CHAPTER 2. Screening plant growth promoting traits of the bacterial isolates INTRODUCTION

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1 CHAPTER 2 Screening plant growth promoting traits of the bacterial isolates INTRODUCTION Bacteria that colonize the rhizosphere and plant roots that can enhance plant growth are referred to as plant growth promoting bacteria (PGPB). In the context of increasing international concern for food and environmental quality and for reducing chemical inputs in agriculture the use of plant growth promoting rhizobacteria (PGPR) for plant growth is a potentially important issue. Greater productivity and competitiveness are anticipated to come from new microbiological and crop production strategies. PGPR have been applied to various crops to enhance plant growth, seed emergence, and crop yield and thus some have been commercialized. Another major benefit of PGPR is to produce antimicrobial compounds that can be effective against certain plant pathogens. (Dey et al., 2004; Kremner et al., 2004). In addition to the improvement of plant growth, PGPR, can contribute to increased nitrogen uptake, phytohormones synthesis, solubilization of minerals such as phosphorus, and production of biocontrol compounds able to suppress plant pathogens (Lee et al., 2003). A renewed interest in the colonization of healthy plants by non-rhizoidal bacteria has also arisen potentially exploitable in agriculture. The role of symbiotic bacteria has already been established and the present study concentrated on nonsymbiotic bacteria that associate and promote plant growth by various means. Diazotrophs belonging to diverse bacterial genera such as Azospirillum, Azatobacter, Arthrobacter, Alcaligenes, Bacillus, Enterobacter, Klebsiella and Pseudomonas can colonize important cereal crops including wheat, rice, sugarcane and maize and promote plant growth. PGPR were reported to improve the growth of maize and were used as biofertilizers (Arshad 1998; Farah, et al., 2000). However, results of these bacteria being used as biofertilizers are variable depending on crops and cultivation methods. In particular variability in root colonization by these bacteria is the most important factor. Hence, there is a need to study the adaptation of diazotrophs to host plants under conditions of cultivation (Cocking, et al., 1992). Seed treatment with 59

2 plant growth promoting rhizobacteria (PGPR) increased the growth of maize and several other crops (Jacoud et al., 1999). There is very little information regarding the use of PGPR as biofertilizer in legume crops. Overall, there is growing interest in PGPR due to their efficacy as biological control and growth promoting agents in many crops (Thakuria et al., 2004). Microorganisms isolated from rhizosphere soil or rhizoplane and composts may be better adapted to crop plants and provide better PGPR than those isolated from other sources, as these are already closely associated with plant systems. Therefore the present study was undertaken to screen for plant growth promoting traits including phosphate solubilization, indole acetic acid production, and ACC deaminase production in the bacteria isolated from composts and soils. 60

3 REVIEW OF LITERATURE There is an important need to enhance the efficiency of limited external inputs in agriculture by employing the best combinations of beneficial microbes to improve crop production. The use of PGPB inoculants as biofertilizers provides a promising alternative to chemical fertilizers and pesticides (Kloepper, 1993, Kloepper & Adesemaye, 2009). Enhancement of plant growth by root colonizing bacteria is well documented by Kloepper et al., (2004); Timmusk et al., (1999). There are several ways by which PGPRs may directly facilitate the proliferation of host plants. They may i) fix atmospheric nitrogen and supply it to plants. ii) synthesize siderophores which can provide iron to plants, iii) synthesize various phytohormones, including auxins and cytokinins that stimulate plant growth, iv) provide mechanisms for the solubilization of minerals such as Ca, S, and phosphorus and v) synthesize enzymes that can modulate plant growth and development (Glick et al., 1995). Rhizobacteria show several plant growth promoting activities that include the production of IAA, ACC deaminase and phosphate solubilization (Chakovskaya, 2001; Naik, 2002). In order to make pigeon pea cultivation sustainable and less dependent on chemical fertilizers it is important to know how to use PGPR that can biologically fix nitrogen, solubilize phosphorus and produce compounds like indole acetic acid (IAA) that can contribute to the improvement of plant growth. Bacterial residents of composts, fields, and rhizosphere soils which are unexplored are of interest to agronomists as new tools for crop productions and improvement (Stutrz et al., 2000). Very few such bacteria have been isolated, characterized and are being used as the bioinoculants in solid and liquid formulations, and have been commercialized for production of plant hormones such as ethylene or non volatile compounds such as gibberillic acid, auxins, cytokinins and abscisic acid (Frankenberg & Poth, 1987). Nitrogen is a major limiting nutrient for crop production. It can be applied through chemical or biological means. To get optimum crop yields, biological means should be explored for acquiring nitrogen for plant growth. PGPR can fix 61

4 atmospheric molecular nitrogen through symbiotic, non-symbiotic or associative nitrogen fixing process (Bashan & Holguin, 1997). The bacterial genera that can be used as PGPR are distributed among different taxa including Acinetobacter, Bacteriodes, Cyanobacteria, Firmicutes and Proteobacteria (Tilak et al., 2005) and proteobacteria belong to the genera Azatobacter, Azospirillum, Bacillus, Arthrobacter, Enterobacter, Pseudomonas, Alcaligenes, Klebsiella and Serratia (Somasegaran & Hoben 1994). Application of bacterial inoculants as biofertilizers has improved growth and yield of cereal crops (Okon & Itzigsohn, 1992). During the last two decades bacterial nitrogen fixation of non legumes has attracted much attention among soil microbiologists (Rao, 2000). Interest in benefical rhizobacteria associated with cereals has increased due to their potential use as biofertilizers (Antoun and Kloepper, 2001). Direct promotion of plant growth by PGPR generally aids in providing a compound to the plant that is synthesized by the bacteria or facilitation of nutrient uptake by the microbe from environment to the plant (Bertrand et al., 2001). Indirect promotion of plant growth can also occur when the bacteria limit or prevent deleterious effects of phytopathogenic organisms (Lim, 1991). At present there are fewer than 20 different PGPR strains that are commercially available (Kloepper et al., 2004). However, this number can be increased by isolating promising strains with screening procedures and obtaining superior genetically engineered strains that can be commercialized (Singleton et al., 2002). Dubey and Maheshwari, (2010) isolated genera of Azatobacter, Bacillus, Burkholderia from soil which supply nitrogen to legume plants. Mobilization of Phosphorus Phosphorus is second to nitrogen in the mineral nutrient most commonly limiting the growth of plants. Soils have large reserves of total P, but the amount available to plants is a small proportion of the total. Many soil microorganisms are able to solubilize unavailable forms of bound P. Visual detection and semiquantitative estimation of phosphate solubilizing ability of microorganism is possible by plate screening methods, that show clearing zones around microbial colonies in media containing insoluble mineral phosphates as sole sources of phosphorus (Stevenson and Cole, 1999). Mineralization of organic phosphorus can be achieved 62

