microbial count.A total of 114 bacterial isolates were isolated from the rhizosphere soils from Middle and North district of Andaman and Nicobar Islands, India (Table 1).In this study, all the isolates were screened for in vitro PGP, extracellular enzyme and antagonistic activity.

Traits of rhizobacterial isolates
Out of 114 rhizobacteria, it was found that 110, 107, 67 and 59 isolates produced siderophore, ammonia, Psolubilization and IAA, respectively, whereas none of the isolates produced HCN.In addition, some isolates produced extracellular enzyme such as protease (92), amylase (73), cellulase (58), chitinase (37), lipase (24) and pectinase (14).Six most promising isolates (NFB3, NPB6, MKP3, MNB1, NNB4 and NTB2) were selected on the basis of having multi-functional properties.The PGP, extracellular enzyme and antagonistic activity of the selected isolates are shown in Table 2.All the isolates utilized a significant amount of iron in siderophore production showing a yellow zone on the CAS agar medium plate, solubilized P in the plate-based assay as evidenced by the formation of a clear halo zone around the colony and tested positive for ammonia production.All the promising isolates exhibited IAA production in qualitative assay.Further the quantitative determination revealed that bacterial strain NTB2 showed maximum significant concentration of IAA followed by NFB3, NNB4, NPB6, MNB1 and MKP3.
It was observed that five isolates except MKP3 were found positive for protease production, four isolates except NNB4 and NTB2 produced cellulase, three isolates except NFB3, NTB2 and NNB4 produced chitinase and three isolates except NFB3, NTB2 and NNB4 produced amylase.However, only one isolates (NFB3) was found positive for pectinase production.In contrast, none of them were found to be positive for lipase production.Among all NPB6 and MNB1 were positive for maximum number of hydrolytic enzymes.Antagonistic activity of the bacterial isolates was evaluated in terms of inhibition zone diameter as an indicator of the reduction in growth of pathogenic fungi.The all selected isolates exhibited most obvious antagonistic activity in vitro against the tested pathogens and showed significant growth inhibition activity against S. rolfsii and Macrophomina sp.In the dual culture assay, NFB3 showed highest inhibition against the plant pathogen Macrophomina spp.(33.3%) and S. rolfsii (23%).Among the isolates, NPB6 and MNB1 were positive to many of the properties tested followed by NFB3, MKP3, NNB4 and NTB2 in PGP and extracellular enzyme activity.

Identification of isolates using Biolog system
The selected isolates were examined for their ability to oxidize different carbon sources using Biolog's automated identification system in which all the potential isolates were revealed as Bacillus and Pseudomonas species.There were large differences in the C utilizations among the Bacillus species, which are arranged according to the order of best C sources utilized (Table 3).Among the multi-trait isolates, the dominant ones were B. cereus (3 strains) followed by Pseudomonas tolaasii (2) and one strain of B. pumilus.Among the isolates B. pumilus (NFB3) and P. tolaasii (NNB4) utilized highest numbers (55) of substrates followed by NTB2 (P.tolaasii).Three strains of Bacillus cereus named NPB6, MKP3 and MNB1 utilized 14, 15 and 22 common carbon.All the strains utilized 1% sodium lactate and sodium chloride up to 4% (w/v) have the ability to grow at pH 5.0.

Sequence analysis of partial 16SrDNA and phylogenetic analysis
About 1.5 kb fragment of 16Sr RNA gene of six selected bacterial isolates were amplified by PCR using universal primers pA and pH.The PCR products were purified and sequenced.The sequence obtained was analyzed using a BLAST search in which six bacterial strains were placed into two genus viz.Bacillus and Pseudomonas.Carbohydrates (28) Among six strains, four strains MNB1, NPB6 and MKP3 were identified as Bacillus cereus, NFB3 was Bacillus stratosphericus, NNB4 was Pseudomonas fluorescens and NTB2 was Pseudomonas simiae (Table 4).Phylogenetic analyses of the strains based on the neighbor joining method resulted into three major clusters (Figure 1).Cluster I formed with Bacillus cereus, cluster II formed with Bacillus stratosphericus and cluster III formed with P. fluorescens and P. simiae.

