Microsoft Word - Paper 5 Patel.docx Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  37  –  52                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   37 ORIGINAL RESEARCH ARTICLE Isolation  and  Characterization  of  Bacterial  Endophytes  From  Lycopersicon  esculentum   Plant  and  Their  Plant  Growth  Promoting  Characteristics     Hardik A. Patel1, Rajesh K. Patel1*, Sunil M. Khristi1, Kruti Parikh1, Geetha Rajendran2 1 Ashok & Rita Patel Institute of Integrated Study and Research in Biotechnology and Allied Sciences (ARIBAS), New Vallabh Vidyanagar, Gujarat-388121, INDIA 2 Sultan Qaboos University, Muscat, Department of Biology, Sultanate of Oman Corresponding author: rkpatel46@yahoo.com Abstract The study was designed to isolate and characterize bacterial endophytes from root and stem of Lycopersicon esculentum plant which was collected form different region of Gujarat. Total 18 isolates of endophytic bacteria were selected in which, all the endophytic bacteria produced one or the other different characteristics involved in plant growth promotion. They either produced phytohormones like indole acetic acid, siderophore, protease, pectinase, organic acid showed antifungal activity, chromium tolerance and solubilized phosphate. Four of the strains among the 18 showed maximum positive results of plant growth promoting regulators (PGPR) test and among them best probable isolate was selected and thus its 16SrDNA was amplified and sequenced. Only HR7 endophyte of tomato turned out to be Pseudomonas aeruginosa. It’s a gram negative coccobacili, sporeforming motile bacilli and show maximum PGPR activity. The results of our present studies indicated that above strains might be endophytic and therefore, were associated with the plant growth. Keywords: Lycopersicon esculentum, endophytic bacteria, PGPR, IAA, 16SrDNA Introduction There are many endophytic and epiphytic bacteria are directly or indirectly involved in plant growth and development. Endophytic bacteria live in plant tissues without causing substantive harm to the host or gaining any benefit other than a noncompetitive environment inside the host. It has recently been demonstrated that bacterial endophytes may also have beneficial effects on host plants, such as growth promotion and biological control of pathogens [10, 25, 28]. Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  37  –  52                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   38 Some studies have indicated that the plant growth-promoting potential of endophytes is higher than that of rhizosphere microbes [23,31], but the role of bacterial endophytes in plant growth are not yet fully understood. Most of these microorganisms are not pathogenic to the host plant. Moreover, the association between the plant and its endophytes is very often mutualistic. In 1926, endophytic growth was recognized as a particular stage in the life of bacteria, described as an advanced stage if infection and as having a close relationship with mutualistic symbiosis [22]. Since then, endophytes have been defined as microorganisms that could be isolated form surface-sterilized plant organ [15]. Although the presence of bacterial endophytes in plants is variable and, occasionally transient [32], they are also often capable of eliciting drastic physiological changes that modulate the growth and development in the plant [8]. The utilization of endophytic and epiphytic bacteria in agriculture production depends on our knowledge of the bacteria-plant interaction and our ability to maintain, manipulate and modify beneficial bacteria population under field condition [14]. Many PGPRs are known to promote plant growth by a variety of mechanisms: fixation of atmospheric nitrogen that is transferred to the plant, production of siderophores that chelate iron and make it available to the plant root, solubilization of minerals such as phosphorus, and synthesis of phytohormones [12]. PGPR have been reported to directly enhance plant growth by the production of plant growth regulators, and improvements in plant nutrient uptake [12,16] or indirectly by the production of metabolites like antibiotics, siderophores etc that decrease the growth of phytopathogens [12]. PGPRs can be of two different types when associated with host tissue that is endophytes or epiphytes; otherwise they can be even rhizospheric bacteria that are present in the root adhering soil. The aim of the present study was to isolate and characterize the endophytic bacteria associated to root and stem part of Lycopersicon