5 by phytases i.e. phosphatases which show high specificity towards phytate or myoinositol hexa phosphate (Gaur, et al., 1990). Various plant species do not possess significant amount of extracellular phytase, and hence cannot use this P source. Hence, growth and P nutrition in plants can be improved by microbial phytases. Phytases have been isolated and characterized from Bacillus subtilis, Klebsiella sps., Pseudomonas sps., Enterobacter sps.,(rodriguez & Fraga, 1999). In the early 1960s native MPS (Mineral Phosphate Solubilizing) microbes were used as biofertilizers and cultures of Bacillus megaterium named Phosphobacterin was used as a phosphate fertilizer in the Soviet Union (Tisdale and Nelson, 1975). Poor understanding of mechanism of phosphate solubilization carried out by this organism discouraged its use (Kucey, 1989). Although many soils have large amounts of complexed phosphate, the use of bacterial organic phosphate solubilizing systems to increase solubilization were considered promising. However, bioinoculants often show varied performances because of their inability to be active in different soil conditions and also due to the nature of native microbial flora. Hence, there is a need to enhance the phosphate solubilizing potential of phosphate solubilizing bacteria, or modify broad host range rhizobacteria to solubilize mineral phosphates using molecular approaches. The level of free phosphorus in naturally occurring soils is often below those of many micronutrients in the range of 10-6 M (Kannaiyan, et al., 2004). Saritha et al., (2006) found that application of phosphate (20-40 ppm) improved the growth and nodulation of pigeon pea. Most of the applied P in fertilizers are reprecipitated into insoluble mineral complexes such as calcium orthophosphate, hydroxyapatite and fluoroapatite in alkaline soils, or crystalline ferric phosphates and crystalline aluminium phosphates in acidic soils, and hence are not efficiently taken up by plants (Pikovskaya, 1948). Farmers are therefore advised to apply four times the phosphate requirement to a particular crop. This over fertilization often leads to an imbalance in the soil and a major environmental concern (Lal, 2002). Various mechanisms have been proposed to solubilize phosphate complexes (Bagyaraj, et al., 1995). Organic acids released by the microorganisms can act as good chelators of divalent cations (Ca +2 ) helping release phosphates from insoluble phosphatic compounds (Gyaneshwar et al., 1998). 63

6 It is generally accepted that the major mechanism of mineral phosphate solubilization is the action of organic acids synthesized by soil microorganisms (Kucey, et al., 1989). The production of gluconic acids by phosphate solubilizing bacteria has been well documented in the work of Chakovskaya, et al., (2001). Strains of Bacillus were found to produce mixtures of lactic, isovaleric, isobutyric and acetic acids. Other organic acids such as glycolic, oxalic, malonic and succinic acids have also been identified among phosphate solubilizers (Leinhos, 1994). IAA PRODUCTION Organic substances capable of regulating plant growth produced either endogenously or applied exogenously are called plant regulators (Wendo, et al., 2002). They regulate plant growth by affecting physiological and morphological processes at very low concentrations (Glick et al., 1995). Among the plant hormones, auxin is the first hormone identified and is known to mediate plant cell tropism, apical dominance, root formation, root elongation, promotion of ethylene production, and subsequent evolution and ripening of fruits. Tilak and Ranganayaki, (2005) described that several microorganisms are capable of producing auxins, cytokinins, gibberillins, ethylene and/or abscisic acid. Bacteria synthesize indole acetic acid (IAA) predominantly by an alternate tryptophan dependent pathway, through indole pyruvic acid. However the role of bacterial IAA in plant growth remains undetermined. Promotion of root growth is one of the major markers by which the beneficial effect of plant growth promoting bacteria is measured (Pattern and Glick, 2002). According to Lebuhn & Hartmann (1993) the ability to synthesize phythohormones is widely distributed among 80% of plant associated bacteria and they regulate growth by affecting physiological and morphological processes at very low concentrations. Most PGPB synthesize IAA and their effect on plants mimics that of exogenous IAA (Frankenberg et al., 1987). The establishment of roots, whether by elongation of primary roots or by proliferation of lateral and adventitious roots, is beneficial to young seedlings as it increases their ability to anchor themselves to the soil and to obtain water and nutrients from the environment, thus enhancing their survival. 64

