Plant Growth Regulation

, Volume 65, Issue 3, pp 449–457

Enhanced biomass and steviol glycosides in Stevia rebaudiana treated with phosphate-solubilizing bacteria and rock phosphate

Authors

  • Mamta Gupta
    • Department of Microbial BiotechnologyPanjab University (PU)
  • Shashi Bisht
    • Plant Pathology and Microbiology Laboratory, Hill Area Tea Science DivisionInstitute of Himalayan Bioresource Technology (CSIR)
  • Bikram Singh
    • Department of Natural Plant ProductsInstitute of Himalayan Bioresource Technology (CSIR)
  • Arvind Gulati
    • Plant Pathology and Microbiology Laboratory, Hill Area Tea Science DivisionInstitute of Himalayan Bioresource Technology (CSIR)
    • Department of Microbial BiotechnologyPanjab University (PU)
Original paper

DOI: 10.1007/s10725-011-9615-9

Cite this article as:
Gupta, M., Bisht, S., Singh, B. et al. Plant Growth Regul (2011) 65: 449. doi:10.1007/s10725-011-9615-9

Abstract

Biofertilizers offer alternative means to promoting cultivation of medicinal plants less dependent on chemical fertilizers. Present study was aimed at evaluating the potential of phosphate-solubilizing bacteria (PSB) Burkholderia gladioli MTCC 10216, B. gladioli MTCC 10217, Enterobacter aerogenes MTCC 10208 and Serratia marcescens MTCC 10238 for utilizing Mussoorie rock phosphate (MRP) to enhance plant growth, and stevioside (ST) and rebaudioside-A (R-A) contents of Stevia rebaudiana. The solubilization of MRP by PSB strains varied from 1.4 to 15.2 μg ml−1, with the highest solubilization by Enterobacter aerogenes 10208. The PSB treatment increased the growth and ST and R-A contents of plants. Plant growth and stevioside contents were more pronounced with plants treated with a mixture of strains and grown in MRP amended soil compared to the unamended soil. The increment in shoot length (47.8%), root length (17.4%), leaf dry weight (164%), stem dry weight (116%), total shoot biomass (136%) resulted in enhanced productivity of ST (291%) and R-A (575%) in plants inoculated with mixture of PSB as compared to the uninoculated plants. The soils of PSB treated plants contained more available P than the soils of uninoculated plants (increase of 86–576%). PSB inoculated plants also recorded higher P content (64–273% increase) compared to uninoculated plants. The PSB strains differed in the extent of rhizosphere colonization, carbon source utilization pattern and whole cell fatty acids methyl esters composition.

Keywords

Stevia rebaudianaSteviosideRebaudioside-AMussoorie rock phosphatePhosphate solubilizing bacteriaFAME

Introduction

Phosphorus is an essential macronutrient required by the plants for their growth and development (Schachtman et al. 1998). Added P fertilizers undergo fixation due to the complex exchanges within the soil (Altomare et al. 1999). Consequently, large quantities of P fertilizers are needed to support the crop production (Sharma et al. 2010). Chemical fertilizers are not only costly but also adversely affect the soil microbial population (Vassilev and Vassileva 2003). Microorganisms may also reduce the fixation of soil nutrients by inorganic soil components (Khan and Joergensen 2009). Several P-solubilizing microorganisms have the ability to convert insoluble low grade rock phosphates into soluble forms available for plant growth (Sulbarán et al. 2009; Vyas and Gulati 2009). P solubilizers have been reported to enhance productivity of food and fodder crops (Selvakumar et al. 2008; Kumar et al. 2009). The PSB are also finding application for promoting growth of the medicinal plants for pharmaceuticals, nutraceuticals and cosmetics (Jaleel et al. 2007; Kaymak et al. 2008).

Stevia rebaudiana is a medicinal plant with a huge demand in pharmaceutical, food and beverage industries as a source of low calorie and high potency natural sweeteners—ent-kaurine stevioside and rebaudiosides. Stevia powder has been reported for hypotensive and heart tonic actions (Ferri et al. 2006). Industry needs large amounts of quality biomass generated with the minimal application of chemical fertilizers. In our previous work, it was reported that the inoculations of PSB along with tricalcium phosphate (TCP) enhanced the plant growth, liberation of P and steviol glycosides in Stevia rebaudiana (Mamta et al. 2010). TCP is an expensive inorganic P fertilizer as compared to rock phosphates. The possibility of using cheaper low-grade rock phosphates by the application of phosphate solubilizing microorganisms has been demonstrated in some plants, including rice, wheat and potato (Sharma and Prasad 2003; Shivay 2010). The present work was carried out to evaluate the effect of PSB in combination with the MRP on the growth and glycosides of S. rebaudiana.