esculentum, to evaluate different characteristics involved in plant growth promotion. Result revealed that four of the strains showed maximum positive results of PGPR test and its 16SrDNA was amplified and sequenced. Materials and methods Isolation of endophytic bacteria form Lycopersicon esculentum Endophytes strains were isolated from root and stem of Lycopersicon esculentum plant (Table 1). Roots and stem part of plant were thoroughly Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  37  –  52                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   39 washed with sterile n-saline (0.85%) and cut down in 1 cm long pieces through sterile forceps with the help of alcohol. The pieces were transferred to sterile N-agar plate and incubated for 24 hrs at 300C. Morphological and physiological characterization of endophytic isolates For the present study, total 18 endophytic bacteria were isolated whose systemic morphological characters done which includes: Size, Shape, Margin, Elevation, Consistency, Opacity, Pigmentation, was done by Systematic Microbiology [3]. Gram’s staining bacterial suspension was prepared in sterile distilled water and from this suspension a smear was prepared on clean & dry glass slide, air dried and then heat fixed. The smear was treated with 1% crystal violet for 1-2 min. Gram’s iodine was applied for 30 sec. to 1 min. Smears were then decolorized with 10% alcohol. The counter stain, saffranin was then applied for 45-60 seconds. The stained slide were washed with tap water, air dried & observed under oil immulsion. For motility test, the culture was inoculated into the Edward’s and Ewing motility agar stab medium by stabbing the medium right into the center of agar. The entire depth of the medium was punctured. The medium was incubated at 28 ± 2ºC for 24 hrs. After incubation, it was observed for the turbid growth across the line of inoculation, which indicates motile organisms. For Antibiotic assay, top agar (1.5%) was prepared and autoclaved. It was cooled to 45º C, 100µl of culture was added to this and overlaid preset N-agar plates. Using sterile forceps, disc containing the antibiotic of interest was placed on the agar and incubated at 28±2ºC for 48 hrs. Estimation of plant growth promoting properties (i) Detection of siderophore production: This was performed by a method described by Schwyn and Neilands [27], which involved the use of chrome azurol S containing indicator plates. Siderophore detection was performed by mixing equal volumes of chrome azurol S (CAS) assay solution and the culture supernatant. Colour change from blue to yellowish orange was indicative of presence of Siderophore. Two percentage of overnight grown culture was inoculated in magnetotactic bacterium Magnetospirillum magneticum AMB-1 (AMB) and grown for 48 hrs. Then the culture was centrifuge at 8000 rpm for 20 min and the supernatant was examined for the presence of siderophore by CAS solution. Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  37  –  52                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   40 (ii) Evaluation of endophytes for chromium tolerance: Isolated strains were tested for resistance to Cr (VI) by plate dilution method using yeast extract mannitol agar (YEMA) medium. In a plate dilution method, agar plates amended with K2Cr2O7 at 50-500 µg/ml were inoculated with 48 hrs grown cultures and incubated at 28 ± 2ºC for 72hrs. The lowest concentration of Cr (VI) inhibiting on YEMA plates was defined as minimum inhibitory concentration [35]. (iii) Phosphate solubilization ability: The phosphate solubilizing ability of the cultures were examined by growing the cultures on Pikovskaya’s agar plate and looking for the zone of clearance after incubating at 28 ± 2°C for 48- 72h. (iv) Antifungal activity: The spores of fungal cultures (Fusarium oxysporium, Alternaria,, Trichoderma and Rhizoctonia solani) grown on Potato dextrose agar (PDA) blocks were placed in the centre of PDA plates and the bacterial cultures were streaked at four ends of the plate. This was incubated at 28 ± 2°C for 48-96 hrs and examined for zone of growth inhibition. (v) Protease production: It is indicated by casein degradation, which was determined by observing clearing zones in Nutrient casein agar plate. All isolated culture was streak on Nutrient casein agar plate and incubated at 28 ± 2°C for 24-48 hrs. Next day flood the plate with Frazier’s reagent to detect clear zone around the colony. (vi) Indole 3-acetic acid (IAA) production test: IAA in presence of FeCl3 develops pink