Environment Health Techniques Efficiency of plant growth-promoting P-solubilizing B. circulans
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Research Paper Efficiency of plant growth-promoting P-solubilizing Bacillus circulans CB7 for enhancement of tomato growth under net house conditions Preeti Mehta1, Abhishek Walia2, Saurabh Kulshrestha3, Anjali Chauhan2 and Chand Karan Shirkot2 1
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Department of Advance Bioenergy Research, Research and Development Centre, Indian Oil Corporation Limited, Government of India, Faridabad, New Delhi, India Department of Basic Sciences (Microbiology Section), Dr. Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India School of Biotechnology, Shoolini University of Biotechnology and Management Sciences, Solan, Himachal Pradesh, India
P-solubilizing bacterial isolate CB7 isolated from apple rhizosphere soil of Himachal Pradesh, India was identified as Bacillus circulans on the basis of phenotypic characteristics, biochemical tests, fatty acid methyl esters analysis, and 16S rRNA gene sequence. The isolate exhibited plant growthpromoting traits of P-solubilization, auxin, 1-aminocyclopropane-1-carboxylate deaminase activity, siderophore, nitrogenase activity, and antagonistic activity against Dematophora necatrix. In vitro studies revealed that P-solubilization and other plant growth-promoting traits were dependent on the presence of glucose in PVK medium and removal of yeast extract had no significant effect on plant growth-promoting traits. Plant growth-promoting traits of isolate CB7 were repressed in the presence of KH2PO4. P-solubilization activity was associated with the release of organic acids and a drop in the pH of the Pikovskaya’s medium. HPLC analysis detected gluconic and citric acid as major organic acids in the course of P-solubilization. Remarkable increase was observed in seed germination (22.32%), shoot length (15.91%), root length (25.10%), shoot dry weight (52.92%) and root dry weight (31.4%), nitrogen (18.75%), potassium (57.69%), and phosphorus (22.22%) content of shoot biomass over control. These results demonstrate that isolate CB7 has the promising PGPR attributes to be developed as a biofertilizer to enhance soil fertility and promote plant growth. Keywords: PGPR / P-solubilization / Siderophore / ACC deaminase / Tomato Received: July 12, 2013; accepted: November 16, 2013 DOI 10.1002/jobm.201300562
Introduction Phosphorus is one of the major essential macronutrients for biological growth and development. On average, soil contains 0.02–0.5% of total phosphorus [1]. It is added to the soil in the form of P- fertilizers, a part of which (1%) is utilized by plants and the rest is rapidly converted into insoluble complexes. Phosphate anions (H2PO4, HPO42) are extremely reactive and form metal complexes with Correspondence: Prof. C. K. Shirkot, Department of Basic Sciences, Dr. Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan 173230, Himachal Pradesh, India E-mail: [email protected] Phone: þ91 9418117399 Fax: þ91 1792252354 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Ca in calcareous soils and Fe3þand Al3þ in acidic soils. These metal ion complexes precipitated the 80% of added P-fertilizer [2]. This leads to the need of frequent application of P-fertilizers, but its use on a regular basis has become a costly affair and environmentally undesirable. Therefore, the necessity to develop economical and eco-friendly technologies is steadily increasing. The use of plant growth-promoting rhizobacteria (PGPR) including phosphate solubilizing bacteria as biofertilizers was suggested as a sustainable solution for the improvement of nutrient availability, plant growth, and yields [3]. The mechanism by which PGPR promote plant growth are not fully understood, but are thought to include: (a) the ability to produce plant hormones, such as auxins, cytokinins, gibberellins, (b) asymbiotic N2 fixation, (c) solubilization of
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inorganic phosphate and mineralization of organic phosphate and/or other nutrients, and (d) antagonism against phytopathogenic microorganisms by production of siderophores, the synthesis of antibiotics, enzymes, and/or fungicidal compounds and competition with detrimental microorganisms [3]. Soil–plant–microbe interactions are complex and there are many ways in which the outcome can influence the plant health and productivity [4]. The interaction may be harmful, beneficial, and neutral to the plants. However, our focus should be to exploit the beneficial interaction of plants and microbes. The use of microbial technologies in agriculture/horticulture is currently expanding quite rapidly with the identification of new bacterial strains, which are more effective in promoting plant growth. Studies have shown that the growth-promoting ability of some bacteria may be highly specific to certain plant species, cultivar, and genotype [5]. Some PGPR exhibit narrow specificity, i.e., Chanway et al. [6] found that six of seven Bacillus subtilis strains enhanced growth of wheat cultivar Katepwa, but none promoted growth of cultivar Neepawa. Other PGPR may exhibit much broader specificity. It appears that PGPR strains vary widely in their responses to specific environmental parameters. For example, PGPR strain GB03 colonizes cotton roots and provides biological control against Ralstonia solani throughout the Cotton Belt, from Texas to the south-eastern United States. Similarly, the same strain acts as a biological control agent against R. solani on peanut from the Southeast to Texas and Oklahoma. The same group of PGPR strains is reported to control soil-borne pathogens throughout the country of China [7]. Knowing the fact that some PGPR strains lack tight host specificity, future selection of new PGPR should be designed to test for broad host ranges for crops colonized, thereby increasing the chances of routine use of PGPR in future biofertilization strategies. Accordingly, this study examined the ability to produce plant growth-promoting metabolites by B. circulans CB7 isolated from apple rhizosphere under in vitro condition, along with changes in the early stages of tomato seedlings growth after inoculation with B. circulans CB7 under net house. Influence of certain factors like carbon and nitrogen sources, availability of free phosphorous on the P-solubilization and other plant growth-promoting traits of the selected PGPR isolate was also determined.
Materials and methods Isolation The soil used for bacterial isolation was collected from rhizosphere of apple trees growing at different locations ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
of Chamba in the Indian trans-Himalayan region of Himachal Pradesh, India, located at an altitude of 2077 m above mean sea level. The processed soil sample was serially diluted, spread plated on full strength nutrient agar and incubated at 30 2 °C for 48 h. A total of 206 different colonies were isolated on nutrient agar and were purified with repeated culturing and maintained in 20% glycerol at 80 °C. A potential isolate CB7 was screened and selected on the basis of halo zone produced in Pikovskaya’s (PVK) agar and assessed for morphology, physiology, and Gram reaction and other characterization. Bacterial identification and characterization Isolates were subsequently differentiated by Gram reaction, salt tolerance, biochemical characterization, and microscopic observation. The ability of the isolates to grow in diverse temperature range was carried out by growing isolates in nutrient broth and incubated at different temperatures ranged from 4 to 65 °C, respectively. Growth was recorded every 24 h at OD600 upto 96 h. The ability of the isolates to grow in different salt concentrations was carried out by inoculating bacterial culture on nutrient agar plates supplemented with 2.5– 10% w/v NaCl and the plates were incubated at 30 2 °C for 3 days. The ability of isolates to grow in alkaline or acidic media was tested in nutrient agar plates in which the pH was adjusted from 5.0 to 11.0 and incubated at 30 2 °C for 3 days. Phenotypic characterization of the isolates was done based on their colony morphology, microscopic observations, and biochemical tests using commercial kits (KB009 Hi carbohydrateTM kit and KB013 Hi BacillusTM Identification kit). A cream whitish colored bacterial colony showing above 15 mm zone of P-solubilization, maximum of 8.5% NaCl tolerance, and growth in wide pH range was selected for further analysis. Among 206 PSBs, CB7 which had a marked P-solubilizing activity on PVK agar medium/broth and also showed maximum effectiveness of multifarious plant growth-promoting attributes (Table 1) was selected in vitro evaluation for tri-calcium phosphate (TCP) solubilization and plant growth-promoting traits under various conditions in PVK broth and its interaction with tomato seedlings under net house condition. Phosphate solubilization and organic acid production Isolates were first screened on PVK agar plates. The P-solubilization was exhibited with a clear zone formed around the colony. Further, quantitative estimation of phosphorus was done in PVK broth amended with 5.0 g L1 TCP by the vanadomolybdate method [8]. For the analysis of organic acids, bacterial culture was filtrated through 0.2 mm filter (Millipore, GTBP) and 20 ml of
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Table 1. Plant growth-promoting attributes of B. circulans CB7 after 72 h of incubation. PGP attribute activity Phosphate solubilization index (PSI) TCP solubilization (mg L1) Organic acid production (%) Oxalic acid Gluconic acid Formic acid 2-Ketogluconic acid Citric acid Fumaric acid Nitrogenase activity (hmole C2H4 h1 mg1 protein) Nitrogen fixation (mg ha1 per day) IAA production (mg ml1) Siderophore production Zone (mm) Units (%) Chitinase activity Zone (mm) Chitinase enzyme index Antagonistic activity against D. necatrix (%) HCN production ACC-deaminase activity
2.7 1.12 828 3.2 0.20 0.02 2.33 0.03 0.18 0.05 0.39 0.08 0.76 0.07 0.05 0.12 267.6 2.8 98 4.32 27.3 1.06 18.31 1.12 83.4 1.17 28.00 1.00 12.61 1.30 73.8 1.4 Positive Positive
Values are the mean of three replicates standard deviation.