Effect of rhizobacteria on growth parameters of vegetables
In in vitro seed germination test of inoculated and un-inoculated seeds, the growth performance of the seedlings were measured and the results are presented in Table 5. Inoculation of rhizobacteria such as B. stratosphericus and B. cereus had resulted in significantly higher seed germination rate in brinjal and okra whereas P. simiae produced similar effect in chilli.In both cases, there was 20% higher seed germination over the control.Radical length was significantly higher due to seed inoculation of all the six selected rhizobacteria and more conspicuous effect was seen in B. cereus in okra (4.06 cm) and P. simiae in brinjal (2.11 cm) and chilli (1.03).Significantly higher plumule length of 2.43, 1.44 and 1.24 cm was observed in brinjal, chilli and okra, respectively in seed bacterization with B. cereus.Seed bacterization also  produced similar effect on the vigor index as that of radian length.P. simiae gave higher vigor index in brinjal (408%) and chilli (235%) whereas in the case of okra B. cereus performed better (510%).Six promising IAA producing isolates with multifunctional properties were tested for their influence on growth parameters on the brinjal, chilli and okra (Figures 2, 3 and 4).In brinjal, isolates NPB6 (75.04 and 58.15%), MKP3 (61.5 and 68.08%) and NTB2 (58.6 and 53.9%) have increased shoot and root length respectively, over control.In chilli, three isolates namely: B. stratosphericus-NFB3, B. cereus-MNB1 and P. simiae-NTB2 showed increased shoot (37.4,32.2 and 39.4%) and root length (52.6, 42.8 and 38.4%) over untreated controls.In okra, P. fluorescens-NNB4, B. cereus -MKP3 and B. cereus-NPB6 showed significant plant growth promotion with respect to increase in root and shoot length.There was no clear trend in increasing secondary root number by all the isolates.It was found that seed bacterisation of P. simiae-NTB2 significantly increased the number of secondary root in chilli followed by NPB6 in brinjal and NNB4 in okra over control.Among theisolates, the overall performance of B. cereus -NPB6 for root length in brinjal, P. fluorescens -NNB4 for shoot length in okra and P. simiae-NTB2 for secondary root number in chilli were highly significant.