Materials and methods

Bacterial isolates and their culture conditions

Burkholderia gladioli MTCC 10216, Burkholderia gladioli MTCC 10217, Enterobacter aerogenes MTCC 10208 and Serratia marcescens MTCC 10238 used as PSB in the present studies were isolated from the rhizosphere of S. rebaudiana plants grown commercially in the agricultural fields at Zirakpur in the State of Panjab, India and identified based on 16S rRNA gene sequencing (Mamta et al. 2010). The strains were compatible with one another and also showed the plant growth promoting attributes of indole acetic acid (B. gladioli 10216 and 10217, and E. aerogenes 10208) and siderophore production (B. gladioli 10216 and 10217, and S. marcescens 10238) (Mamta et al. 2010). The cultures stored in 30% glycerol at −80°C were revived for the present studies by growing in nutrient broth at 30°C for 24 h.

Characterization of PSB

The PSB strains were analyzed for utilization of 95 carbon sources using BIOLOG system (BIOLOG Microstation) and BIOLOG Microlog version 4.20.05 software (Gulati et al. 2009).

The PSB strains were analyzed for whole-cell fatty acids, derivatized to methyl esters using the Sherlock Microbial Identification System (MIS-MIDI, USA). The FAME profiles were compared using ITSA1 aerobe library general system software v5.0 (Sasser and Wichman 1991). The dendrogram was constructed based on the matrix generated by FAME analyses using STATISTICA software version 7.

Mussoorie rock phosphate solubilization by PSB

The phosphate solubilization by PSB was studied by inoculating the strains separately into 100 ml modified PVK broth contained in 250 ml conical flask and incubating at 30°C on a rotary shaker (130 rev min−1). The composition of medium was (g l−1): yeast extract, 0.50; dextrose, 10.0; MRP, 5.0; (NH4)2SO4, 0.50; KCl, 0.20; Mg2SO4·7H2O, 0.10; Mn2SO4·H2O, 0.0001; Fe2SO4·7H2O, 0.0001; pH adjusted to 7.0 (Mamta et al. 2010). MRP was obtained from M/S Pyrites, Phosphates and Chemicals Ltd., Noida, India as 100-mesh size powder with compostion (%): CaO, 38.5; P2O5, 21.2; F, 2.30; CO3, 13.8; Na2O, 0.17; MgO, 5.60; K2O, 0.25; Al2O3, 0.73; SiO2, 6.60; Fe2O3, 4.41; sulfide-sulfur, 4.0; organic-C, 1.14; chlorides, 0.015; SO4S, 0.10; and neutral ammonium citrate (C6H14N2O7), 2.20 (Mittal et al. 2008). A 2 ml aliquot was withdrawn at regular interval of 24 h for 7 days from each culture vessel, centrifuged (10,000g), and supernatant analyzed for soluble P content by colorimetric chlorostannous reduced molybdo-phosphoric acid blue method (Jackson 1973).

Pot experiments

Experiment on the influence of PSB on plant growth was done in the Green House in the Department of Botany, Panjab University, Chandigarh, India. The inoculum was prepared by growing PSB strains separately in 300 ml nutrient broth in 1L conical flasks at 30°C on a rotary shaker (150 rev min−1, 2.5 cm radius of rotation) for 24 h (Mamta et al. 2010). The cultures were centrifuged at 6,000 g for 15 min and pellets re-suspended in phosphate buffer saline, pH 7.2, and optical densities (OD) adjusted to values equivalent to 108 CFU ml−1 for various PSB. The culture suspensions adjusted to equal cell density (108 CFU ml−1) were used for inoculating the experimental plants. For making a mixed inoculum, individual cultures of PSB were mixed together in equal quantities.

Pots of size 24 cm diameter × 30 cm height were filled with 3.5 kg of unamended soil or soil amended with MRP (300 mg kg−1 soil). The roots of tissue culture plantlets were surface-sterilized by dipping in 2% NaOCl solution for 10 min, washed three times with sterilized distilled water, dipped in the culture inoculum for 15 min and planted in pots filled with unamended soil or soil amended with MRP. Each soil treatment consisted of six different sets in three replications: (1) Uninoculated soil (S) or MRP amended soil; inoculated treatments in S or S + MRP as follows: (2) B. gladioli 10216; (3) B. gladioli 10217; (4) S. marcescens 10238; (5) E. aerogenes 10208; and (6) Mixture of all four strains. Experiments were performed in a complete randomized block design.