filtrates were injected to HPLC (Waters 996 HPLC) equipped with Photo Diode Array detector. The organic acid separation was carried out on RP-18 column (Merck, Germany) with 0.1% orthophosphoric acid (Merck) as mobile phase. Retention time of each signal was recorded at a wavelength of 210 nm and compared with the standard acids. Indole-acetic acid, aminocyclopropane-1-carboxylate deaminase, and nitrogenase activity Estimation of indole-acetic acid was done by inoculation of bacterial suspension in PVK broth not amended with Ltryptophan (500 mg ml1) and incubating it at 30 2 °C for 72 h. The indole-acetic acid content in the culture suspension was estimated by the standard procedure [9]. Aminocyclopropane-1-carboxylate (ACC)-deaminase activity of the isolate was assessed by quantifying ammonia liberated by the hydrolysis of ACC. ACC-deaminase activity was detected on plates with DF minimal medium containing ACC as the sole source of nitrogen [10]. Nitrogen fixation of isolate was determined in nitrogen free medium by the acetylene reduction assay [11]. Siderophore, hydrocyanic acid production Siderophore production was detected by the standard Chrome Azurol-S assay [12] and the diameter of the clearing zone was measured. Hydrocyanic acid ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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production was inferred by the qualitative method of Bakker and Schipper [13]. The change in the color of the filter paper previously dipped in 2% sodium carbonate prepared in 0.05% picric acid, from yellow to dark brown was rated visually depending on the intensity of the color change. Chitinolytic and antifungal activity The bacterial cultures were spotted on to the prepared minimal agar medium amended with 0.3% colloidal chitin and the plates were incubated at 30 2 °C for 4 days. Development of clear halo zone around the colony after addition of iodine was considered as positive for chitinase enzyme production [14]. Antagonistic activity of the culture filtrate (10%) of the isolate was tested against Dematophora necatrix (the fungal pathogen causing white root rot of apple) on potato dextrose agar using agar dilution technique [15]. Percentage of growth inhibition was calculated using the formula proposed by Vincent [16]. All the studies were repeated on three independent dates to confirm the results. DNA extraction, 16S rRNA gene sequencing, and phylogenetic analysis The bacterial isolate was grown in nutrient broth at 30 2 °C overnight. Bacterial cells were harvested by centrifugation at 5000g for 5 min and DNA was isolated from these bacterial cells by using a Real Genomics DNA Extraction Kit (Life Technologies, Delhi, India). The isolated DNA was finally suspended in 100 ml elution buffer and quantified on a 1% agarose gel. The total genomic DNA was kept at 20 °C before use. Species level identification of the isolate was conducted by 16S rRNA sequence comparison. PCR reactions were carried out in 20 ml reaction buffer containing 50 ng template DNA, 20 pmol of each primer forward (50 -GCAAGTCGAGCGGACAGATGGGAGC-30 ) and reverse primer (50 -AACTCTCGTGGTGTGACGGGCGGTG-30 ), 0.2 mM dNTPs, and 1 U Taq polymerase (MP Biomedicals, Santa Ana, CA) in 1 PCR buffer. Reaction were cycled 35 times as 94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min 30 s followed by final extension at 72 °C for 10 min. The PCR products were analyzed on a 1% agarose gel in 1 TAE buffer, run at 100 V for 1 h. Gels were stained with ethidium bromide and photographed. Amplified PCR products were eluted from the gel using a gel extraction kit (Real Genomics Hi Yield TM Gel/PCR DNA Extraction Kit); the eluted fragment was then sequenced using PCR primers. Based on 1375 bp long 16S rRNA gene sequences, phylogenetically-related bacteria were aligned by using a BLAST search [17] against the GenBank database. Multiple alignments with sequences of related taxa of the genus
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Bacillus were implemented by using CLUSTAL_X. A neighbor-joining phylogenetic tree was constructed by using PHYLIP v3.6. The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein [18] with 1000 replicates. The NCBI GenBank accession number for the isolate is KF278973.
per pot were maintained. Six replicated pots per treatment with three plants in each pot were placed in a randomized block design in net house. After 2 months, % germination, vigor index, seedling traits, nitrogen, phosphorus, and potassium (NPK) content of soil and shoot biomass were studied [20]. The vigor index was calculated using the following formula [21].
Whole cell fatty acids methylester analysis The isolate was identifies based on whole-cell fatty acids, derivatized to methyl esters, and analyzed by gas chromatography using the Sherlock Microbial Identification System (MIS-MIDI, USA). The fatty acids methyl ester (FAME) profiles were compared with the TSBA50 aerobe library general software v5.0. Qualitative and quantitative differences in the fatty acid profiles were used to compute the distance for each strain relative to the strains in the library [19].
Vigour index ¼ ðmean root length þ mean shoot lengthÞ germination ð%Þ
Effect of various ingredients of PVK broth, different concentrations of yeast extract and KH2PO4 on tri-calcium phosphate solubilization, and plant growth-promoting attributes P-solubilization, indole-acetic acid, siderophore, and antifungal activity of isolate CB7 was determined under the influence of each ingredients of PVK medium, different concentrations of yeast extract (0, 0.05, 0.10, 1.0, 1.5, 2.0, and 2.5%) and various concentration of soluble P in KH2PO4 (0.05, 0.1, 0.125, 0.15, 0.20, and 0.25%) in PKV broth after 72 h of incubation. Two hundred and fifty milliliter-Erlenmayer flask containing 50 ml of PKV broth medium was inoculated with 0.1 ml of culture (106 cells ml1). Uninoculated flasks served as a control. The flasks were incubated at a range of temperature and pH with shaking (150 rpm) on a rotary shaker. The culture was harvested by centrifugation at 10,000 rpm for 20 min. Supernatant was used to assess phosphate and secondary metabolites released into the solution. Plant growth-promotion ability in tomato The plant growth-promotion ability of the isolate was conducted using tomato seedlings in sterile soil under net house conditions. Soil obtained was sieved through 2 mm sieve and used for pot culture experiment. The sand, soil, and farm-yard manure was mixed in a ratio of 1:1:1 in order to make the potting mixture. The mixture was then filled in the 20 cm 20 cm pots at the rate of 2 kg per pot and moistened to one-third saturation capacity. Twentyfive seeds treated or untreated with cell suspension of 1.5 OD were sown at equidistance and covered with moss grass till emergence of plumule. After 3–4 days of seedlings emergence thinning was done and three plants ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Germination energy index was computed using the formula: GEI ¼ ½A1 ðA1 þ A2Þ þ ðA1 þ A2 þ A3Þ þ þ ðA1 þ þ AaÞ=N þ n 100 where A1, A2, and A3 is the number of seeds newly germinated up to nth day respectively, N is the total number of seeds used for treatment, and n is the number of days of observations. Statistical analysis Data were statistically analyzed by analysis of variance using the general linear model developed by the SAS Institute (version 9.1; Cary, NC), and means were compared using the least significant difference (LSD) method; p 0.05 was considered significant.