7 ACC Deaminase Ethylene is essential for plant growth and development but has different inhibitory effects on plants. In stress conditions such as drought and flooding, plants synthesize the precursor of ethylene ACC (1-Amino cyclopropane, 1-carboxylate) and which is converted to ethylene (Chen et al., 2005; Glick et al., 2007). A number of PGPB contain the enzyme 1-amino cyclopropane 1-carboxylate deaminase (ACC deaminase) and this enzyme cleaves ACC into ammonia and α-ketobutyrate and thus prevents high levels of ethylene production (Jacobson M.B, 1991; Glick et al., 1998; Penrose et al., 2001). Shaharoona (2006) stated that for most plants, ethylene is required to break seed dormancy but after germination, high levels of ethylene can inhibit root elongation. PGPB that contain the enzyme ACC deaminase, when bound to the seed coat of a developing seedling can act as a mechanism for ensuring that ethylene levels do not become elevated to the point where initial root growth is impaired (Jacobson, 1994). Shaharoona et al., (2006) showed that ACC deaminase activity can decrease ethylene production in the roots of host plants and results in root elongation. These bacteria facilitate the formation of longer roots and enhance the survival of seedlings especially during the first few days after the germination (Hirsch & Fang, 1997). Plants treated with ACC deaminase producing bacteria are dramatically more resistant to the deleterious effects of ethylene stress synthesized as a consequence of adverse conditions such as flooding, high heavy metals, the presence of phytopathogens, drought and high salt conditions (Arshad et al., 2008). These PGPB bacteria are beneficial to plant growth since in the natural environments plants are often subjected to stress that can lead to high ethylene production (Grichko et al., 2000). The present study investigates i. Qualitative in vitro screening of PGPR traits e.g. phosphate solubilization, IAA production and ACC deaminase production by the bacterial isolates. ii. Quantitative phosphate solubilization of inorganic tricalcium phosphate with potential bacteria and changes in ph during phosphate solubilization. iii. Quantitative IAA production in submerged bacterial cultivations. iv. ACC deaminase producing ability of the bacterial isolates. 65

8 Materials and Methods Sixteen bacterial cultures belonging to eight genera; Azatobacter (RB27), Bacillus (RB1, RB6, RB8, RB13), Pseudomonas (RB11, RB15, RB22), Klebsiella (RB19), Serratia (RB3, RB24), Enterobacter (RB9, RB17), Sinorhizobium (RB30) and Azospirillum (RB23, RB32), were tested for plant growth promoting traits including phosphate solubilization, IAA production, and ACC deaminase activities both qualitatively and quantitatively. Preparation of Bacterial Inoculums: Requirements: Nutrient broth, vortex mixture (Eltek) Sterile Nutrient broth (5ml) was dispensed into sterile test tubes and plugged with non absorbent cotton and labeled. Bacterial inocula (0.01ml) (10 4 cfus) were taken from stock cultures and transferred aseptically into sixteen culture tubes with nutrient broth. The tubes were vortexed (Eltek) and incubated in the orbital shaker incubator at 37 o C for 24 hours at 150rpm till the turbidity reached 0.5 O.D at 620nm. Preparation of Nutrient broth: As given in 1 chapter. Phosphate solubilization: (Qualitative test) The potential of bacterial cultures for phosphate solubilization was estimated by using the phosphate solubilization assay as described by Nautiyal (1999). Requirements: Pikovaskaya agar (PVK), Bacteriological loop Procedure: Bacterial culture in log phase (O.D 0.5 at 620nm) was spot inoculated with a bacteriological loop onto the PVK agar plates containing precipitated tricalcium phosphate in a laminar flow cabinet under aseptic conditions. Four bacterial cultures per plate were inoculated and incubated in the incubator at 37 o C for 24 and 48 hours. The plates were incubated in an inverted manner to prevent contamination. The formation of transparent zones of clearance around bacterial colonies after overnight incubation was used as an indicator for positive P- solubilization. The zone of clearance was measured with a ruler and the colony diameter was also measured. This experiment was performed in triplicate for each of 66

9 the sixteen isolates and was repeated 3x. Phosphate solubilization efficiency (PSE) was calculated by using the formula and recorded. Solubilization diameter PSE = x 100 Bacterial culture growth diameter Preparation of Pikovaskaya agar (PVK): Glucose-10.0g, Tricalcium phosphate- 5.0g, Sodium chloride- 0.2g, Magnesium sulphate- 0.1g, Potassium chloride- 0.2g, Yeast extract-0.5g, MnSO 4 - trace, Ferrous sulphate- trace, Agar-15.0g, Distilled water-1000ml, ph- 6.8 All ingredients were weighed and dissolved in distilled water (1000ml). The ph adjusted to 6.5, and the solution was sterilized in an autoclave at 15lbs for 15minutes. Preparation of PVK agar plates: The PVK agar is poured into sterile Petri plates under aseptic conditions in a laminar flow chamber and were allowed to solidify without water condensation and stored for future use. Quantitative Assay of Phosphate solubilization: The quantitative assay was done using turbidometric method described by Subba Rao and Sinha (1999). Effect of different tricalcium phosphate concentrations on the PSE: The quantitative assay for phosphate solubilization with different concentrations of tricalcium phosphate (TCP) was tested using the method of Subba Rao and Sinha (1999). The assay was performed for promising isolates obtained after qualitative assay; specifically Bacillus pumilis (RB8), Bacillus cereus (RB13), Enterobacter cancerogenus (RB17), Serratia marcescens (RB24), Klebsiella oxytoca (RB19). Requirements: PVK broth supplemented with tricalcium phosphate 2.0g/l, 4.0g/l, 6.0g/l, 8.0g/l, 10g/ respectively, Bartons reagent 67

