characterize microorganisms from the rhizosphere soil of. 0.5 ml of FCR was ... manual of determinative bacteriology. The identified ..... Subba Rao, N.S., 1982.
Advances in Biological Research 9 (4): 230-235, 2015 ISSN 1992-0067 © IDOSI Publications, 2015 DOI: 10.5829/idosi.abr.2015.9.4.1115
Efficiency of Plant Growth Promoting Rhizobacteria for the Enhancement of Sesbania grandiflora and Moringa oleifera Growth 1
B. Harinathan, 1S. Sankaralingam, 2T. Shankar and 2D. Prabhu Saraswathi Narayanan College, Madurai, Tamilnadu, India Ayya Nadar Janaki Ammal College, Sivakasi, Tamilnadu, India 1
2
Abstract: In this study, eight phosphate solubilizing bacteria from rhizosphere soil sample of local weed plants were isolated in Senbagathoppu hills at Srivilliputtur, Tamilnadu, India. Among the eight phosphate solubilizing bacterial strains, only one bacterium was finally selected for further experimental analysis due to high amount of phosphate solubilizing capacity. The selected bacterial strain was indentified through biochemical and 16S rRNA gene sequencing. The identified strain was inoculated in two crop plants to find out the growth and development of Agathi and Drumstick. The plant growth parameters were analyzed from the two crop plants by the inoculation of potential strain isolated from weed plants. The estimation of carotenoids, protein, phenol, catalase, peroxidase, nitrate reductase, gibberellic acid, siderophore production were done in the PSB inoculated and control plantlets. The inoculation of PSB in Agathi (Sesbania grandiflora) and Drumstick (Moringa oleifera) proved the potential to increase carotenoids, protein, phenol, catalase, peroxidase, nitrate reductase, gibberellic acid and siderophore production. Key words: Phosphate solubilizing bacteria
Sesbania grandiflora and Moringa oleifera
INTRODUCTION
Burkholderia, Bacillus, Enterobacter, Rhizobium, Erwinia, Serratia, Alcaligenes, Arthrobacter, Acinetobacter and Flavobacterium. Bacterial inoculants have been used to increase plant yields in several countries and commercial products are currently available. For example, in several biofertilizers are commercially produced and employed with different crops, mostly using strains of Azotobacter, Rhizobium, Azospirillum and Burkholderia. The mechanisms by which PGPR can exert a positive effect on plant growth can be of two types: direct and indirect [4]. Indirect growth promotion is the decrease or prevention of deleterious effect of pathogenic microorganisms, mostly due to the synthesis of antibiotics [5], or production of siderophores [6] by the bacteria. Direct growth promotion can be through the synthesis of phytohormones [7]. N 2 fixation [8], reduction of membrane potential of the roots [4], synthesis of some enzymes (such as ACC deaminase) that modulate the level of plant hormones [2]. As well as the solubilization of inorganic phosphate and mineralization of organic phosphate, which makes phosphorous available to the plants.
Phosphorus (P) is an essential element for plant growth and development making up about 0.2% of plant dry weight. After nitrogen, phosphorus is the second most important element, plays a significant role in plant metabolism by supplying energy required for metabolic processes. Plants acquire phosphorus from soil solution as phosphate anions. However, phosphate anions are extremely reactive and may be immobilized through precipitation with cations such as Ca2+, Mg2+, Fe3+ and Al3+ depending on the properties of soil. In these forms, phosphorus is highly insoluble and unavailable to plants [1]. It is well known that a considerable number of bacterial species, mostly those associated with the plant rhizosphere, are able to exert a beneficial effect upon plant growth. Therefore, their use as biofertilizers or control agents for agriculture improvement has been a focus of numerous researchers for a number of years [2]. This group of bacteria has been termed ‘plant growth promoting rhizobacteria’ (PGPR) [3] and among them are strains from genera such as Pseudomonas, Azospirillum,
Corresponding Author: S. Sankaralingam, S.N. College, M.K. University, Madurai, Tamilnadu, India.
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The objective of the present study is to isolate and characterize microorganisms from the rhizosphere soil of local plants; and, to find out its role in mechanism of phosphate solubilization and production of plant growth promoting substances by these microorganisms. MATERIALS AND METHODS Isolation and Screening: The rhizosphere soil samples were collected and transferred under aseptic conditions to laboratory. The soil sample was spread on modified Pikovskaya agar plates for phosphate solubilization assay as described by [9]. Bacterial colonies forming halo zones were considered to be phosphate solubilizers. Then it was identified by standard procedures described in Bergy’s manual of determinative bacteriology. The identified organism was utilized for further experimental analysis. Siderophore Production: PSB isolates were assayed for siderophore production on the chrome azurole S agar (CAS) described by [10]. Chrome azurole S agar plates were prepared and spot inoculated with test organism and incubated at 30°C for 5 days. Development of yelloworange halo around the colony was considered as positive for siderophore production.