Results Isolation and characterization of the bacterial isolate Among 206 phosphate solubilizing bacterial isolates, CB7 showed high P-solubilization and production of multifarious plant growth-promoting attributes in tandem. A bacterial isolate CB7 producing about 18-mm zone of P-solubilization after 48 h incubation on PVK agar and morphologically showed a resemblance to Bacillus in major phenotypic characteristics. The bacterial isolate was Gram-positive, motile rods, with round wavy, convex rough surface, and cream in color. The isolate was positive for utilization of citrate, casein hydrolysis, catalase, gelatin hydrolysis, arginine dihydrolase cytochrome oxidase, ONPG, esculin hydrolysis, negative for nitrate reduction, hydrogen sulfide, methyl red test, indole production, tyrosine utilization, urea hydrolysis, oxidation/fermentation (O/F), starch hydrolysis, and utilizes number of carbon sources (Table 2). It was able to grow over a wide range of temperatures 10–42 °C, with optimum at 30 2 °C. It had a pH tolerance over the range of 5–11, with optimum
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Table 2. Morphological, biochemical, and physiological characterization of B. circulans CB7. Morphological characteristics Colony morphology Grams reaction/cell shape/cell size Spore shape/position
Cream, round wavy, convex rough surface þ/Rods/long Oval/central
Physiological characteristics Growth in NaCl concentration (2.5–8.5%) Growth at different temperature (10–42 °C) Growth at different pH (5.0–11) Biochemical tests/carbohydrates utilization Catalase þ Malonate Voges Proskauer’s Citrate utilization þ ONPG þ Nitrate reduction Arginine dihydrolase þ Hydrogen sulfide production Gelatin hydrolysis þ Starch hydrolysis Casein decomposition þ Indole production Esculin hydrolysis þ Tyrosine utilization Lactose Xylose
þ þ þ Fructose Dextrose Galactose Raffinose Trehalose Melibose Sucrose Mannose Inulin Sodium glucanate Glycerol Salicin Dulcitol Inositol Sorbitol Arabitol
þ þ þ þ þ
Erythritol a-Methyl-D-glucoside Rhamnose Cellobiose Melezilose a-Methyl-D-mannoside Xylitol D-Arabinose Sorbose Adonitol Oxidation/fermentation (O/F) Urea hydrolysis Cytochrome oxidase
þ
þ, tested positive/utilized as substrate; , tested negative/not utilized as substrate.
7.0 0.5 and could tolerate 8.5% of NaCl concentration (w/v). Isolate CB7 was initially identified using the biochemical analysis and was confirmed by 16S rRNA gene sequencing. The identification was also confirmed at the Microbial Type Culture Collection (MTCC) and Gene Bank, Institute of Microbial Technology (IMTECH), Chandigarh, India and the isolate was identified as B. circulans MTCC 8983. Molecular analysis based on 16S rRNA homology of 1394-bp partial sequence confirmed that isolate CB7 belongs to Bacillus genus. In the phylogenetic tree, isolate CB7 clustered closely with B. circulans (EU733231Bc, EU 373401Bc) with high boot strap value (100%) (Fig. 1). Isolate contained straight-chain and terminally branched saturated and mono-unsaturated fatty acids with a composition of 6.53, 72.63, and 15.42%, respectively. The fatty acids iso-C14:0, C14:0, iso-C15:1F, isoC15:0, anteiso-C15:0, C16:1 w7c alcohol, iso-C16:0, C16:1 w11c, C16:0, iso-C 17:1 w10c, and iso-C17:0 were present in the isolate CB7 (Table 3). Among the fatty acids measured, tetradecanoic acid (14:0), 13-methyl pentadecanoic acid (15:0 iso), and 12-methyl tetradecanoic acid (15:0 anteiso) were the major fatty acids of the isolate CB7. The analysis showed the highest similarity of the isolate with B. circulans as per MIDI system (Microbial Identification System, Inc.). ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Effects of various ingredients of PVK on tricalcium phosphate solubilization and plant growth-promoting trails The results (Fig. 2a and b) showed that maximum P-solubilization (832 mg/L), % siderophore unit (85.21%), % growth inhibition (76.67%), and indole-acetic acid production (26.2 mg ml 1 ) were found in control PVK where all the ingredients were present. Minimum P-solubilization (40.26%), % siderophore unit (37.59%), % growth inhibition (48.08%), and indoleacetic acid production (11.6 mg ml 1 ) were observed in PVK broth without glucose. P-solubilization ability of the isolate CB7 decreased by about 59.74% in the absence of glucose in PVK broth as compared to control. The statistical analysis revealed that the difference in % P-solubilization between PVK without glucose and FeSO 4 was statistically at par with each other. Indole-acetic acid, % siderophores unit, and antifungal activity were significantly lower in medium PVK glucose. The final pH decreased from original 7.4 to minimum 4.4 in control PVK broth. The difference in pH between PVK-glucose and PVK-MgSO4; PVK-KCl, and extract; PVKMnSO4 and (NH4)2SO4 was statistically at par with each other (data not shown).
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Figure 1. Neighbor-joining phylogenetic dendrogram based on 16S rRNA sequences showing relationships between isolate CB7 (MTCC 8983) and related taxa. Only the bootstrap percentages higher than 50% are shown at branching points.
Effects of different concentrations of yeast extract on tri-calcium phosphate solubilization, and plant growth-promoting traits The results (Fig. 3a and b) revealed that P-solubilization, indole-acetic acid, % siderophore unit, and % growth inhibition against D. necatrix were significantly higher in control PVK containing 0.05% yeast extract and thereafter decreased gradually with increase in concentration of yeast extract in PVK medium. P-solubilization ability of B. circulans CB7 decreased by about only 1.6% in the absence of yeast extract. The deletion of yeast extract from PVK media had non-significant effect on P-solubilization. % P-solubilization decreased gradually with increase in concentration of yeast extract from 0.1 to 5%. Minimum % P-solubilization (21.2%) was found ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
in PVK medium containing 5.0% of yeast extract and P-solubilization decreased by 78.8% as compared to control. There was a gradual increase in the final pH with increase in the concentration of yeast extract and with statistical significant difference among each yeast extract concentration. The deletion of yeast extract from PVK media had non-significant effect on indole-acetic acid production and was found to be at par with control PVK containing 0.05% of yeast extract. Effect of addition of soluble phosphorus (KH2PO4) on tri-calcium phosphate solubilization and plant growth-promoting traits The results (Fig. 4a and b) revealed that P-solubilization decreased gradually from 832.0 mg L1 in PVK medium
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Table 3. Cellular fatty acid composition of B. circulans CB7. Fatty acids
Composition (%)
C14:0 C16:0 Iso-C14:0 Iso-C15:1F Iso-C15:0 Iso-C16:0 Iso-C17:0 Anteiso-C15:0 Anteiso-C17:0 C16:1 w7c alcohol C16:1 w11c 16:1 w7c/15 iso 2OH Iso-C 17:1 w10c 17:1 ISO I/ANTEI B
1.31 5.22 2.12 0.64 9.22 2.86 3.36 50.03 4.4 0.89 5.22 2.13 5.15 2.03
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without KH2PO4 to 26.00 mg L1 at highest concentration of 0.25% of KH2PO4. There was a gradual increase in the final pH with increase in the concentration of KH2PO4 and with statistical significant difference among each soluble P-concentration. Indole-acetic acid, % siderophore unit, and % growth inhibition of D. necatrix also decreased significantly with increase in concentration of KH2PO4. Organic acid production in PVK broth The six major peaks were identified as oxalic acid, gluconic acid, formic acid, 2-ketogluconic acid, citric acid, and fumaric acid by comparing the retention times with those of the authentic standards (data not shown). Gluconic acid (2.33%) and citric acid (0.76%) were detected as the major organic acids with small
Figure 2. Effects of various ingredients of PVK broth by B. circulans CB7 on TCP solubilization and IAA (a), siderophore production and antifungal activity against D. necatrix. (b). Each value represents the mean of three replicates and the error bars represent the standard deviations of the average.