10 Assay Procedure: Sterile PVK broths were prepared by keeping all the ingredients constant except for tricalcium phosphate of concentration (2.0g/l, 4.0g/l, 6.g/l, 8.0g/l, 10g/liter) as the sole source of P and the ph adjusted to 6.8. The PVK broth (25ml) with different concentrations of TCP was dispensed into sterile flasks and inoculated with 0.1 ml of growing bacterial cultures (10 8 cfus /ml). Culture tubes without any bacterial inoculation were used as controls. All isolates were maintained at 37 o C in an orbital shaker (200rpm) for 5 days and sampled every 24 hours. About 2ml of sample was taken and centrifuged at 10,000 rpm for 10 min and the supernatant was assayed for a solubilized P in the media by calculating the absorbance at 540nm using spectrophotometer (Systronics) b) the ph for every culture tube was determined using a ph meter (Elico) by taking about 2ml culture broth from the culture tubes at intervals of every 24h till the end of the experiment. The mean values of phosphate solubilized were calculated by using a standard constructed using known concentration of KH 2 PO 4. A drop in ph over time of incubation was also recorded for all five isolates. All experiments were conducted in triplicate for all samples and repeated 3X. Preparation of PVK broth: Glucose-10.0g, tricalcium phosphate (TCP) - 5.0g, sodium chloride- 0.2g, magnesium sulphate- 0.1g, potassium chloride- 0.2g, yeast extract-0.5g, MnSO 4 - trace, ferrous sulphate- trace, distilled water-1000ml, ph- 6.5 glucose, TCP, sodium chloride, magnesium sulphate, yeast extract, and potassium chloride were weighed and dissolved in 250ml of distilled water. Trace amounts of manganese sulphate and ferrous sulphate were added to the media. The ph was adjusted to 6.8. Media were made up to 1000ml with distilled water and sterilized in an autoclave at 15lbs for 15minutes. Preparation of Barton s reagent: Solution A: Ammonium molybdate (25gms) was dissolved in 400ml distilled water. Solution B: Ammonium metavanadate (1.25gms) was dissolved in 300ml of boiling water and cooled and then 250ml of concentrated HNO 3 was added. Solution A and Solution B were mixed and the volume was made up to 1 liter with distilled water. 68

11 Preparation of Standard KH 2 PO 4 (200 µgms/ml): Dissolve KH 2 PO 4 (200mg) in distilled water and make up the volume to 1 litre (1ml = 0.2mg) using distilled water. IAA PRODUCTION Qualitative test for the ability of IAA productions Volatile indoles produced by bacteria were tested using the Salkowski reagent and method as described by Fischer et al., (2007). Requirements: Tryptone broth, Salkowski reagent. Procedure: Sterile tryptone broth was dispensed (5ml) into sterile screw cap tubes and 0.1ml (10 8 cfus/ml) of exponentially growing bacterial pure cultures were inoculated aseptically. The tubes were incubated at 37 0 C for 48 hours. The experiment was performed for sixteen culture isolates. Each culture was maintained in triplicates in tryptone broth tubes and the experiment repeated 3x for detection of indole compounds. The presence of indole was detected by a pink color formed after the addition of Salkowski reagent drop wise in the edges of the test tube. The sixteen cultures detected for indole production were recorded and were also tested for quantification abilities by recording the absorbance at 530nm. The amount of IAA produced by each culture was calculated using a standard graph constructed using pure standard IAA. The experiment was repeated 3x for quantification. Preparation of Tryptone broth: Tryptone (Hi-media)-2gms/l, NaCl-0.85gms, Distilled water-100ml. Tryptone (2gms) was dissolved in 100ml distilled water, ph adjusted to 7.2, sterilized in an autoclave at 15lbs for 15minutes. Preparation of Salkowsi reagent (H 2 SO 4 -FeCl 3 ): Perchloric acid 35% (35gms of Perchloric acid dissolved in 100ml of distilled water), 0.5% FeCl 3 (0.5gms of FeCl 3 dissolved in 100ml of distilled water). To 50 ml of perchloric acid solution (35%) 1ml of 0.5% Ferric chloride was mixed. The reagent was always prepared fresh. 69

12 Preparation of Standard IAA: (250µg/ml) 250mg of IAA is dissolved in distilled water and the volume made up to 100ml in volumetric flask. Effect of tryptophan concentration, time of incubation and ph on IAA production IAA produced by bacteria Serratia marcescens (RB24), Bacillus cereus (RB13), Enterobacter cancerogenus (RB17), Klebsiella oxytoca (RB19), Sinorhizobium meliloti (RB30) was assayed colorimetrically using the ferric chloride-perchloric acid reagent (FeCl 3 -HClO 4 ) following Glickman & Dessaux (1995) method, modified by Fischer et al., (2007). Requirements: Nutrient broths supplemented with L-tryptophan stock solutions Assay Procedure: The quantity of IAA produced was determined with different concentrations of L-tryptophan i.e. 0.5, 0.7, 0.9, 1.2 g/l in the culture broths. Bacterial isolates in exponential phase were inoculated into 10ml of Nutrient Broth and incubated in the incubator at 32 o C for 24 hours in an orbital shaker incubator at 150 rpm. To measure the amount of IAA produced, the bacterial broth cultures were centrifuged at 10,000 rpm for 5 minutes. One ml of supernatant was added to 2 ml of Salkowski reagent and after 25 minutes the O.D was recorded in UVspectrophotometer at 530 nm absorbance. The sterile broth without inoculated culture was used as a blank. A calibration curve was constructed using standard IAA (250µgms /ml) and absorbance measured at 530 nm with UV-spectrophotometer. The amounts of IAA produced per milliliter culture for five cultures, were estimated using the standard curve of IAA and was recorded. The experiment was repeated 3x using three replicates for each bacterium. Preparation of Nutrient broth: As prepared earlier and supplemented with tryptophan stock solutions of (0.5g/l, 0.7g/l, 0.9g/l. 1.2 g/l) respectively in each of the sterile conical flasks and used for testing IAA production. Preparation of L-tryptophan stock solution: Glucose-10gms, L-tryptophan- 0.5g/l, 0.7g/l, 0.9g/l. 1.2 g/l respectively for each broth preparation, distilled water-1000ml. 70