Plant analyses

Dried plant parts were analyzed for P uptake, ST and R-A contents. P content in shoots was determined using vanadomolybdo phosphoric yellow color method (Koeing and Johnson 1942). ST and R-A contents were estimated using HPLC (Mamta et al. 2010). Samples of 50 mg dried and powdered leaves were extracted with 10 ml methanol and filtered (Whatman filter paper, Sartorius, grade 389) into 50 ml distillation flasks. The filtrate was distilled on a rotary evaporator at 45°C, 50 rev min−1 and 70 mbar vacuum. The residue was defatted with hexane and dissolved in 5.0 ml acetonitrile:water (80:20). ST and R-A contents were determined on Waters 996 High Performance Liquid Chromatogram (HPLC) system equipped with PDA detector, 7,725 Rheodyne injector (Auto sampler 717), Lichrocart® 100 NH2 column (250 mm × 4.6 mm, 5 μM) from E. Merck (Germany), and EM Power Pro Software. Acetonitrile and water (80:20) was used as the mobile phase at flow rate 1 ml min−1. The eluates were detected at wavelength 210 nm and identified by retention time and co-chromatography of the samples spiked with ST and R-A standards supplied by Sigma Chemicals.

Soil analyses

Available P content in soil was determined by colorimetric sodium bicarbonate-extractable P method (Olsen et al. 1954).

Rhizosphere colonization by PSB

The inoculated PSB in each treatment were analyzed for their ability to colonize the plant rhizosphere by plate count and identification of P-solubilizing colonies by random amplification of polymorphic DNA banding pattern (Mamta et al. 2010). The soil adhering to the roots of harvested plants separated by gentle tapping was serially diluted in PBS (pH 7.2) and tenfold serial dilutions spread on PVK agar medium, and incubated at 30°C for 5 days. Bacterial colonies showing P solubilizing zone on PVK agar were counted to determine CFU g−1 soil. The detection limit was 10–100 CFU g−1 soil. Representative colonies of isolated PSB were analyzed for DNA banding pattern using the primer OPA-04 (5′-AATCGGGCTG-3′). PSB strains were identified as being one of the inoculated strains by similarity in banding pattern to the inoculated PSB.

Statistical analyses

Statistical analysis was done using one-way ANOVA statistical package for social sciences (SPSS) software, version 30. Means were compared by the LSD test at P ≤ 0.05.

Results

Identification of PSB

These PSB were identified by 16S rRNA gene sequence analysis reported in our previous publication (Mamta et al. 2010). In the present report, PSB were also analyzed for carbon source utilization pattern and fatty acid methyl esters composition. All PSB strains were positive for utilization of l-arabinose, d-galactose, a-d-glucose, mannose, methyl pyruvate, monomethyl succinate, acetic acid, citric acid, DL-lactic acid, malonic acid, succinic acid, bromosuccinic acid, d-alanine, l-alanine, l-asparagine, l-glutamic acid, l-histidine, l-phenyl alanine and l-proline, and negative for the utilization of α-cyclodextrin, N-acetyl-d-galactosamine, erthyritol, lactulose, DL-carnitine, phenyl ethylamine and 2,3-butanediol. B. gladioli 10216, E. aerogenes 10208 and S. marcescens 10238 also utilized dextrin, cellobiose, gentiobiose, melibiose, D-psicose and glucuronamide. S. marcescens 10238 was also positive for α-keto glutaric acid, α-keto valeric acid, α-keto butyric acid and 2-amino ethanol utilization. Only B. gladioli 10216 was positive for utilization of glycogen, Tween 40, α-d-lactose, maltose, l-rhamnose, d-raffinose, sucrose, turanose, d-galactonic acid lactone, d-galacturonic acid, d-glucuronic acid, p-hydroxy phenylacetic acid, siccinamic acid, alaninamide, l-alanyl glycine, glycyl-l-aspartic acid, glycyl-l-glutamic acid, l-ornithine, l-threonine, urocanic acid, inosine, uridine, thymidine, glycerol and glucose-1-phosphate.