Figure 3. Effects of different concentrations of yeast extract by B. circulans CB7on TCP solubilization and IAA (a), siderophore production and antifungal activity against D. necatrix (b). Each value represents the mean of three replicates and the error bars represent the standard deviations of the average. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Figure 4. Effects of different concentration of KH2PO4 by B. circulans CB7 on TCP solubilization and IAA (a), siderophore production and antifungal activity against D. necatrix (b). Each value represents the mean of three replicates and the error bars represent the standard deviations of the average.
percentages of 2-ketogluconic acid (0.39%), formic acid (0.18%), oxalic acid (0.20%), and fumaric acid (0.05%) during TCP solubilization by B. circulans CB7 (Table 1). Plant growth-promotion potential The plant growth-promotion potential of B. circulans CB7 was determined in tomato seeds. A significant influence on growth was resulted with treatment of B. circulans CB7 in tomato seedlings grown in pots under net house conditions. It was observed that the bacterized seedlings recorded 30.24 and 16.91% higher root and shoot lengths compared to uninoculated control (Table 4). Seed bacterization resulted in greater enhancement of the root growth, as compared to the shoot growth. Increase in shoot dry weight (63.50%) and root dry weight (54.08%) was also observed. It was observed that NPK content of soil showed significant difference over untreated control. Significant higher level of nitrogen (382.59 kg ha1), phosphorus (143.759 kg ha1), and potassium (326.2 kg ha1) as compared to untreated control was observed (Fig. 5a). NPK content of the soil before sowing of tomato seed was 194.4, 108.0, and
201.9 kg ha1, respectively. The shoot analysis of NPK revealed a significantly higher level of nitrogen (0.76%), phosphorus (0.44%), and potassium (2.87%) content in seedlings treated with B. circulans CB7 as compared to untreated control (Fig. 5b).
Discussion In the present study, the efficacy of P-solubilizing rhizobacteria CB7 isolated from apple rhizosphere was observed for growth-promoting effect on tomato seedlings under net house conditions to test whether selected P-solubilizing PGPR is crop specific or not. Phosphorous nutrition influences overall plant growth and root development. In view of environmental concerns and current developments in sustainability, research efforts are concentrated on elaboration of techniques that involve the use of less expensive, though less bio-available sources of plant nutrients such as rock and by application of PSB and the agronomic effectiveness can be enhanced [22]. A potential B. circulans CB7
Table 4. Effect of inoculation of B. circulans CB7 on tomato seedlings after 60 days of sowing. Treatments
Control
Bacillus sp. CB7
CD0.05
Germination (%) Germination energy index Vigor index Root length (cm) Shoot length (cm) Root dry weight (g) Shoot dry weight (g)
65.67 1.20 288.33 4.41 1706.67 12.02 7.25 0.05 18.92 0.06 0.70 0.03 3.08 0.05
80.33 1.45 413.33 7.27 2375 16.07 9.07 0.06 21.93 0.06 0.92 0.03 4.71 0.04
5.38 24.23 57.21 0.23 0.06 0.03 0.05
Values are means of three replicates of three independent experiments standard deviation, control was used without culture.
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Figure 5. Effect of inoculation of B. circulans CB7 on percent increase in nitrogen, phosphorus, and potassium content in soil (a) and percent uptake in shoot biomass (b) in net house (60 days after sowing). Each value represents the mean of three replicates and the error bars represent the standard deviations of the average.
isolated from apple rhizosphere possessed multiple PGPTs, like P-solubilization, indole-acetic acid, ACC deaminase activity, nitrogenase activity, siderophore production, hydrocyanic acid production, and antifungal activity against D. necatrix (Table 1), which may promote plant growth directly, indirectly, or synergistically, suggesting that the application of this PGPR with multifaceted PGPTs could be more beneficial biofertilizer. The results suggested that the bacterium could be indirectly augmenting the availability of phosphorus because the siderophore production also is one of the mechanisms involved in the solubilization of iron-bound phosphorus by the microorganisms. Hydrocyanic acid production by rhizospheric bacterium has been variably viewed, while it is considered effective from the biocontrol point of view [23]. The study suggests that the occurrence of higher P-solubilizing siderophore producers in apple rhizosphere is of direct significance to plants as it helps in iron sequestering near the roots, especially in iron deficient conditions. In addition, siderophores and hydrocyanic acid producing microorganisms protects plants at two levels: first, limiting growth of plant pathogens and secondly triggering plants defensive mechanism [24]. In the present work, P-solubilization was dependent on the presence of glucose in the medium and removal of yeast extract had no significant effect on TCP solubilization (Fig. 2). These observations agree with those of Nautiyal [25], who reported that glucose and TCP were essential and yeast extract non-essential components of the medium. It was also reported that at the yeast extract concentration of 0.1 g L1 (0.01%), the P-solubilization ability of Pseudomonas sp. increased by 44%. The yeast concentration of 0.01% was not included in the present experimentation. Therefore, it is premature to assume that B. circulans CB7 may also act like Pseudomonas sp. at concentration of yeast extract below 0.05% in PVK ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
medium. However, the observation that the concentration of yeast extract above 0.05% decreased P-solubilization (Fig. 3) further prove that the presence of yeast extract in PVK medium may be inhibitory to the P-solubilization. The noticeable observation that the exclusion of 0.05% yeast extract from PVK medium did not affect the P-solubilization has led to the formulation of a defined medium to elucidate the role of bacteria in P-solubilization. Also, the yeast extract can be replaced with 0.05% ammonium sulfate as nitrogen source in this synthetic medium. Therefore, the synthetic medium developed in this study may include glucose as carbon source and ammonium sulfate as nitrogen source. This modified synthetic medium is similar to the NBRIP medium [25] with variation in concentration of ingredients and differ from PVK with replacement of yeast extract by ammonium sulfate as nitrogen source. Antifungal activity and production of secondary metabolites were dependent on nutrients in the assay medium (Fig. 2). These observations are in agreement with those of Vijayakumari et al. [26] who observed that production of secondary metabolites was dependent on the composition of the growth medium. Small deviations in medium components can make a significant difference in the production of metabolites. Our study shows that when glucose was deleted from PVK medium, production of secondary metabolites (indole-acetic acid, siderophore, and antifungal activity) were significantly decreased as compared to PVK having glucose, suggesting that glucose is essential ingredient in PVK broth. Previous study [27] showed that the addition of glucose resulted in dramatic increase of pyochelin and salicylic acid, indicating that the ratio of carbon source to nutrient concentration played a key role in the metabolic flow. P-solubilization ability of B. circulans CB7 was repressed in the presence of additional soluble phosphorus (Fig. 4)
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indicating a role of source of soluble phosphorus in the TCP solubilization ability of B. circulans CB7. Our results are in agreement with previous study that P-solubilizing activity of Erwinia herbicola was negatively correlated with the amount of soluble phosphorus in the medium and 0.1% soluble phosphorus suppressed visible P-solubilization in dicalcium phosphate plate [9] and 0.5% soluble phosphorus suppressed P-solubilization by rhizobial strain [28]. In cultivated soils, soluble phosphorus ranges from 0.3 to 1 mg ml1 in solution phase and extreme value ranged from
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Research Paper Efficiency of plant growth-promoting P-solubilizing Bacillus circulans CB7 for enhancement of tomato growth under net house conditions Preeti Mehta1, Abhishek Walia2, Saurabh Kulshrestha3, Anjali Chauhan2 and Chand Karan Shirkot2 1
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Department of Advance Bioenergy Research, Research and Development Centre, Indian Oil Corporation Limited, Government of India, Faridabad, New Delhi, India Department of Basic Sciences (Microbiology Section), Dr. Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India School of Biotechnology, Shoolini University of Biotechnology and Management Sciences, Solan, Himachal Pradesh, India