13 The stock solution was prepared by dissolving the contents and filtering through a sterile 0.2 µm membrane filter (Millipore). Preparation of Salkowski Reagent (HClO 4 -FeCl 3 ): As prepared before. Preparation of Standard IAA: (250µg/ml) 250mg of IAA was dissolved in 25ml of distilled water and the volume made up to 100ml with distilled water. Qualitative ACC Deaminase production The qualitative assay of ACC deaminase was determined by the method of Jacobson, (1994). Requirements: Dworkin Foster minimal media (DF), standard ACC (Sigma chemicals USA), (NH 4 ) 2 SO 4, MgSO 4. Assay Procedure: DF media supplemented with ACC (3mM), (NH 4 ) 2 SO 4 (0.1M), MgSO 4 (0.1M) respectively, were used to test ACC metabolism by bacterial isolates. The ACC metabolism assay for each of the sixteen bacterial isolates was performed by growing each bacterial culture isolate on two different nitrogen sources and one mineral source. ACC was one nitrogen source, ammonium sulphate another nitrogen source and magnesium sulphate was the mineral source. Culture broths with ACC and ammonium sulphate respectively, were used as test cultures and MgSO 4 in culture broth was used as the control. Culture tubes were incubated for 48hours at 28 o C on orbital shaker incubator. Optical Density was measured at 550nm and O.D values of ACC were compared with ammonium sulphate and MgSO 4 to determine the ability of bacteria to metabolize ACC. The difference between O.D values of MgSO 4, ammonium sulphate and ACC sources was measured. The strains were characterized as O.D >0.7 with high ACC metabolizing ability, O. D= as medium, and low ACC metabolizing ability with O.D<0.5. Bacteria with high absorbance in ACC supplemented media were considered as ACC deaminase producers. The experiment was repeated 3x and results were recorded. 71

14 Preparation of Dworkin Foster minimal media - Glucose -1gm, KH 2 PO g, Na 2 HPO g, FeSO , H 3 BO 3-10mg MnSO4-10mg, ZnSO4-70 µgms, CuSO4-50 µgms, MoO 3-10 µgms, distilled water-1000ml All ingredients were weighed and dissolved in distilled water, and sterilized in an autoclave at 15lbs for 15minutes. Preparation of DF media supplemented with ACC (3mM): gms of ACC was dissolved in 100ml of sterile DF media and is further sterilized by membrane filteration (0.2µ). Preparation of DF Minimal media supplemented with (NH 4 ) 2 SO 4 (0.1M): 2gms of ammonium sulphate was dissolved in 1000ml of DF minimal media and sterilized in an autoclave at 15lbs pressure for 15minutes. Preparation of DF media supplemented with MgSO 4 (0.1M): 0.2 gms of magnesium sulphate was dissolved in 1000ml of DF minimal media and sterilized in an autoclave at 15 lbs pressure for 15 minutes. Quantitative ACC production The production of ACC deaminase was determined as described by Honma and Shimomura (1978), modified by Glick et al., (1995). The quantitative production of ACC demaminase potential for five bacterial isolates (Enterobacter agglomerans (RB9), Enterobacter cancerogenus (RB17), Klebsiella oxytoca (RB19), Serratia marcescens (RB23) and Pseudomonas fluorescence (RB15) was tested. Requirements: Dworkin Foster minimal media (DF), dinitrophenyl hydrazine reagent (DNPH), α-ketobutyrate Procedure: Bacterial test cultures were inoculated in 10ml of DF minimal media amended with ACC as the sole nitrogen source and incubated in a shaker incubator at 150rpm, 37 o C for 48hours. DF media without a bacterial inoculum served as control. Culture broths were subjected to centrifugation at 1500rpm for 5min. Cell pellets were collected and 72

15 suspended in 0.1ml of Tris HCl (ph 7.6) and centrifuged at 1500rpm for 5 min. Pellets were resuspended in Tris HCl ph (8.5) and 30µl of toluene was added and the sample vortexed for 15min. Thereupon, 2ml of the 2-4-dinitrophenyl hydrazine reagent was added, and the tubes were incubated for 30 minutes. The color of phenyl hydrazine was developed by addition of 2ml (2M) NaOH and the absorbance read at 540nm. Micromoles of α-ketobutyrate produced upon hydrolysis of ACC were recorded and the experiment was repeated 3x with three replications for each bacterial isolate. Preparation of ACC: 3gms of ACC dissolved in 1000ml of DF media. Preparation of DF media: As prepared before. Preparation of dinitrophenyl hydrazine reagent (DNPH) 0.2% (Used for detecting α-ketobutyrate produced in the media by ACC deaminase activity on substrate ACC): 0.2gms of DNPH was weighed and dissolved in 100ml of 2M HCl. Preparation of HCl (2M): 16.5ml of concentrated HCl made up to 100ml with distilled water. Preparation of NaOH (2M): 8gms of NaOH was weighed and dissolved in 100ml of distilled water to obtain 2M NaOH. Preparation of Stock solution: 100mM α-ketobutyrate (For constructing standard graph) Stock solution of 100mM, α-ketobutyrate was prepared in 0.1M Tris HCl by dissolving 100mg of α-ketobutyrate in 100ml 0.1M Tris HCl and stored at 4 o C. The stock solution was diluted with same buffer to make a 10mM solution. Statistical analysis: Mean values and standard error for each of the bacterial isolates for quantitative production of P, IAA, and ACC were calculated using SPSS-17, Software system analyzer. 73