color. This fact is utilized in determination of IAA. Different mineral acids like hydrochloric acid, perchloric acid, phosphoric acid, nitric acid and sulphuric acid can be used to develop the color. FeCl3 –HClO4 reagent is the most sensitive and shows least interference by other iodole compounds like, tryptophan, skatole, acetyletryptamine etc. Loopful of each culture was inoculated in luria broth (LB) 2ml containing 50µg/ml tryptophan and incubated at 28oC for 24 hrs on shaking condition, centrifuged at 9000 rpm for 15min, 2ml of supernatant was taken in fresh tube and 2-3 drops of orthophosphoric acid was added. A quantity of 4ml of reagent (1ml of 0.5 M FeCl3 in 50 ml of 35% HClO4) was added to this aliquot and incubated for 25 min at RT. Absorbance was measured at 530 nm. Auxin quantification values were recorded by preparing standard calibration curve made by using IAA standard in the range of 10-100 µg/ml. IAA stock solution was prepared as 100 µg/ml in 50% ethanol. Standard graph of IAA concentration was plotted against O.D 530 and Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  37  –  52                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   41 the concentration of IAA in samples used was calculated. Organic acid production: It was studied by growing the cultures in Calcium carbonate agar plate and observing for a clear zone around the colony. Chitinase production: It was observed by spotting the culture on chitin agar plate and observing zone of clearance after incubating at 28 ± 2°C for 48- 72h. Chitinase activity (degradation of β- 1,4- N- acetylglucosamine polumer) were tested in a minimal medium. There were clear zones were detected after 5 days incubation period at 30°C. Pectinase production: It was detected by spotting the culture on pectin agar plate and observing zone of clearance after incubating at 28 ± 2°C for 48- 72h. 16S-rDNA sequencing of PGPR isolates Well isolated colonies (2-3 colonies) of the culture whose 16S-rDNA had to be amplified were suspended in 20µl-30µl of sterile distilled water. The suspension was heated at 95ºC for 20 min and centrifuged at 9000 rpm for 1min. The supernatant was used as template DNA in the PCR system [26]. The 16S-rDNA gene fragment was amplified using universal eubacterial full- length primers. The amount of DNA taken for amplification was 10ng. Primer sequences Forward Primer (PF) 5’ AGA GTT TGA TCC TGG CTC AG 3’ Reverse Primer (PR 5’ ACG GCT ACC TTG TTA CGA CTT 3’ The PCR components and conditions (to set a system of 30 µl) used for amplification. Amplifications were performed in Eppendroff gradient thermal cyclers programmed for 30cycles. The PCR thermal cycle consist of an initial denaturation step of 3 min at 94°C, Then 30 sec at 94°C for denaturation, 30 sec at 57°C for primer annealing and in last step primer extension done by 2 min at 72°C. Steps 2, 3, 4 repeated for 30 cycles followed by a final extension of 10 min at 72°C. The amplified products were then examined by an aliquot of the DNA (2µl) was analyzed on a 1.0 % agarose gel along with 500bp ladder and stained with ethidium bromide (0.5µg/ml). The gels were visualized under UV light in a transilluminator and photographed subsequently. Sequence Analysis: The product was sequenced and matched with the already available sequences in the Gene Bank by uploading the obtained sequence in its FASTA format in nucleotide sequence match available at the online tool of RDP Database Project II. Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  37  –  52                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   42 Results and discussion Isolation of endophytic bacteria We have isolated endophytic bacteria from the Lycopersicon esculentum (tomato) plants from different field areas on the Nutrient agar (NA) medium. Colonies showing different morphological characteristics on the Nutrient agar plates were selected for further characterization. About 18 strains were isolated. The number of isolates, the source of their plant and field from where the samples were procured are mentioned in the Table 1. Table1: Bacterial endophytes isolates form Lycopersicon esculentum Sample Location No. of isolate s Name of the isolates 1 AAU (Anand) 5 HR 1 HR 2 HR 3 HR 4 HR 5 2 Mansa (Gandhina gar) 7 HR 6 HR 7 HR 8 HR 9 HR 10 HR 11 HR 12 3 Gana (Anand) 6 HR 13 HR 14 HR 15 HR 16 HR 17 HR 18 Morphological and physiological Characterization In this work all the 18 isolates strains were picked on the basis of different morphological characteristics. The morphological characteristics