P-solubilizing bacterial isolate CB7 isolated from apple rhizosphere soil of Himachal Pradesh, India was identified as Bacillus circulans on the basis of phenotypic characteristics, biochemical tests, fatty acid methyl esters analysis, and 16S rRNA gene sequence. The isolate exhibited plant growthpromoting traits of P-solubilization, auxin, 1-aminocyclopropane-1-carboxylate deaminase activity, siderophore, nitrogenase activity, and antagonistic activity against Dematophora necatrix. In vitro studies revealed that P-solubilization and other plant growth-promoting traits were dependent on the presence of glucose in PVK medium and removal of yeast extract had no significant effect on plant growth-promoting traits. Plant growth-promoting traits of isolate CB7 were repressed in the presence of KH2PO4. P-solubilization activity was associated with the release of organic acids and a drop in the pH of the Pikovskaya’s medium. HPLC analysis detected gluconic and citric acid as major organic acids in the course of P-solubilization. Remarkable increase was observed in seed germination (22.32%), shoot length (15.91%), root length (25.10%), shoot dry weight (52.92%) and root dry weight (31.4%), nitrogen (18.75%), potassium (57.69%), and phosphorus (22.22%) content of shoot biomass over control. These results demonstrate that isolate CB7 has the promising PGPR attributes to be developed as a biofertilizer to enhance soil fertility and promote plant growth. Keywords: PGPR / P-solubilization / Siderophore / ACC deaminase / Tomato Received: July 12, 2013; accepted: November 16, 2013 DOI 10.1002/jobm.201300562
Introduction Phosphorus is one of the major essential macronutrients for biological growth and development. On average, soil contains 0.02–0.5% of total phosphorus [1]. It is added to the soil in the form of P- fertilizers, a part of which (1%) is utilized by plants and the rest is rapidly converted into insoluble complexes. Phosphate anions (H2PO4, HPO42) are extremely reactive and form metal complexes with Correspondence: Prof. C. K. Shirkot, Department of Basic Sciences, Dr. Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan 173230, Himachal Pradesh, India E-mail: [email protected] Phone: þ91 9418117399 Fax: þ91 1792252354 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Ca in calcareous soils and Fe3þand Al3þ in acidic soils. These metal ion complexes precipitated the 80% of added P-fertilizer [2]. This leads to the need of frequent application of P-fertilizers, but its use on a regular basis has become a costly affair and environmentally undesirable. Therefore, the necessity to develop economical and eco-friendly technologies is steadily increasing. The use of plant growth-promoting rhizobacteria (PGPR) including phosphate solubilizing bacteria as biofertilizers was suggested as a sustainable solution for the improvement of nutrient availability, plant growth, and yields [3]. The mechanism by which PGPR promote plant growth are not fully understood, but are thought to include: (a) the ability to produce plant hormones, such as auxins, cytokinins, gibberellins, (b) asymbiotic N2 fixation, (c) solubilization of
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inorganic phosphate and mineralization of organic phosphate and/or other nutrients, and (d) antagonism against phytopathogenic microorganisms by production of siderophores, the synthesis of antibiotics, enzymes, and/or fungicidal compounds and competition with detrimental microorganisms [3]. Soil–plant–microbe interactions are complex and there are many ways in which the outcome can influence the plant health and productivity [4]. The interaction may be harmful, beneficial, and neutral to the plants. However, our focus should be to exploit the beneficial interaction of plants and microbes. The use of microbial technologies in agriculture/horticulture is currently expanding quite rapidly with the identification of new bacterial strains, which are more effective in promoting plant growth. Studies have shown that the growth-promoting ability of some bacteria may be highly specific to certain plant species, cultivar, and genotype [5]. Some PGPR exhibit narrow specificity, i.e., Chanway et al. [6] found that six of seven Bacillus subtilis strains enhanced growth of wheat cultivar Katepwa, but none promoted growth of cultivar Neepawa. Other PGPR may exhibit much broader specificity. It appears that PGPR strains vary widely in their responses to specific environmental parameters. For example, PGPR strain GB03 colonizes cotton roots and provides biological control against Ralstonia solani throughout the Cotton Belt, from Texas to the south-eastern United States. Similarly, the same strain acts as a biological control agent against R. solani on peanut from the Southeast to Texas and Oklahoma. The same group of PGPR strains is reported to control soil-borne pathogens throughout the country of China [7]. Knowing the fact that some PGPR strains lack tight host specificity, future selection of new PGPR should be designed to test for broad host ranges for crops colonized, thereby increasing the chances of routine use of PGPR in future biofertilization strategies. Accordingly, this study examined the ability to produce plant growth-promoting metabolites by B. circulans CB7 isolated from apple rhizosphere under in vitro condition, along with changes in the early stages of tomato seedlings growth after inoculation with B. circulans CB7 under net house. Influence of certain factors like carbon and nitrogen sources, availability of free phosphorous on the P-solubilization and other plant growth-promoting traits of the selected PGPR isolate was also determined.
Materials and methods Isolation The soil used for bacterial isolation was collected from rhizosphere of apple trees growing at different locations ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
of Chamba in the Indian trans-Himalayan region of Himachal Pradesh, India, located at an altitude of 2077 m above mean sea level. The processed soil sample was serially diluted, spread plated on full strength nutrient agar and incubated at 30 2 °C for 48 h. A total of 206 different colonies were isolated on nutrient agar and were purified with repeated culturing and maintained in 20% glycerol at 80 °C. A potential isolate CB7 was screened and selected on the basis of halo zone produced in Pikovskaya’s (PVK) agar and assessed for morphology, physiology, and Gram reaction and other characterization. Bacterial identification and characterization Isolates were subsequently differentiated by Gram reaction, salt tolerance, biochemical characterization, and microscopic observation. The ability of the isolates to grow in diverse temperature range was carried out by growing isolates in nutrient broth and incubated at different temperatures ranged from 4 to 65 °C, respectively. Growth was recorded every 24 h at OD600 upto 96 h. The ability of the isolates to grow in different salt concentrations was carried out by inoculating bacterial culture on nutrient agar plates supplemented with 2.5– 10% w/v NaCl and the plates were incubated at 30 2 °C for 3 days. The ability of isolates to grow in alkaline or acidic media was tested in nutrient agar plates in which the pH was adjusted from 5.0 to 11.0 and incubated at 30 2 °C for 3 days. Phenotypic characterization of the isolates was done based on their colony morphology, microscopic observations, and biochemical tests using commercial kits (KB009 Hi carbohydrateTM kit and KB013 Hi BacillusTM Identification kit). A cream whitish colored bacterial colony showing above 15 mm zone of P-solubilization, maximum of 8.5% NaCl tolerance, and growth in wide pH range was selected for further analysis. Among 206 PSBs, CB7 which had a marked P-solubilizing activity on PVK agar medium/broth and also showed maximum effectiveness of multifarious plant growth-promoting attributes (Table 1) was selected in vitro evaluation for tri-calcium phosphate (TCP) solubilization and plant growth-promoting traits under various conditions in PVK broth and its interaction with tomato seedlings under net house condition. Phosphate solubilization and organic acid production Isolates were first screened on PVK agar plates. The P-solubilization was exhibited with a clear zone formed around the colony. Further, quantitative estimation of phosphorus was done in PVK broth amended with 5.0 g L1 TCP by the vanadomolybdate method [8]. For the analysis of organic acids, bacterial culture was filtrated through 0.2 mm filter (Millipore, GTBP) and 20 ml of
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Table 1. Plant growth-promoting attributes of B. circulans CB7 after 72 h of incubation. PGP attribute activity Phosphate solubilization index (PSI) TCP solubilization (mg L1) Organic acid production (%) Oxalic acid Gluconic acid Formic acid 2-Ketogluconic acid Citric acid Fumaric acid Nitrogenase activity (hmole C2H4 h1 mg1 protein) Nitrogen fixation (mg ha1 per day) IAA production (mg ml1) Siderophore production Zone (mm) Units (%) Chitinase activity Zone (mm) Chitinase enzyme index Antagonistic activity against D. necatrix (%) HCN production ACC-deaminase activity
2.7 1.12 828 3.2 0.20 0.02 2.33 0.03 0.18 0.05 0.39 0.08 0.76 0.07 0.05 0.12 267.6 2.8 98 4.32 27.3 1.06 18.31 1.12 83.4 1.17 28.00 1.00 12.61 1.30 73.8 1.4 Positive Positive
Values are the mean of three replicates standard deviation.