16 RESULTS Sixteen bacterial isolates belonging to eight genera were studied in detail for their PGPB characteristics. Out of sixteen isolates, four of them were Bacillus sps (RB1, RB6, RB8, RB13) three Pseudomonas species ( RB11, RB15, RB22), two each of Serratia sps,(rb3, RB24), Enterobacter species (RB9, RB17), Azospirillum (RB23, RB32) and one each of Klebsiella (RB19), Sinorhizobium (RB30), Azatobacter (RB27). They were characterized and tested for phosphate solubilization, IAA production and ACC deaminase production. Qualitative and quantitative studies were performed to identify putative promising bacteria for plant growth promoting traits. 2.1 Qualitative screening of Bacteria for PGPR a. Phosphate solubilization The bacterial isolates that showed zones of clearance on PVK agar media were considered as phosphate solubilizers and the phosphate solubilization efficiency of all cultures is reported in Table 2.1. The phosphate solubilization efficiency for the sixteen isolates was tabulated via formation of zones of clearance in diameter (mm) (Fig. a & b in Plate 6). The zone of clearance was highest with Serratia marcescens (RB24) and Bacillus cereus (RB13), moderate with Klebsiella oxytoca (RB19) and Enterobacter cancerogenus (RB17), and lowest with Bacillus licheniformis (RB6), Pseudomonas alkaligenes (RB11) and Sinorhizobium meliloti (RB30). b. IAA production Indole acetic acid production was detected by formation of a pink color using the Salkowski reagent. Sixteen isolates were reported to be IAA positive and hence were tested quantitatively for IAA production. Bacillus cereus (RB13) and Klebsiella oxytoca (RB19) produced the highest amounts of IAA, moderate amounts by Sinorhizobium meliloti (RB30) and Serratia marcescens (RB 24) and Bacillus licheniformis (RB6) produced the least amount of IAA among the sixteen isolates (Table 2.1). 74

17 c. ACC deaminase production The sixteen isolates were different in their ability to utilize ACC as a sole source of nitrogen. Bacillus cereus, Klebsiella oxytoca, Serratia protemaculans Pseudomonas fluorescence and Enterobacter agglomerans and Enterobacter cancerogenus produced the highest levels of ACC deaminase, by showing O.D values higher than 0.7 (Table 2.1). Table 2.1 Phosphate solubilization, IAA, ACC deaminase production by 16 bacterial isolates *Phosphate *IAA **ACC deaminase production Bacterial solubilizati productio Isolates O.D on (mm) n (mg/l) O.D>0.7 O.D<O Bacillus subtilis (RB1) 411± ± Bacillus licheniformis(rb6) 308± ± Bacillus pumilis(rb8) 526± ± Bacillus cereus (RB13) 954± ± Enterobacter agglomerans(rb9) 384± ± Enterobacter cancerogenus(rb17) 616± ± Klebsiella oxytoca(rb19) 733± ± Sinorhizobium meliloti(rb30) 320± ± Serratia proteamaculans(rb3) 484± ± Serratia marcescens(rb24) 1100± ± Pseudomonas alkaligens(rb11) 316± ± Pseudomonas fluorescens(rb15) 450± ± Azospirillum lipoferum(rb32) 483± ± Azatobacter beijerinckii(rb27) 350± ± Azospirillum brasilense(rb23) 494± ± Pseudomonas mallei(rb22) 366± ± * Values are means of triplicate values. **O.D > 0.7- High ACC producer; O.D : Medium ACC producer, O.D < O.5 Low ACC metabolim 75

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19 2.2 Quantitative phosphate solubilization assay Five promising bacterial strains Bacillus pumilis (RB8), Bacillus cereus (RB13), Enterobacter cancerogenus (RB17), Serratia marcescens (RB24), and Klebsiella oxytoca (RB19) were inoculated in PVK broth media to monitor drop in ph, production of organic acids and thus promotion of phosphate solubilization. Maximum phosphate solubilization in liquid media was shown with Enterobacter cancerogenus followed by Bacillus pumilis, Bacillus cereus, and Serratia marcescens. Very low amounts of P were solubilized with Klebsiella oxytoca (Table 2.2). There was an increase in phosphate solubilization with time of incubation over five days. Phosphate solubilization increased with the increase in TCP concentration. All the isolates showed increase in P solubilization when TCP concentration was 10g/l. 77

20 Table 2.2 Phosphate solubilization efficiency of Bacteria with different TCP concentrations during submerged cultivation (Incubation for 5 days) Conc of *PO 2-4 Solubilized (in mg/l) Bacterial TCP Isolates 24hrs 48hrs 72hrs 96hrs 120 g/l ± ±10 260± ± ±10 Bacillus pumilis RB ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±10 Bacillus cereus RB ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±5.77 Enterobacter cancerogenus RB 17 Serratia marcescens RB ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±10 Klebsiella oxytoca RB ± ± ± ± ± ± ±20 420± ± ± ± ± ± ± ±11.54 *Values are Mean ±S.D of triplicate values. *TCP - Tricalcium Phosphate 78

21 2.3 Effect of ph during Phosphate solubilization: All the isolates except Klebsiella oxytoca (RB19) showed a decrease in ph within 24 hours that favored solubilization of phosphorus. Enterobacter cancerogenus (RB17) showed highest decrease in ph and a considerably smaller decrease in ph was observed with Klebsiella oxytoca (Table 2.3). Table 2.3 Change of ph during phosphate solubilization in submerged fermentation (initial ph of media 6.8) Bacterial Isolates Concn. of TCP (g/l) *Change of ph 24hrs 48hrs 72hrs 96hrs 120hrs ± ± ± ± ±0.17 Bacillus pumilis (RB8) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.14 Bacillus cereus (RB13) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.07 Enterobacter cancerogenus (RB 17) Serratia marcescens (RB24) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.11 4±0.14 4± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.07 Klebsiella oxytoca RB ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.1 * Values are Mean±S.D of triplicate values 79