of the final four short-listed isolates are shown in Table 2. Gram’s staining and Motility Result showed that out of 18 isolates tested 9 were gram negative coccobacilli, 5 were gram positive bacilli and only 4 were gram negative cocci. This indicated that majority (50%) of the bacteria in our studies belonged to gram negative coccobacilli strains followed by 22.22% gram positive bacilli and gram negative cocci seemed to be the most uncommon one constituting only 27.77% of the total isolates. While in case of motility 66.66% were motile and remaining 43.44 % were non-motile (Table 3). Antibiotic assay The endophytic isolates were also checked for their sensitivity (S) and resistance (R) against antibiotics like Ampicillin, Gentamycin, Spectinomycin, Tetracycline. The result of the antibiotic assay of the rhizospheric isolates is tabulated (Table 4). Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  37  –  52                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   43 Table 2: Morphologyical and Physiological characteristics of 18 isolates Colony character Size Shape Margin Elevation Texture Opacity Pigmentation HR1 Medium Round Entire Raised Smooth Transparent No pigmentation HR2 Medium Round Entire Flat Smooth Transparent No pigmentation HR3 Small Round Entire Slightly Raised Smooth Transparent Yellow pigmentation HR4 Small Round Entire Slightly Raised Smooth Opaque Yellow pigmentation HR5 Small Round Entire Flat Rough Transparent No pigmentation HR6 Small Round Entire Slightly Raised Smooth Transparent Yellow pigmentation HR7 Medium Round Entire Flat Smooth Opaque Pitch pigmentation HR8 Medium Round Entire Raised Smooth Opaque Yellow pigmentation HR9 Medium Irregular Irregular Flat Rough Opaque White pigmentation HR10 Medium Round Entire Flat Smooth Transparent Yellow pigmentation HR11 Small Round Entire Raised Smooth Transparent Yellow pigmentation HR12 Medium Irregular Irregular Flat Rough Opaque White pigmentation HR13 Small Round Entire Flat Rough Transparent No pigmentation HR14 Medium Round Entire Flat Smooth Transparent Yellow pigmentation HR15 Medium Irregular Irregular Flat Rough Opaque White pigmentation HR16 Medium Round Entire Flat Smooth Transparent Yellow pigmentation HR17 Medium Irregular Irregular Raised Rough Transparent Golden yellow pigmentation HR18 Small Irregular Irregular Flat Rough Opaque White pigmentation Siderophore production Assay of siderophore production performed by CAS agar plate method in which following isolates HR1, HR3, HR4, HR7, HR18 showed production of siderophore. So further estimation of siderophore was performed to determine which types of siderophores are produced, either cathecolate or hydroxymates type of sideophore. Unfortunately we could not obtain the result. IAA production test All the isolates were tested for their IAA production. After 24 hrs of incubation with tryptophan all the strains exhibited a significant amount of IAA production. The production of IAA by isolates indicated that the tested strains Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  37  –  52                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   44 Table 3: The Gram nature and Motility of the 18 isolated strains. Isolates from Tomato plant Gram’s Nature Motility HR 1 Gram –ve cocco bacilli + HR 2 Gram –ve cocco bacilli + HR 3 Gram –ve cocco bacilli + HR 4 Gram –ve cocco bacilli + HR 5 Gram –ve cocco bacilli + HR 6 Gram –ve cocco bacilli + HR 7 Gram –ve cocco bacilli + HR 8 Gram –ve cocco bacilli + HR 9 Gram -ve cocci - HR 10 Gram -ve cocci - HR 11 Gram +ve bacilli + HR 12 Gram +ve bacilli - HR 13 Gram +ve bacilli + HR 14 Gram +ve bacilli - HR 15 Gram +ve bacilli - HR 16 Gram -ve cocci + HR 17 Gram –ve cocco bacilli + HR 18 Gram –ve cocco bacilli + + : Indicates motile organism, - : Indicates non-motile organism. utilized tryptophan as a precursor for growth and produced IAA, the primary auxins in the majority of plant species as a plant growth promoter. Data indicated that all the bacterial endophytes from plant were able to produce IAA in the presence of tryptophan (Table 5). Production of IAA is widespread among bacteria-plant associated. Several bacteria having the ability to anabolise indole-3-acetic acid (IAA) with supplemented L- tryptophan have been isolated from the plant surfaces. Bacterial IAA producers (BIPs) have the potential to interfere with any of these processes by input of IAA into the plant's auxin pool [1]. Patten and Glick [20,21] have shown that bacterial IAA stimulates the development of the root system of the host plant