filtrates were injected to HPLC (Waters 996 HPLC) equipped with Photo Diode Array detector. The organic acid separation was carried out on RP-18 column (Merck, Germany) with 0.1% orthophosphoric acid (Merck) as mobile phase. Retention time of each signal was recorded at a wavelength of 210 nm and compared with the standard acids. Indole-acetic acid, aminocyclopropane-1-carboxylate deaminase, and nitrogenase activity Estimation of indole-acetic acid was done by inoculation of bacterial suspension in PVK broth not amended with Ltryptophan (500 mg ml1) and incubating it at 30 2 °C for 72 h. The indole-acetic acid content in the culture suspension was estimated by the standard procedure [9]. Aminocyclopropane-1-carboxylate (ACC)-deaminase activity of the isolate was assessed by quantifying ammonia liberated by the hydrolysis of ACC. ACC-deaminase activity was detected on plates with DF minimal medium containing ACC as the sole source of nitrogen [10]. Nitrogen fixation of isolate was determined in nitrogen free medium by the acetylene reduction assay [11]. Siderophore, hydrocyanic acid production Siderophore production was detected by the standard Chrome Azurol-S assay [12] and the diameter of the clearing zone was measured. Hydrocyanic acid ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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production was inferred by the qualitative method of Bakker and Schipper [13]. The change in the color of the filter paper previously dipped in 2% sodium carbonate prepared in 0.05% picric acid, from yellow to dark brown was rated visually depending on the intensity of the color change. Chitinolytic and antifungal activity The bacterial cultures were spotted on to the prepared minimal agar medium amended with 0.3% colloidal chitin and the plates were incubated at 30 2 °C for 4 days. Development of clear halo zone around the colony after addition of iodine was considered as positive for chitinase enzyme production [14]. Antagonistic activity of the culture filtrate (10%) of the isolate was tested against Dematophora necatrix (the fungal pathogen causing white root rot of apple) on potato dextrose agar using agar dilution technique [15]. Percentage of growth inhibition was calculated using the formula proposed by Vincent [16]. All the studies were repeated on three independent dates to confirm the results. DNA extraction, 16S rRNA gene sequencing, and phylogenetic analysis The bacterial isolate was grown in nutrient broth at 30 2 °C overnight. Bacterial cells were harvested by centrifugation at 5000g for 5 min and DNA was isolated from these bacterial cells by using a Real Genomics DNA Extraction Kit (Life Technologies, Delhi, India). The isolated DNA was finally suspended in 100 ml elution buffer and quantified on a 1% agarose gel. The total genomic DNA was kept at 20 °C before use. Species level identification of the isolate was conducted by 16S rRNA sequence comparison. PCR reactions were carried out in 20 ml reaction buffer containing 50 ng template DNA, 20 pmol of each primer forward (50 -GCAAGTCGAGCGGACAGATGGGAGC-30 ) and reverse primer (50 -AACTCTCGTGGTGTGACGGGCGGTG-30 ), 0.2 mM dNTPs, and 1 U Taq polymerase (MP Biomedicals, Santa Ana, CA) in 1 PCR buffer. Reaction were cycled 35 times as 94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min 30 s followed by final extension at 72 °C for 10 min. The PCR products were analyzed on a 1% agarose gel in 1 TAE buffer, run at 100 V for 1 h. Gels were stained with ethidium bromide and photographed. Amplified PCR products were eluted from the gel using a gel extraction kit (Real Genomics Hi Yield TM Gel/PCR DNA Extraction Kit); the eluted fragment was then sequenced using PCR primers. Based on 1375 bp long 16S rRNA gene sequences, phylogenetically-related bacteria were aligned by using a BLAST search [17] against the GenBank database. Multiple alignments with sequences of related taxa of the genus
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Bacillus were implemented by using CLUSTAL_X. A neighbor-joining phylogenetic tree was constructed by using PHYLIP v3.6. The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein [18] with 1000 replicates. The NCBI GenBank accession number for the isolate is KF278973.
per pot were maintained. Six replicated pots per treatment with three plants in each pot were placed in a randomized block design in net house. After 2 months, % germination, vigor index, seedling traits, nitrogen, phosphorus, and potassium (NPK) content of soil and shoot biomass were studied [20]. The vigor index was calculated using the following formula [21].
Whole cell fatty acids methylester analysis The isolate was identifies based on whole-cell fatty acids, derivatized to methyl esters, and analyzed by gas chromatography using the Sherlock Microbial Identification System (MIS-MIDI, USA). The fatty acids methyl ester (FAME) profiles were compared with the TSBA50 aerobe library general software v5.0. Qualitative and quantitative differences in the fatty acid profiles were used to compute the distance for each strain relative to the strains in the library [19].
Vigour index ¼ ðmean root length þ mean shoot lengthÞ germination ð%Þ
Effect of various ingredients of PVK broth, different concentrations of yeast extract and KH2PO4 on tri-calcium phosphate solubilization, and plant growth-promoting attributes P-solubilization, indole-acetic acid, siderophore, and antifungal activity of isolate CB7 was determined under the influence of each ingredients of PVK medium, different concentrations of yeast extract (0, 0.05, 0.10, 1.0, 1.5, 2.0, and 2.5%) and various concentration of soluble P in KH2PO4 (0.05, 0.1, 0.125, 0.15, 0.20, and 0.25%) in PKV broth after 72 h of incubation. Two hundred and fifty milliliter-Erlenmayer flask containing 50 ml of PKV broth medium was inoculated with 0.1 ml of culture (106 cells ml1). Uninoculated flasks served as a control. The flasks were incubated at a range of temperature and pH with shaking (150 rpm) on a rotary shaker. The culture was harvested by centrifugation at 10,000 rpm for 20 min. Supernatant was used to assess phosphate and secondary metabolites released into the solution. Plant growth-promotion ability in tomato The plant growth-promotion ability of the isolate was conducted using tomato seedlings in sterile soil under net house conditions. Soil obtained was sieved through 2 mm sieve and used for pot culture experiment. The sand, soil, and farm-yard manure was mixed in a ratio of 1:1:1 in order to make the potting mixture. The mixture was then filled in the 20 cm 20 cm pots at the rate of 2 kg per pot and moistened to one-third saturation capacity. Twentyfive seeds treated or untreated with cell suspension of 1.5 OD were sown at equidistance and covered with moss grass till emergence of plumule. After 3–4 days of seedlings emergence thinning was done and three plants ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Germination energy index was computed using the formula: GEI ¼ ½A1 ðA1 þ A2Þ þ ðA1 þ A2 þ A3Þ þ þ ðA1 þ þ AaÞ=N þ n 100 where A1, A2, and A3 is the number of seeds newly germinated up to nth day respectively, N is the total number of seeds used for treatment, and n is the number of days of observations. Statistical analysis Data were statistically analyzed by analysis of variance using the general linear model developed by the SAS Institute (version 9.1; Cary, NC), and means were compared using the least significant difference (LSD) method; p 0.05 was considered significant.