FAME analyses showed differences in the composition of cell-wall fatty acids of PSB. The fatty acid-profiles of B. gladioli 10216 and 10217 showed 14:00, 16:00, 16:1 iso I/14:0 3OH and 18:1w7c as major fatty acids and exhibited 56.4 and 57.8% similarity respectively, with B. gladioli, while S. marcescens 10238 with 14:00, 14:0 2OH, 16:1 iso I/14:0 3OH, 16:00, 16:1w7c, 17:0 cyclo, and 18:1 w7c as the major fatty acids showed 81.6% similarity with S. marcescens in the data base ITSA 1 library. E. aerogenes 10208 showed the presence of major fatty acids 12:00, 14:00, 16:00, 16:1 iso I/14:0 3OH, 16:1 w7c and 18:1w7c and 63.0% similarity with E. aerogenes. Cluster analysis generated one group containing B. gladioli 10216, B. gladioli 10217 and E. aerogenes 10208, while S. marcescens 10238 stood outside the group (Fig. 1).
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Fig. 1

Dendrogram constructed on the basis of whole-cell fatty acid composition by GC-FAME analysis of bacterial isolates using MIDI Sherlock analysis software version 6.0

MRP solubilization by PSB

The rate of solubilization during 7 days varied from 1.4 to 15.2 μg ml−1 by PSB (Fig. 2). The maximum solubilization was by E. aerogenes 10208 (15.2 μg ml−1 on seventh day), followed by S. marcescens 10238 (9.6 μg ml−1 on first day), B. gladioli 10217 (8.4 μg ml−1 on seventh day) and B. gladioli 10216 (1.4 μg ml−1 on third day). The pH of the medium decreased with the increase in phosphate solubilization (Fig. 2). A significant (P = 0.05) negative correlation coefficient of −0.8 was observed between P solubilization and pH of the medium.
https://static-content.springer.com/image/art%3A10.1007%2Fs10725-011-9615-9/MediaObjects/10725_2011_9615_Fig2_HTML.gif
Fig. 2

P liberated (solid line with open square) and change in pH (dotted line with filled circle) of the medium by Burkholderia gladioli 10216 (a), Burkholderia gladioli 10217 (b), Serratia marcescens 10238 (c) and Enterobacter aerogenes 10208 (d) for 7 days in MRP supplemented modified PVK broth. Values are mean of three replications. Error bars represent standard deviation

Plant growth promotion by PSB

The plants inoculated with mixed PSB inoculum showed significantly higher growth over the uninoculated plants (Table 1). The maximum increase in plant growth was observed in the mixed treatment of PSB in MRP amended soil, which resulted in an increase of 47.8, 17.4, 164, 116 and 136% in shoot length, root length, leaf dry weight, stem dry weight and total shoot biomass respectively, compared to the control plants.
Table 1

Effect of phosphate-solubilizing bacteria on growth of Stevia rebaudiana

Treatment

Shoot length (cm)

Root length (cm)

Leaf dry weight (g)

Stem dry weight (g)

Total shoot biomass (g)

S (control)

55.7 ± 3.1 (a)

10.0 ± 0.2 (a)

0.30 ± 0.1(a)

0.36 ± 0.02 (a)

0.66 ± 0.05 (a)

S + B. gladioli (MTCC 10216)

58.1 ± 2.0 (a)

10.4 ± 0.3 (a, b)

0.47 ± 0.1 (b)

(55.1)

0.48 ± 0.02 (b)

(33.3)

0.95 ± 0.1 (b)

(43.3)

S + B. gladioli (MTCC 10217)

64.9 ± 1.4 (b)

(16.5)a

10.7 ± 0.4 (b, c)

(7.0)

0.44 ± 0.1 (b)

(47.5)

0.53 ± 0.03 (b, c)

(48.1)

0.98 ± 0.05 (b, c)

(47.8)

S + E. aerogenes (MTCC 10208)

58.8 ± 1.1 (a)

10.9 ± 0.5 (b, c)

(8.8)

0.47 ± 0.04 (b)

(55.8)

0.54 ± 0.02 (c)

(51.4)

1.01 ± 0.05 (b, c)

(53.2)

S + S. marcescens (MTCC 10238)

67.2 ± 1.8 (b, c)

(20.7)

10.9 ± 0.2 (b, c)

(9.3)

0.47 ± 0.1 (b)

(57.8)

0.58 ± 0.02 (c)

(62.8)

1.06 ± 0.1 (b, c)

(60.6)

S + consortium

74.1 ± 2.9 (d)

(33)

11.3 ± 0.2 (c)