22 2.4 IAA production Five promising bacterial strains; Bacillus cereus (RB13), Enterobacter cancerogenus (RB17), Serratia marcescens(rb24), Klebsiella oxytoca (RB19) and Sinorhizobium meliloti (RB30) were inoculated in Tryptone broth medium to monitor decrease in ph, and production of indole acetic acid. Different quantities of IAA were produced with different concentrations of tryptophan (0, 0.1, 0.3, 0.7, 1 g/l) that were supplemented into the broth (Table 2.4). Each organism produced IAA in different quantities with increase in period of incubation for over four days and with increase of tryptophan quantities supplemented in the broth. Bacillus cereus, Klebsiella oxytoca and Serratia marcescens showed high levels of IAA when incubated for a period of four days and with Tryptophan at 1g/l, while the lowest amount of IAA was recorded by the other isolates, e.g. Sinorhizobium meliloti and Enterobacter cancerogenus. Serratia marcescens (RB24) was producing IAA more or similar when the concentration of tryptophan was 0.7g/l and 1g/l. Drop in ph during IAA production was maximum with Serratia and Sinorhizobium species (Table.2.4) and drop in ph was minimum with Enterobacter sps. 80

23 Table 2.4 Amount of IAA produced in mg/l by the bacterial isolates and changes of ph during submerged fermentation Bacterial Isolate Serratia marcescens (RB24) Bacillus cereus (RB13) Enterobacter cancerogenus (RB17) Klebsiella oxytoca (RB19) Sinorhizobium meliloti (RB30) Concn. of *Amount of IAA Produced in mg/l *Change of ph Tryptophan ( g/l) 24hrs 48hrs 72hrs 96hrs 24hrs 48hrs 72hrs 96hrs ± ± ± ± ± ±0.54 6± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1 22.4± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.34 *Values are Mean ±S.D of triplicate values 81

24 2.5 ACC deaminase Six promising bacterial strains; Bacillus cereus (RB13), Enterobacter agglomerans (RB9), Enterobacter cancerogenus (RB17), Serratia proteamaculans (RB3), Klebsiella oxytoca (RB19), Pseudomonas fluorescens (RB15) were tested for ACC deaminase activity. The results were recorded as micromoles of alpha ketobutyrate produced by hydrolysis of ACC by the isolates in ACC supplemented DF medium (Table 2.5). Enterobacter cancerogenus, E.agglomerans and Pseudomonas fluorescence showed high ACC deaminase activity, however the differences were not that large. Table 2.5: ACC deaminase activity of Bacterial isolates Bacterial Isolates *ACC deaminase activity µmol /gm Bacillus cereus (RB13) 12.1±1.12 Enterobacter agglomerans (RB9) 16.9±2.13 Enterobacter cancerogenus RB ±1.18 Klebsiella oxytoca (RB19) 11.4±2.43 Serratia proteamaculans (RB3) 13.6±1.14 Pseudomonas fluorescens (RB15) 15.4±2.12 *All the values are Mean and Standard deviation of three replicates. 82

25 DISCUSSION Sixteen bacterial cultures isolated from soils and composts were tested for PGPR traits in vitro including phosphate solubilization, IAA and ACC deaminase production. The bacteria belonged to eight genera, Bacillus, Enterobacter, Serratia, Klebsiella, Pseudomonas, Azatobacter, Sinorhizobium, and Azospirillum. The genera selected were non-symbiotic diazotrophs that could play significant roles in plant growth promotion according to Kennedy et al., (2004). These genera are found in soils and composts and were considered as PGPB by Correa et al., (2004). Some were also isolated from the rhizosphere, by Dey et al., (2004); and were identified to aid in soil fertility by enhancing soil nutrients. Phosphate solubilization Microbial communities influence soil fertility through processes including, decomposition, mineralization, and storage or release of nutrients. Microorganisms enhance the phosphorus (P) availability to plants by mineralizing organic P in soil & by solubilizing precipitated phosphates (Chen et al., 2006 and Khan et al., 2009). In this study Serratia marcescens (RB24) showed three fold high phosphate solubilization on PVK agar plate than Serratia proteamaculans (RB3) that showed less phosphate solubilization. Among the bacterial isolates examined belonging to the genera Bacillus, Bacillus cereus showed three fold more phosphate solubilization than Bacillus licheniformis and two fold more phosphate solubilization than Bacillus subtilis and B. pumilis. Bacteria belonging to Enterobacter, Enterobacter cancerogenus was high phosphate solubilizer than Enterobacter agglomerans. This phenomenon indicates that the mineral phosphate solubilization (MPS) is an inherent metabolic tendency of individual bacterial species, strains and not a generalized trait of individual genera. Lal et al., (2002), also reported similar results and stated that it is the individual ability of each bacterial strain capable of solubilizing phosphates. In our study, P solubilization on PVK agar plates started within 24 hours and reached a maximum after the third day, indicating P solubilization is dependent on incubation time. The solubilization and size of halo formation on PVK plates may be due to bacterial phosphatase enzyme production, and/or, acidification of the media by lowering of the ph which can solubilize inorganic phosphate. Utilization of 83

26 tricalcium phosphate and production of organic acids into the media may be the result of P solubilization as seen by halo formation. Thus, parameters such as enzyme phosphatase activity, ph of the media, and time of bacterial incubation can play roles in phosphate solubilization. The colony size of the bacteria did not seem to have any effect on zone formation, as Klebsiella with large colonies had a smaller Phosphate Solubilization Efficiencies (PSE) than Serratia which had pin sized colonies but higher PSE values. Our data indicate that solubilization of phosphorus was different between bacteria belonging to different genera and isolates showed different phosphate solubilzation efficiencies on solid media. Similar results were reported by Gaur, (1990), namely that PSE on solid media was time dependent and solubilization increased with the time of incubation. Rodriguez, (1999) derived similar observations, again that bacteria aid in phosphate solubilization via decreases in ph on PVK agar plates. Abd Alla, (1994); Whitelaw et al., (2000) and Stephen et al., (2009) reported that phosphate solubilization may be due to combined effect of decreased of ph, carboxylic acid synthesis, microbial growth and phosphatase activity. Five promising bacterial strains Bacillus pumilis (RB8), Bacillus cereus (RB13), Enterobacter cancerogenus (RB17), Serratia marcescens (RB24), Klebsiella oxytoca (RB19) with good phosphate solubilization efficiencies on solid PVK agar plates were further tested for phosphate solubilization in submerged liquid cultivations in PVK broth. Among these bacteria phosphate solubilization in Bacillus pumilis, and Enterobacter cancerogenus was high whereas Klebsiella oxytoca was found to be a low phosphate solubilizer in liquid media. Bacillus cereus (RB13) which showed high phosphate solubilization efficiency on solid media did not perform similarly in liquid media, thus consistent results were not obtained for phosphate solubilization when examining solid medium to submerged liquid media. Such results noting differences in P solubilization on solid and liquid media were reported by Gyaneshwar et al., (1999), Srivatsav et al., (2004). Our study indicated that Enterobacter cancerogenus showed a three fold and Serratia marcescens a two fold greater P solubilizations than Klebsiella oxytoca and Bacillus cereus that showed equal P solubilization. Possible differences in ph maintenance and nutrient availability and exposure to toxic compounds that vary in solid and liquid media, may be the cause of this difference in results. The results do show that decreases in ph, 84