Results Isolation and characterization of the bacterial isolate Among 206 phosphate solubilizing bacterial isolates, CB7 showed high P-solubilization and production of multifarious plant growth-promoting attributes in tandem. A bacterial isolate CB7 producing about 18-mm zone of P-solubilization after 48 h incubation on PVK agar and morphologically showed a resemblance to Bacillus in major phenotypic characteristics. The bacterial isolate was Gram-positive, motile rods, with round wavy, convex rough surface, and cream in color. The isolate was positive for utilization of citrate, casein hydrolysis, catalase, gelatin hydrolysis, arginine dihydrolase cytochrome oxidase, ONPG, esculin hydrolysis, negative for nitrate reduction, hydrogen sulfide, methyl red test, indole production, tyrosine utilization, urea hydrolysis, oxidation/fermentation (O/F), starch hydrolysis, and utilizes number of carbon sources (Table 2). It was able to grow over a wide range of temperatures 10–42 °C, with optimum at 30 2 °C. It had a pH tolerance over the range of 5–11, with optimum
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Table 2. Morphological, biochemical, and physiological characterization of B. circulans CB7. Morphological characteristics Colony morphology Grams reaction/cell shape/cell size Spore shape/position
Cream, round wavy, convex rough surface þ/Rods/long Oval/central
Physiological characteristics Growth in NaCl concentration (2.5–8.5%) Growth at different temperature (10–42 °C) Growth at different pH (5.0–11) Biochemical tests/carbohydrates utilization Catalase þ Malonate Voges Proskauer’s Citrate utilization þ ONPG þ Nitrate reduction Arginine dihydrolase þ Hydrogen sulfide production Gelatin hydrolysis þ Starch hydrolysis Casein decomposition þ Indole production Esculin hydrolysis þ Tyrosine utilization Lactose Xylose
þ þ þ Fructose Dextrose Galactose Raffinose Trehalose Melibose Sucrose Mannose Inulin Sodium glucanate Glycerol Salicin Dulcitol Inositol Sorbitol Arabitol
þ þ þ þ þ
Erythritol a-Methyl-D-glucoside Rhamnose Cellobiose Melezilose a-Methyl-D-mannoside Xylitol D-Arabinose Sorbose Adonitol Oxidation/fermentation (O/F) Urea hydrolysis Cytochrome oxidase
þ
þ, tested positive/utilized as substrate; , tested negative/not utilized as substrate.
7.0 0.5 and could tolerate 8.5% of NaCl concentration (w/v). Isolate CB7 was initially identified using the biochemical analysis and was confirmed by 16S rRNA gene sequencing. The identification was also confirmed at the Microbial Type Culture Collection (MTCC) and Gene Bank, Institute of Microbial Technology (IMTECH), Chandigarh, India and the isolate was identified as B. circulans MTCC 8983. Molecular analysis based on 16S rRNA homology of 1394-bp partial sequence confirmed that isolate CB7 belongs to Bacillus genus. In the phylogenetic tree, isolate CB7 clustered closely with B. circulans (EU733231Bc, EU 373401Bc) with high boot strap value (100%) (Fig. 1). Isolate contained straight-chain and terminally branched saturated and mono-unsaturated fatty acids with a composition of 6.53, 72.63, and 15.42%, respectively. The fatty acids iso-C14:0, C14:0, iso-C15:1F, isoC15:0, anteiso-C15:0, C16:1 w7c alcohol, iso-C16:0, C16:1 w11c, C16:0, iso-C 17:1 w10c, and iso-C17:0 were present in the isolate CB7 (Table 3). Among the fatty acids measured, tetradecanoic acid (14:0), 13-methyl pentadecanoic acid (15:0 iso), and 12-methyl tetradecanoic acid (15:0 anteiso) were the major fatty acids of the isolate CB7. The analysis showed the highest similarity of the isolate with B. circulans as per MIDI system (Microbial Identification System, Inc.). ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Effects of various ingredients of PVK on tricalcium phosphate solubilization and plant growth-promoting trails The results (Fig. 2a and b) showed that maximum P-solubilization (832 mg/L), % siderophore unit (85.21%), % growth inhibition (76.67%), and indole-acetic acid production (26.2 mg ml 1 ) were found in control PVK where all the ingredients were present. Minimum P-solubilization (40.26%), % siderophore unit (37.59%), % growth inhibition (48.08%), and indoleacetic acid production (11.6 mg ml 1 ) were observed in PVK broth without glucose. P-solubilization ability of the isolate CB7 decreased by about 59.74% in the absence of glucose in PVK broth as compared to control. The statistical analysis revealed that the difference in % P-solubilization between PVK without glucose and FeSO 4 was statistically at par with each other. Indole-acetic acid, % siderophores unit, and antifungal activity were significantly lower in medium PVK glucose. The final pH decreased from original 7.4 to minimum 4.4 in control PVK broth. The difference in pH between PVK-glucose and PVK-MgSO4; PVK-KCl, and extract; PVKMnSO4 and (NH4)2SO4 was statistically at par with each other (data not shown).
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Figure 1. Neighbor-joining phylogenetic dendrogram based on 16S rRNA sequences showing relationships between isolate CB7 (MTCC 8983) and related taxa. Only the bootstrap percentages higher than 50% are shown at branching points.
Effects of different concentrations of yeast extract on tri-calcium phosphate solubilization, and plant growth-promoting traits The results (Fig. 3a and b) revealed that P-solubilization, indole-acetic acid, % siderophore unit, and % growth inhibition against D. necatrix were significantly higher in control PVK containing 0.05% yeast extract and thereafter decreased gradually with increase in concentration of yeast extract in PVK medium. P-solubilization ability of B. circulans CB7 decreased by about only 1.6% in the absence of yeast extract. The deletion of yeast extract from PVK media had non-significant effect on P-solubilization. % P-solubilization decreased gradually with increase in concentration of yeast extract from 0.1 to 5%. Minimum % P-solubilization (21.2%) was found ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
in PVK medium containing 5.0% of yeast extract and P-solubilization decreased by 78.8% as compared to control. There was a gradual increase in the final pH with increase in the concentration of yeast extract and with statistical significant difference among each yeast extract concentration. The deletion of yeast extract from PVK media had non-significant effect on indole-acetic acid production and was found to be at par with control PVK containing 0.05% of yeast extract. Effect of addition of soluble phosphorus (KH2PO4) on tri-calcium phosphate solubilization and plant growth-promoting traits The results (Fig. 4a and b) revealed that P-solubilization decreased gradually from 832.0 mg L1 in PVK medium
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Table 3. Cellular fatty acid composition of B. circulans CB7. Fatty acids
Composition (%)
C14:0 C16:0 Iso-C14:0 Iso-C15:1F Iso-C15:0 Iso-C16:0 Iso-C17:0 Anteiso-C15:0 Anteiso-C17:0 C16:1 w7c alcohol C16:1 w11c 16:1 w7c/15 iso 2OH Iso-C 17:1 w10c 17:1 ISO I/ANTEI B
1.31 5.22 2.12 0.64 9.22 2.86 3.36 50.03 4.4 0.89 5.22 2.13 5.15 2.03
7
without KH2PO4 to 26.00 mg L1 at highest concentration of 0.25% of KH2PO4. There was a gradual increase in the final pH with increase in the concentration of KH2PO4 and with statistical significant difference among each soluble P-concentration. Indole-acetic acid, % siderophore unit, and % growth inhibition of D. necatrix also decreased significantly with increase in concentration of KH2PO4. Organic acid production in PVK broth The six major peaks were identified as oxalic acid, gluconic acid, formic acid, 2-ketogluconic acid, citric acid, and fumaric acid by comparing the retention times with those of the authentic standards (data not shown). Gluconic acid (2.33%) and citric acid (0.76%) were detected as the major organic acids with small
Figure 2. Effects of various ingredients of PVK broth by B. circulans CB7 on TCP solubilization and IAA (a), siderophore production and antifungal activity against D. necatrix. (b). Each value represents the mean of three replicates and the error bars represent the standard deviations of the average.