(12.5)

0.76 ± 0.1(c)

(150)

0.73 ± 0.02 (d)

(104)

1.49 ± 0.1(e)

(126)

S + MRP (control)

60.1 ± 2.2 (a, b)

10.1 ± 0.3 (a)

0.3 ± 0.03 (a)

0.4 ± 0.03 (a)

0.7 ± 0.1 (a)

S + MRP + B. gladioli (MTCC 10216)

73.0 ± 3.4 (c, d)

(21.6)

11.1 ± 0.3 (c)

(10.4)

0.58 ± 0.1 (d)

(93.3)

0.67 ± 0.03 (e)

(64.9)

1.25 ± 0.1 (d)

(76.9)

S + MRP + B. gladioli (MTCC 10217)

66.5 ± 1.7 (b)

(10.7)

10.9 ± 0.13 (b, c)

(7.6)

0.42 ± 0.1 (a, b)

0.54 ± 0.02 (c)

(33.5)

0.97 ± 0.1 (b, c)

(37.4)

S + MRP + E. aerogenes (MTCC 10208)

71.6 ± 3.5 (c, d)

(19.2)

11.2 ± 0.3 (c)

(10.6)

0.54 ± 0.1 (b)

(80)

0.59 ± 0.02 (c)

(45.3)

1.13 ± 0.1 (c, d)

(60)

S + MRP + S. marcescens (MTCC 10238)

67.4 ± 1.2 (c, d)

(12.2)

10.8 ± 0.2 (b, c)

(7.27)

0.53 ± 0.1 (b)

(76.7)

0.58 ± 0.01 (c)

(42.5)

1.11 ± 0.1 (b, c, d)

(56.9)

S + MRP + consortium

88.8 ± 2.1 (e)

(47.8)

11.8 ± 0.1 (d)

(17.4)

0.79 ± 0.1 (c)

(164)

0.88 ± 0.05 (f)

(116)

1.67 ± 0.1(f)

(136)

Values are mean of three replications, mean values (mean ± SD) with common letters in each column do not differ statistically by LSD at P = 0.05, S uninoculated soil, MRP Mussoorie rock phosphate

aValue in parenthesis represents percentage increase over the respective control

Effect of PSB on P content in plants and soil

Inoculation with PSB resulted in increased P content in soil as well as in plant shoots as compared to the control (Table 2). PSB treatments showed an increase of 86–576% in available P content of soil and 63.9–273% P content in plant shoots. The maximum increase of P content in both soil and plants was observed by inoculating mixed inoculum of all PSB as compared to the control treatments. The increase in amount of available P in soil was followed by increase in P content in plant shoots and total shoot biomass (Fig. 3).
Table 2

Effect of phosphate-solubilizing bacteria on available P content in soil and P content in shoots of Stevia rebaudiana

Treatment

Available P content in soil (mg kg−1)

Total P content in shoots (mg plant−1)

S (control)

0.89 ± 0.2 (a)

1.51 ± 0.2 (a)

S + B. gladioli (MTCC 10216)

2.32 ± 0.2 (c) (160)a

2.61 ± 0.3 (b) (71.9)

S + B. gladioli (MTCC 10217)

2.32 ± 0.04 (c) (160)

2.63 ± 0.3 (b) (73.6)

S + E. aerogenes (MTCC 10208)

2.90 ± 0.1 (d) (225)

2.79 ± 0.31 (b, c) (84.2)

S + S. marcescens (MTCC 10238)

3.36 ± 0.1 (e) (277)

3.03 ± 0.41 (b, c) (99.8)

S + consortium

6.02 ± 0.1 (g) (576)

5.14 ± 0.31(d) (239)

S + MRP (control)

1.80 ± 0.1 (b)

1.64 ± 0.3 (a)

S + MRP + B. gladioli (MTCC 10216)

5.01 ± 0.1 (f) (179)

3.72 ± 0.2 (c) (127)

S + MRP + B. gladioli (MTCC 10217)

3.34 ± 0.1 (e) (86)

2.69 ± 0.1 (b) (63.9)

S + MRP + E. aerogenes (MTCC 10208)

5.0 ± 0.2 (f) (178)

3.36 ± 0.4 (c) (105)

S + MRP + S. marcescens (MTCC 10238)

5.0 ± 0.1 (f) (178)

3.13 ± 0.4 (c) (90.4)

S + MRP + consortium

7.69 ± 0.1 (h) (327)