DISCUSSION
The PGPR isolates promote plant growth and induce resistance in different crops (Podile and Kishore, 2006).PGPR or potential biological control strains isolated from one region may not produce similar results in other soils  and climatic conditions as in the case of its original habitat (Duffy et al., 1997).Therefore, isolation of resident microbial strains and their utilization constitute the best alternate strategy.This is more pertinent to Andaman and Nicobar Islands, which is a repository of biodiversity by virtue of its nature and location.In the present study, six potential PGPR isolates were selected on the basis of their performance with multi-functional properties such as production of IAA, siderophore, ammonia, P-Solubilization, extracellular enzyme and antagonistic activity.Among the bacterial isolates, P. simiae (NTB2) showed significant concentration of IAA followed by B. stratosphericus (NFB3), P. fluorescens (NNB4), B. cereus (NPB6), B. cereus (MNB1) and B. cereus (MKP3) sugges-ting that these   isolates could be used for plant growth promotion.A higher amount of IAA was produced in the presence of Ltryptophan by P. simiae (NTB2) because L-tryptophan acts as suitable precursor for IAA biosyn-thesis.Similarly, higher level of IAA production in the presence of Ltryptophan by Pseudomonas and Bacillus sp. was reported by Xie et al. (1996) and Srinivasan et al. (1996).
All isolates showed solubilization zone on Pikovskaya medium, indicating its potential role as a P-solubilizer.The phosphate solubilization by the isolate could increase the availability of phosphorous in the rhizospheric region.It is a well established fact that improved phosphorous nutrition results in the overall plant growth and root development (Jones and Darrah, 1994).All the selected isolates utilized a significant amount of iron by siderophore production, which is indicative of their ability to suppress fungal pathogens in the rhizosphere by chelating iron.It also exhibited production of ammonia, which could be taken up by plants as a source of nitrogen for their growth (Ahmad et al., 2008).
It was also shown that rhizobacteria inhibits phytopathogens by producing chitinase, β-1, 3-glucanase and pectinase, which degrade the fungal cell wall (Friedlander et al., 1993;Bloemberg and Lugtenberg, 2001;Persello-Cartieaux et al., 2003).In the present study five isolates were found positive for protease production followed by four isolates for cellulase, three each for chitinase and amylase and only one isolate produced pectinase whereas, none of the isolates produced lipase.This could be a principal reason for the observed antagonistic activity against S. rolfsii and Macrophomina sp.Characterization of the isolates on the basis of carbon source utilization in Biolog system revealed that they are grampositive Bacillus and gram-negative Pseudomonas sp.Compared to other bacterial groups, Bacillus group were found to have the ability to survive in hot humid climate of Andaman and Nicobar Islands because of its ubiquitous nature like multilayered cell wall, stress resistant, endospore formation, secretion of peptide antibiotics, peptide signal molecules and extracellular enzyme productions.Among the identified isolates, three isolates of Bacillus sp.followed by two isolates of Pseudomonas sp. were found to exhibit multi-trait properties.There were large differences in the C-utilizations among the Bacillus sp., which are arranged according to the order of best C sources utilized.The data obtained showed a greater abundance of Gram-positive bacteria in the tropical soils of Andaman and Nicobar Islands.This was in agreement with the previous studies (Garbeva et al., 2003;Rau et al., 2009;Kumar et al., 2011;Amaresan et al., 2012) that showed a higher level of Gram-positive Bacillus and Paenibacillus species in the rhizosphere soils of cultivated vegetable crops and wild grass.GEN III microplates contain some substrates that commonly occur in root exudates, which represent a primary source of carbon and energy and are likely to favor fast-growing microbes in the rhizosphere (Alisi et al., 2005).Thus, the ability to metabolize root exudates at high rates has been often related to the colonization of roots by bacteria (Baudoin et al., 2003).
Bacteria are known to produce different metabolites like IAA, gibberellins and cytokinin like substances, which can exert positive effect on seed germination and radicle length (Tien et al., 1979).In the present study, under laboratory conditions, seed treatment with the test strain improved seed germination and seedling emergence of brinjal, okra and chilli over the control, which might be due to the production of IAA by the rhizobacteria (Mirza et al., 2001).Similar improvement of seed germination parameters by rhizobacteria was reported in rice seedlings, cowpea seedlings (Minaxi et al., 2011) and sorghum (Raju et al., 1999) due to increased synthesis of gibberellins, which triggered the activity of specific enzymes such as α-amylase that promoted early germination by increasing the availability of starch assimilation.
IAA-producing bacteria are known to promote root elongation and plant growth (Patten and Glick, 2002).The significant increase on the root and shoot length of brinjal, chilli and okra crops could be attributed to the production of growth promoting substances by the inoculated bacteria that carry out an important role in the stem expansion process (Burd et al., 2000).In a similar study, Adesemoye et al. (2008) reported that inoculation of tomato, okra and African spinach with Bacillus subtilis and P. aeruginosa enhanced percent emergence and growth of seedlings.In addition, the isolates significantly increased the secondary roots production prominently.B. stratosphericus (NFB3) showed increase in secondary roots in all the crops.As these isolates were observed to produce significant amount of IAA, which positively influenced the root growth and development thereby enhancing nutrient uptake (Khalid et al., 2004).In addition, healthy plants may exhibit greater resistance to pathogens than that which are under stress.Among the isolates, B. cereus (NPB6) in brinjal, B. stratosphericus (NFB3) in chilli and P. fluorescens (NNB4) in okra significantly improved the growth parameters.
These IAA producing multi-functional Bacillus and Pseudomonas sp. with broad range of carbon sources has the potential to be used as bio-inoculants to attain the desired plant growth promotion in brinjal, okra and chilli seedling.Also, it showed the usefulness of PGPRinoculated treatment for improving crop productivity of tropical crops growing in hot humid climate of Andaman and Nicobar Islands, which help to minimize the uptake of fertilizers, reduce environmental pollution and promote sustainable agriculture.These isolates having the property to tolerate 8% NaCl stress and pH 5, can also be used as a bio-inoculants in alkaline and acidic agriculture soil conditions of Andaman and Nicobar Islands.

Figure 1 .
Figure 1.Neighbour-joining phylogenetic tree-based 16S rDNA sequences and their closest phylogenetic neighbours.Bootstrap values are indicated at nodes.Scale bar represents observed number of changes per nucleotide position.

Figure 2 .
Figure 2. Inoculation effect of PGPR strains on shoot length of seedlings after one month.

Figure 3 .
Figure 3. Inoculation effect of PGPR strains on root length of seedlings after one month.

Figure 4 .
Figure 4. Inoculation effect of PGPR strains on secondary root numbers seedlings after one month.

Table 1 .
Effect of crop and cropping history on the CFU in rhizosphere soil samples.

Table 2 .
Determination of substrates utilization as carbon sources by selected strains.

Table 3 .
Characterization of selected bacterial isolates for plant growth promoting and antagonistic traits.

Table 4 .
Identification of bacterial isolates using carbon source utilization and 16SrDNA partial sequences.