27 correlate with phosphate solubilization e.g. a decrease in ph was seen in Klebsiella oxytoca (RB19), and lower phosphate solubilization was also observed. Sharma, (2005) illustrated that mineral phosphate solubilizing (MPS) bacteria utilize direct oxidation pathways to produce gluconic acid and 2-keto gluconic acid. Typically a decrease in ph indicates the production of acids, which is considered an important parameter responsible for P solubilization (Pandey, et al., 2004). There was a drop in ph during P solubilization for the first 24 hours but the change in ph was almost negligible with further incubation periods upto 120hours. These minor changes of ph after 24 hrs of incubation may be a result of buffering capacity that might have occurred during the longest bacterial incubation periods as an adaptive mechanism or production of basic metabolites. i.e. ammonia, carbonate etc. by bacteria. Bacteria that produce less organic acids reduce phosphate solubilization (Hameeda, 2006). The observed lower P solubilization even accompanying a drop in ph seen with Bacillus cereus (RB13) and Serratia marcescence (RB24) when compared to Enterobacter cancerogenus might be due to lower bacterial proliferation or lower phosphatase activity by these isolates. Reyes (2007) and Khan et al., (2009) reported that nutritional and physiological aspects of bacterial isolates play a role in microbial proliferation and in turn, P solubilization. Increase in P solubilization was observed at increased TCP concentrations in our study, which was consistent with results of Richardson, (2001) and Fankem et al., (2006). Similar results were also obtained by Barik, (1998); Rodriguez et al., (2000) who showed that higher P availability in the medium correlated with bacterial strains able to enhance P solubilization. Some of the variation seen in P solubilization by Serratia marcescens and other bacteria showed a high standard error might be due to reprecipitaion of phosphates during certain growth phases of the bacteria. In our study, Enterobacter cancerogenus (RB17) a gram negative bacteria was showing two to three fold more P solubilization than gram positive bacteria Bacillus cereus (RB13). There are reports of the high P solubilization ability of Bacillus megaterium, Enterobacter agglomerans (Kim, 1998a, b) and Bacillus asburiae (Hameeda et al., (2006) being used for growth promotion of pearl millet. Preethi Mehta et al., (2010) reported high P solubilization by Bacillus circulans (MTCC 8983) among various Bacillus species. Ten Bacillus spp., and six Serratia isolates were characterized by Chen et al., (2005) as phosphate solubilizers from semi arid tropics. Pseudomonas, 85

28 Bacillus, Enterobacter and B.circulans, B.polymyxa were reported as potential strains for P solubilization (Khan et al., 2009; Whitelaw et al., 2000). IAA production Bacillus cereus (RB 13 ) and Serratia marcescens (RB23), showed high IAA production along with phosphate solubilization, ranking them with multiple PGPR traits in this study This is in accordance with results of Barea et al., (1978) and Lal, (2002), who showed PSB bacteria isolated from soils produce regulatory substances including IAA. In our study, Klebsiella oxytoca showed high IAA production at different levels of tryptophan in the media. The amount of IAA produced was greater with increased tryptophan concentrations and increased during the time of incubation. These results were consistent with work by Pattern and Glick, (2002) that illustrated tryptophan as a main precursor of IAA biosynthesis in bacteria via indole pyruvic acid pathway (IPA). IAA is a secondary metabolite produced by bacteria mostly during stationary phase. IAA production differed in various isolates presumably due to genetic and physiological properties. Small ph decreases were seen in culture media with Bacillus cereus and Klebsiella oxytoca (RB19), but these strains produced IAA levels. Thus IAA production did not correlate to acidification as seen for P solubilization. Hence, cultures with acidic ph showed low IAA production, potentially because acidification of the media resulted in growth retardation of the bacteria and/or inhibited enzyme activity. High amounts of IAA were seen Bacillus cereus irrespective of ph change. Similarly high IAA production with B.circulans (MTCC 8983) among sixteen bacterial strains isolated by Preethi Mehta, et al., (2010) has been reported. According to reports by Khalid and Arshad, (2004), IAA concentration increases with tryptophan concentration as tryptophan is the precursor of IAA. Pattern and Glick (2002) reported higher IAA production by Pseudomonas putida. According to Sachdev, (2009) Klebsiella was found to be the best strain for IAA production. Our study reported Sinorhizobium meliloti to be a good IAA producer at higher tryptophan concentrations after 96 hour of incubation. In contrast, to a study by Piyush Pandey, (2007) Sinorhizobium meliloti produced IAA only after 160 hour period of incubation. When tryptophan concentrations were reduced, auxin concentrations were also reduced (Leinhos 1994). According to Wani et al., (1996) and Wani et al., (2003), Bacillus macerans produced IAA and also helped in nitrogen 86