the extract was taken and made up to 3 ml with water and 0.5 ml of FCR was added. After 3 min of incubation 2 ml of sodium carbonate was added. It was shaken well and kept in boiling water bath for one minute and cooled. The blue solution obtained was diluted to 25 ml with water and the absorbance was read at 650 nm. The reagent was served as blank. The amount of phenol was determined using catachol as the standard. Catalase Assay: One gram of leaves was macerated with 0.1 M phosphate buffer and it was centrifuged at 6000 rpm for 10 mins. Then the supernatant was used for enzyme assay. An enzyme substrate mixture was prepared by adding 3 ml of 0.1 M phosphate buffer, 2 ml of 0.005M H2O2 and 1 ml of enzyme. The enzyme mixture was incubated at 20°C for 1 min. After 1 min the reaction was stopped by adding 10 ml of 0.7 N H2SO4. The reaction mixture was titrated against 0.01 N KMNO4 to find out the residual H2O2 until a faint purple color persists for at least 15 sec. Similarly, the blank was prepared without enzyme extract. Nitrate Reductase Assay: 100 mg of leaves were placed with 5 ml of assay medium in a first tube. It was incubated in dark for 30 min, after incubation the tubes were added with 0.5 ml assay medium and 4.5 ml distilled water. Then it was mixed with 1 ml sulphanilamide and 1 ml napthyl ethylene diamene dihydrochloride. The tubes were incubated at room temperature for 10 min for the formation of pink color. The absorbance was read at 540 nm. The blank contains 2 ml distilled water and 1 ml napthyl ethylene diamene dihydrochloride.
Biochemical Changes in Crop Plants Estimation of Carotenoids: One gram of leaves were macerated with 10ml of acetone and filtered through Whatmann No.1 filter paper. Extraction was repeated until the residue turned colourless. Then the extract was transferred into a separating funnel containing equal volume of petroleum ether and mixed gently. The ether layer was evaporated at 35°C. The residue was dissolved in ethanol and 60% of aqueous KOH was added at the rate of 1 ml for every 10 ml of ether extract. It was boiled for 5-10 min. To this equal volume of water and ether was added and it was evaporated. Then the residue was dissolved in ethanol. The absorbance was read at 450 nm.
Peroxidase Assay: One gram of leaves was macerated with 0.1 M phosphate buffer and it was centrifuged at 6000 rpm for 10 min. 0.1 ml enzyme was added with 3 ml phosphate buffer and 0.5 ml H2O2. Then the absorbance was read at 420 nm for every 20 seconds for 3 mins. Gibberllic Acid: One gram shoot was homogenized with 10 ml of prechilled methanol and it was kept at 4°C overnight. Then it was filtered through the Whatman No. 1 filter paper. It was re-extracted with cold ethanol and methanol until the residue turned colorless. The extract was collected and evaporated at 40°C. The extract was freezed and thawed to room temperature. Finally it was filtered through the Whatman No. 1 filter paper to remove chlorophyll and protein precipitates. The extract was stirred with poly vinyl pyrilidone (PVP) and the pH of the
Estimation of Protein: The protein content was determined by the procedure described by [6]. Estimation of Phenol: The phenolic content was estimated by Folin-Ciocalteu method. 100 mg fresh leaves was taken and macerated with 10 ml of 80% ethanol and centrifuged at 10,000 rpm for 20 min. The collected supernatant was evaporated and the remaining residue was dissolved with 5 ml distilled water. About 0.2 ml of 231
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They have reported that the inoculation of Azotobacter and Azospirillum treated plants had the highest protein contents (Fig. 1). In the present study, the catalase and peroxidase activity of agathi (Sesbania grandiflora) and drumstick (Moringa oleifera) were determined for both the plants (bioinoculated and uninoculated) and the values were recorded. The catalase activity was recorded high in PSB inoculated plants. Bacterial strains showing catalase activity must be highly resistant to environmental, mechanical and chemical stress (Fig. 2 & 3). Some of the above tested isolate could exhibit more than two or three PGP traits, which promote plant growth directly or indirectly synergistically. This is well correlated with earlier studies on Vigna mungo by Pseudomonas fluorescens and Bacillus subtilis [13]. Results demonstrated that the phosphate solubilizing Enterobacter cancerogenus enhanced the nitrate reductase activity of agathi (Sesbania grandiflora) and drumstick (Moringa oleifera) was determined for both the plants (bioinoculated and uninoculated) and the values were recorded (Fig. 4). Our findings are in accordance with the earlier work of many researchers who have confirmed the bacterial inoculation influence the growth of crops and its development [14, 15, 16, 17]. PGPR having the potential of phosphate solubilization enhanced the growth hormone production, availability of phosphorus and rate of nitrogen fixation [18]. In this study, the gibberellin of agathi (Sesbania grandiflora) and drumstick (Moringa oleifera) was determined for both the plants (bioinoculated and uninoculated) and the values were recorded (Fig. 5). Enterobacter cancerogenus inoculation influenced the growth of agathi and drumstick at each level of P fertilizer might be attributed to rhizosphere colonization, better nutrient availability and biosynthesis of growth hormones. Microbe having the potential of synthesizing plant hormones might be responsible for expansion of root surface area and enhanced plant-microbe interaction resulted in more nutrient uptake [15, 19, 11]. The carotenoid content of agathi (Sesbania grandiflora) and drumstick (Moringa oleifera) carotenoid was determined for both the plants (Bioinoculated and inoculated) and the values were recorded. In our present study, we observed that the amount of carotenoid was predominantly high in bioinoculated plants (Fig. 6). This result is similar to the earlier result [12]. They have reported that the inoculation of Azotobacter and Azospirillum treated plants had the highest caroternoid contents.