and Brandi and Lindow (1998) have studied the contribution of IAA for bacterial epiphytic fitness, observation supported by the investigation of other workers [12,20,2,9,33]. Chromium tolerance of the endophytic strains Almost 17 out of 18 isolates from Lycopersicon esculentum tolerated a chromium concentration upto 500µg/ml. One of the isolate HR11 tolerated upto 300µg/ml, wheres all the isolates showing tolerance above 450µg/ml. There are reports of certain Bacillus spp. tolerating upto Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  37  –  52                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   45 Table 4: Antibiotic assay of isolated strains Isolates Ampicillin Streptomycin Tetracycline Chloramphenicol HR1 10 16 18 23 HR2 R 19 13 22 HR3 14 21 19 24 HR4 R 9 15 R HR5 R 17 R 14 HR6 R 9 R 13 HR7 R 18 R 17 HR8 R 11 R 8 HR9 28 29 27 38 HR10 R R 7 19 HR11 7 16 18 21 HR12 R 13 11 19 HR13 R R 9 16 HR14 13 21 15 14 HR15 8 R 11 14 HR16 R R 8 18 HR17 R 12 R R HR18 21 20 19 24 Resistance microorganism- R, Number mentioned is zone of inhibition in mm 550 µg/ml [35] and Bacilli spp. is a well known PGPR strain. All the standard strains except R. leguminosarum and S. meliloti showed very less tolerance to chromium. Both the strains R. leguminosarum and S. meliloti are well known for their PGPR activity in leguminous plants. A Rhodococcus erythropolis MTCC 7905 strain has been shown to be resistant to 300 mg l-1 of Cr6+ isolated from metal contaminated soil samples from a site near Indian Himalayan region has been reported to reduce substantial amounts of Cr6+ to Cr3+ as well as showed to have plant growth promotion of pea (Pisum sativum) in the presence of toxic Cr6+ concentration [30]. Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  37  –  52                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   46 Table 5: Indole Acetic Acid production by endophytic bacterial isolates Isolates OD at 530nm HR1 0.061 HR2 0.020 HR3 0.050 HR4 0.241 HR5 0.199 HR6 0.067 HR7 0.057 HR8 0.114 HR9 0.056 HR10 0.097 HR11 0.181 HR12 0.007 HR13 0.029 HR14 0.270 HR15 0.112 HR16 0.094 HR17 0.166 HR18 0.079 Phosphate solubilisation Phosphorous is one of the most important plant nutrient and a large portion of inorganic phosphate applied to soil as fertilizer is rapidly immobilized [19,24]. Endophytic bacteria possess the capacity to solubilize immobilized mineral phosphates. In this study all the 18 isolates were tested for their phosphate solubilizing activity on Pikovasky agar medium. It was interesting to note that out of 18 endophytic isolates, 8 showed phosphate solubilisation activity (Table 6). Result revealed that majority of the PGPR strains do have phosphate solubilizing activity and such organisms play a major role in plant growth promotion [24]. Table 6: Phosphate Solubilization by endophytic bacterial isolates Isolate No. Growth on PV Zone (mm) HR 1 Full growth 20 HR 2 Full growth - HR 3 Full growth 21 HR 4 No growth - HR 5 No growth - HR 6 No growth - HR 7 Full growth 17 HR 8 No growth - HR 9 No growth 21 HR10 No growth - HR 11 Less growth 8 HR 12 Less growth 11 HR 13 No growth - HR 14 No growth - HR 15 No growth - HR 16 No growth - HR17 Full growth 31 HR 18 Less growth 9 mm zone of clearance (Pink colour Zone) Organic acid production Out of the 8 endophytic isolates showing phosphate solubilization, all 8 showed organic acid production. The isolates number HR7, HR8, HR9, HR10, HR14, HR15 showed slight organic acid production by forming a very thin zone of clearance on the plates of Pikovasky with methyl red as pH indicator dye. This gave the pink coloured zone that indicated shift in pH change from alkaline to acidic. Some isolates like HR4, HR7, HR13, HR16 were unable to solubilize phosphate and also did not produce organic acid. This could be because the amount Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  37  –  52                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   47 of organic acid produced might be very less to do so (Table 7). Chitinase production In the present study none of the strain revealed a clear zone, but 5 isolates out of 18 showed growth on the chitin agar plate, remaining 13 strains did not show any growth (Table 8). Biological control of plant pests and diseases is much more attractive than chemical treatment methods due to its greater specificity and less harmful impact on the environment [34,35]. Major component of fungal cell is chitin. Thus organism having the ability to produce chitinase might have antifungal property. Pectinase