Figure 3. Effects of different concentrations of yeast extract by B. circulans CB7on TCP solubilization and IAA (a), siderophore production and antifungal activity against D. necatrix (b). Each value represents the mean of three replicates and the error bars represent the standard deviations of the average. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Figure 4. Effects of different concentration of KH2PO4 by B. circulans CB7 on TCP solubilization and IAA (a), siderophore production and antifungal activity against D. necatrix (b). Each value represents the mean of three replicates and the error bars represent the standard deviations of the average.
percentages of 2-ketogluconic acid (0.39%), formic acid (0.18%), oxalic acid (0.20%), and fumaric acid (0.05%) during TCP solubilization by B. circulans CB7 (Table 1). Plant growth-promotion potential The plant growth-promotion potential of B. circulans CB7 was determined in tomato seeds. A significant influence on growth was resulted with treatment of B. circulans CB7 in tomato seedlings grown in pots under net house conditions. It was observed that the bacterized seedlings recorded 30.24 and 16.91% higher root and shoot lengths compared to uninoculated control (Table 4). Seed bacterization resulted in greater enhancement of the root growth, as compared to the shoot growth. Increase in shoot dry weight (63.50%) and root dry weight (54.08%) was also observed. It was observed that NPK content of soil showed significant difference over untreated control. Significant higher level of nitrogen (382.59 kg ha1), phosphorus (143.759 kg ha1), and potassium (326.2 kg ha1) as compared to untreated control was observed (Fig. 5a). NPK content of the soil before sowing of tomato seed was 194.4, 108.0, and
201.9 kg ha1, respectively. The shoot analysis of NPK revealed a significantly higher level of nitrogen (0.76%), phosphorus (0.44%), and potassium (2.87%) content in seedlings treated with B. circulans CB7 as compared to untreated control (Fig. 5b).
Discussion In the present study, the efficacy of P-solubilizing rhizobacteria CB7 isolated from apple rhizosphere was observed for growth-promoting effect on tomato seedlings under net house conditions to test whether selected P-solubilizing PGPR is crop specific or not. Phosphorous nutrition influences overall plant growth and root development. In view of environmental concerns and current developments in sustainability, research efforts are concentrated on elaboration of techniques that involve the use of less expensive, though less bio-available sources of plant nutrients such as rock and by application of PSB and the agronomic effectiveness can be enhanced [22]. A potential B. circulans CB7
Table 4. Effect of inoculation of B. circulans CB7 on tomato seedlings after 60 days of sowing. Treatments
Control
Bacillus sp. CB7
CD0.05
Germination (%) Germination energy index Vigor index Root length (cm) Shoot length (cm) Root dry weight (g) Shoot dry weight (g)
65.67 1.20 288.33 4.41 1706.67 12.02 7.25 0.05 18.92 0.06 0.70 0.03 3.08 0.05
80.33 1.45 413.33 7.27 2375 16.07 9.07 0.06 21.93 0.06 0.92 0.03 4.71 0.04
5.38 24.23 57.21 0.23 0.06 0.03 0.05
Values are means of three replicates of three independent experiments standard deviation, control was used without culture.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Efficiency of plant growth-promoting P-solubilizing B. circulans
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Figure 5. Effect of inoculation of B. circulans CB7 on percent increase in nitrogen, phosphorus, and potassium content in soil (a) and percent uptake in shoot biomass (b) in net house (60 days after sowing). Each value represents the mean of three replicates and the error bars represent the standard deviations of the average.
isolated from apple rhizosphere possessed multiple PGPTs, like P-solubilization, indole-acetic acid, ACC deaminase activity, nitrogenase activity, siderophore production, hydrocyanic acid production, and antifungal activity against D. necatrix (Table 1), which may promote plant growth directly, indirectly, or synergistically, suggesting that the application of this PGPR with multifaceted PGPTs could be more beneficial biofertilizer. The results suggested that the bacterium could be indirectly augmenting the availability of phosphorus because the siderophore production also is one of the mechanisms involved in the solubilization of iron-bound phosphorus by the microorganisms. Hydrocyanic acid production by rhizospheric bacterium has been variably viewed, while it is considered effective from the biocontrol point of view [23]. The study suggests that the occurrence of higher P-solubilizing siderophore producers in apple rhizosphere is of direct significance to plants as it helps in iron sequestering near the roots, especially in iron deficient conditions. In addition, siderophores and hydrocyanic acid producing microorganisms protects plants at two levels: first, limiting growth of plant pathogens and secondly triggering plants defensive mechanism [24]. In the present work, P-solubilization was dependent on the presence of glucose in the medium and removal of yeast extract had no significant effect on TCP solubilization (Fig. 2). These observations agree with those of Nautiyal [25], who reported that glucose and TCP were essential and yeast extract non-essential components of the medium. It was also reported that at the yeast extract concentration of 0.1 g L1 (0.01%), the P-solubilization ability of Pseudomonas sp. increased by 44%. The yeast concentration of 0.01% was not included in the present experimentation. Therefore, it is premature to assume that B. circulans CB7 may also act like Pseudomonas sp. at concentration of yeast extract below 0.05% in PVK ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
medium. However, the observation that the concentration of yeast extract above 0.05% decreased P-solubilization (Fig. 3) further prove that the presence of yeast extract in PVK medium may be inhibitory to the P-solubilization. The noticeable observation that the exclusion of 0.05% yeast extract from PVK medium did not affect the P-solubilization has led to the formulation of a defined medium to elucidate the role of bacteria in P-solubilization. Also, the yeast extract can be replaced with 0.05% ammonium sulfate as nitrogen source in this synthetic medium. Therefore, the synthetic medium developed in this study may include glucose as carbon source and ammonium sulfate as nitrogen source. This modified synthetic medium is similar to the NBRIP medium [25] with variation in concentration of ingredients and differ from PVK with replacement of yeast extract by ammonium sulfate as nitrogen source. Antifungal activity and production of secondary metabolites were dependent on nutrients in the assay medium (Fig. 2). These observations are in agreement with those of Vijayakumari et al. [26] who observed that production of secondary metabolites was dependent on the composition of the growth medium. Small deviations in medium components can make a significant difference in the production of metabolites. Our study shows that when glucose was deleted from PVK medium, production of secondary metabolites (indole-acetic acid, siderophore, and antifungal activity) were significantly decreased as compared to PVK having glucose, suggesting that glucose is essential ingredient in PVK broth. Previous study [27] showed that the addition of glucose resulted in dramatic increase of pyochelin and salicylic acid, indicating that the ratio of carbon source to nutrient concentration played a key role in the metabolic flow. P-solubilization ability of B. circulans CB7 was repressed in the presence of additional soluble phosphorus (Fig. 4)
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Preeti Mehta et al.
indicating a role of source of soluble phosphorus in the TCP solubilization ability of B. circulans CB7. Our results are in agreement with previous study that P-solubilizing activity of Erwinia herbicola was negatively correlated with the amount of soluble phosphorus in the medium and 0.1% soluble phosphorus suppressed visible P-solubilization in dicalcium phosphate plate [9] and 0.5% soluble phosphorus suppressed P-solubilization by rhizobial strain [28]. In cultivated soils, soluble phosphorus ranges from 0.3 to 1 mg ml1 in solution phase and extreme value ranged from