extract was reduced to 2.5 with 3.2 M HCl and it was extracted with equal volume of ethyl acetate and then it was evaporated at 40°C. Then the residue was dissolved in methanol. This will contain free acidic fraction of gibberellin like substances. One ml of the gibberellin extract was added with 15 ml of phosphomolybdic acid and the mixture was boiled for one hour in boiling water bath. Then it was cooled to room temperature and the volume was made up to 25 ml with distilled water. The absorbance was read at 780 nm. Siderophore Production: The leaves of the plant agathi (Sesbania grandiflora) were crushed well and the bioinoculated plant extracts was centrifuged, from that 0.5 ml supernatant was added with 2% 0.5 ml ferric chloride. The positive result was observed by the color change of the supernatant from yellow to orange or brown. RESULTS AND DISCUSSION In the present study, Enterobacter cancerogenus was isolated by preparing the serial dilutions from the rhizosphere of weed plants at Shenbahathoppu hills in Srivillliputhur, Tamilnadu and characterized for their giberellin biosynthesis, catalase activity, NR activity and phosphate solubilization potential. Among the eight isolates, only one potential bacterial strain was used in this study depending upon their phosphate solublization efficiency. Based on the morphological, physiological and biochemical characteristics the suspected bacterial strain was identified as Enterobacter cancerogenus by the following standard keys of Bergey’s Manual of Determinative Bacteriology. 16S rRNA gene sequences of phosphate solubilizing bacterial isolate revealed that it is more closely related to bacterial genomic sequences of Enterobacter cancerogenus. The dendrogram constructed based on their phylogenetic relationship revealed that all the isolates distinctly placed under separate clusters. Similarly [11] collected the samples from local weed plants from Shenbagathoppu hills in Srivilliputhur. They have reported that the Enterobacter cancerogenus had influence the growth and development of Sesbania grandiflora and Moringa oleifera. In the present study, the protein content of agathi (Sesbania grandiflora) and drumstick (Moringa oleifera) was determined in both the plants (bioinoculated and uninoculated) and the values were recorded. The protein content was predominantly high in bioinoculated plants. This result is identical to the previous result of [12]. 232
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Fig. 1: Effect of Phosphate solubilizing bacteria on Protein in Sesbania grandiflora and Moringa oleifera after 30 days
Fig. 5: Effect of Phosphate solubilizing bacteria on Gibberellins in Sesbania grandiflora and Moringa oleifera after 30 days
Fig. 2: Effect of Phosphate solubilizing bacteria on Catalase in Sesbania grandiflora and Moringa oleifera after 30 days
Fig. 6: Effect of Phosphate solubilizing bacteria on Carotenoid in Sesbania grandiflora and Moringa oleifera after 30 days
Fig. 3: Effect of Phosphate solubilizing bacteria on Peroxidase in Sesbania grandiflora and Moringa oleifera after 30 days
Fig. 7: Effect of Phosphate solubilizing bacteria on Phenol in Sesbania grandiflora and Moringa oleifera after 30 days
Fig. 4: Effect of Phosphate solubilizing bacteria on Nitrate reductase in Sesbania grandiflora and Moringa oleifera after 30 days
Fig. 8: Siderophore Production 233
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The phenolic content of agathi (Sesbania grandiflora) and drumstick (Moringa oleifera) was determined for both the plants (bioinoculated and uninoculated) and the values were recorded. In our present study we observed that the amount of phenol was predominantly high in bioinoculated plants. In addition to this replication was also observed high proliferation was noticed in bioinoculated plants (Fig. 7). Phenolic compounds are secondary plant metabolites and are involved in a wide range of specialized physiological functions. They are very important for the normal growth, development and defense mechanisms of plants [20]. Siderophore production was done in agathi (Sesbania grandiflora). We observed that the bright brown coloration was noticed in bioinoculated plants while in the uninoculated plants no coloration was noticed. In other crop plant drumstick (Moringa oleifera) siderophore production was not observed (Fig. 8). PSB inoculated plantlets produced high amount of secondary metabolites such as phytohormones, antibiotics and siderophores and aid in plant growth and development [16, 11].
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CONCLUSION According to this result, PSB is beneficial for plants under certain conditions. At the same time, it also provides natural and harmless means of improving the growth, yield of crops and minimizes the use of agrochemicals. PSB use one or more direct mechanism to improve the plant growth. These mechanisms include improvement of nutrient uptake by phosphatesolubilization, N2 fixation and phytohormone production like gibberellins and also increase the amount of carotenoids, protein, phenol, catalase, peroxidase, nitrate reductase and also siderophore production. Use of phosphate solubilizing bacteria as inoculants increases Phosphorus uptake, crop yield, induces resistance against salinity and pathogens.
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