production For pectinase production, 17 out of 18 isolates of Lycopersicon esculentum revealed the production of pectinase. The strains showing Table 7: Isolates showing organic acid production Sample No. Isolate No. Production of Organic acid Zone (mm) Pink colour of Zone Sample 1. HR 1 Medium 13 +++ HR 2 Medium 10 ++ HR 3 Very less 8 ++ HR 4 No - - HR 5 No - - Sample 2. HR 6 Very less 7 - HR 7 Medium 11 ++ HR 8 Medium 13 - HR 9 Medium 10 - HR 10 Very less 9 - HR 11 Medium 12 + HR 12 Very less 8 ++ HR 13 No - - Sample 3. HR 14 Medium 11 - HR 15 Very less 8 - HR 16 No - - HR 17 Medium 11 +++ HR 18 Medium 13 + + : 1.0 mm ZOC (zone of clearance), ++: 1.2 mm ZOC, +++: 1.4 mm ZOC, -: No zone production of pectinase on pectin agar plate are listed in Table 9. Maximum research indicated that pectin methyl esterase (PME) (EC 3.1.1.11) catalyzes the hydrolysis of methyl-ester groups of cell wall pectins. It has been found in all plant tissues and in some of plant cell wall-degrading microorganisms or insects [5,6] and has been implicated in a number of processes including cell growth [18], fruit ripening [11, 29], abscission and senescence [17], pathogenesis [7] and cambial cell differentiation [13]. Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  37  –  52                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   48 Table 8: Isolates showing Chitinase production Isolates Chitinase Production HR1 - HR2 + HR3 - HR4 + HR5 - HR6 - HR7 + HR8 - HR9 - HR10 - HR11 - HR12 - HR13 - HR14 + HR15 - HR16 - HR17 - HR18 + + : positive - : negative Table 9: Isolates showing Pectinase production Isolates Pectinase production HR1 + HR2 + HR3 + HR4 + HR5 + HR6 + HR7 + HR8 + HR9 + HR10 + HR11 + HR12 + HR13 + HR14 + HR15 + HR16 - HR17 + HR18 + + : positive - : negative 16S-rDNA sequencing of PGPR isolates Colony PCR All the plant growth promoting results when compiled together showed one strain (HR7) showed maximum positive features and thus the 16SrDNA of the strain was amplified using universal full length primers. An amplicon of 1.5kb was obtained and sent for sequencing to Bangalore Genei, Pvt, Ltd India. The sequence obtained was matched with the online available sequences in RDP (Ribosomal Database Project II) bioinformatics tool. Multiple sequence alignment phylogenetic analysis BLAST (Basic local alignment search tool) search was done for partial 16s rDNA of the isolates HR7 by submitting queries to NCBI- BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi.) and homologous sequences obtained by standard nucleotide-nucleotide BLAST (blastn) were aligned with the different 16s rDNA isolates after sequencing and various related sequence were retrieve after blasting the partial sequence of the isolates obtained after sequencing. Accession No. of the related species was retieved and Multiple sequence alignment (Fig 1) was performed using CLC free protein workbench 5.0. Evolutionary tree for the same Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  37  –  52                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   49 data was obtained by neighbor joining method with Bootstrap values (expressed as percentages of 100 replications) as shown in (Fig 2). Except HR7 other do not give the sequencing results. Accession no. of some isolates used for multiple alignment with HARDIKSEQ-1(HR7) isolate were, JF423918, JF281099, HQ995502, HQ268732, HQ202541, HQ202540, HQ259948, FM995816, FM995815, FM995811, FM995802, FM995800, FM995798, FM995797, FM995796. Fig. 1: Multiple sequence alignment for the partial 16s rDNA sequence of hardik seq-1 (HR7) isolate with other related species retrieve after BLAST, resulted in versatile coloring scheme that highlighted the conserved sequence in Aligned sequences. Fig. 2: Phylogenetic tree of partial 16S rRNA genes of Hardik seq-1(HR7) islolates from closely related of resistant bacteria obtained after BLAST. The tree was constructed based on partial 16S rRNA sequences of the isolates and the reference strains. Bootstrap values (expressed as percentages of 100 replications) are shown at branch points. Bootstrap values over 50% are shown. The scale bar 0.500 indicates 50% nucleotide sequence substitution Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  37  –  52                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   50 Precisely, the research concluded that endophytic bacteria isolated form Lycopersicon esculentum produced one or the other different characteristics involved in plant growth promotion. 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