6.13 ± 0.2 (d) (273)

Values are mean of three replications, values with common letters in each column do not differ statistically by LSD at P = 0.05, mean values (mean ± SD), S uninoculated soil, MRP Mussoorie rock phosphate

aValue in parenthesis represents percentage increase over the respective control

https://static-content.springer.com/image/art%3A10.1007%2Fs10725-011-9615-9/MediaObjects/10725_2011_9615_Fig3_HTML.gif
Fig. 3

Effects of phosphate-solubilizing bacteria on the available P in soil, P content in plant shoots and total shoot biomass. Error bars represent standard deviations

Effect of PSB on stevioside and rebaudioside-A contents

The plants treated with mixed PSB inoculum and grown in MRP soil showed the highest increase in ST and R-A contents of 291 and 575% on whole plant basis as compared to the control plants (Table 3). The combined treatment of all PSB strains in unamended soil resulted in 252% increase in ST content and 566% in R-A content. The increases in ST and R-A contents in plants treated with mixed PSB inoculum were only due to increases in biomass rather than concentration of steviol glycosides as non-significant increases were observed in ST and R-A contents over control plants (Table 3). Treatment with individual PSB strains did not cause a significant change in steviol glycosides (Table 3).
Table 3

Effect of phosphate-solubilizing bacteria on stevioside and rebaudioside-A contents of Stevia rebaudiana

Treatment

ST (mg g−1 leaves)

ST (mg plant−1)

R-A (mg g−1 leaves)

R-A (mg plant−1)

S (control)

25.3 ± 1.8 (a)

7.71 ± 1.2 (a)

4.8 ± 2.3 (a)

1.49 ± 0.9 (a)

S + B. gladioli (MTCC 10216)

34.2 ± 4.1 (a)

16.0 ± 1.4 (a, b)

14.2 ± 2.5 (a)

6.6 ± 2.0 (a, b)

S + B. gladioli (MTCC 10217)

33.1 ± 7.6 (a)

14.8 ± 3.3 (a, b)

1.4 ± 0.2 (a)

0.62 ± 0.1 (a)

S + E. aerogenes (MTCC 10208)

22.7 ± 4.6 (a)

10.6 ± 1.7 (a)

12.0 ± 3.5 (a)

5.59 ± 1.3 (a, b)

S + S. marcescens (MTCC 10238)

21.5 ± 9.4 (a)

10.4 ± 5.1(a)

10.5 ± 3.2 (a)

5.06 ± 1.8 (a, b)

S + consortium

35.7 ± 2.0 (a)

27.2 ± 2.6 (b)

(252)a

13.2 ± 3.3 (a)

9.92 ± 2.0 (b)

(566)

S + MRP (control)

27.4 ± 0.3 (a)

8.20 ± 0.3 (a)

5.83 ± 1.0 (a)

1.75 ± 0.4 (a)

S + MRP + B. gladioli (MTCC 10216)

32.4 ± 2.0 (a)

18.8 ± 1.2 (a, b)

15.0 ± 3.6 (a)

8.69 ± 2.1 (a, b)

S + MRP + B. gladioli (MTCC 10217)

34.6 ± 4.3 (a)

14.8 ± 1.5 (a, b)

2.97 ± 0.9(a)

1.26 ± 0.3 (a)

S + MRP + E. aerogenes (MTCC 10208)

22.5 ± 3.6 (a)

12.2 ± 2.2 (a)

14.0 ± 2.6 (a)

7.49 ± 0.9 (a, b)

S + MRP + S. marcescens (MTCC 10238)

31.9 ± 10.3 (a)

17.0 ± 5.8 (a, b)

12.2 ± 4.2 (a)

6.41 ± 2.1 (a, b)

S + MRP + consortium

40.6 ± 5.0 (a)

32.1 ± 2.8 (b)

(291)

15.0 ± 1.7 (a)

11.9 ± 1.6 (b)

(575)

Values are mean of three replications, mean values (mean ± SD), with common letters in each column do not differ statistically by LSD at P = 0.05, S uninoculated soil, MRP Mussoorie rock phosphate

aValue in parenthesis represents percentage increase over the respective control

Rhizosphere colonization potential of PSB

All PSB inoculants were able to survive in the rhizosphere of treated plants (Table 4). The highest rhizosphere soil colonization was by Burkholderia gladioli 10216 in MRP amended soil (3.0 × 109 CFU g−1) and unamended soil (2.6 × 109 CFU g−1). The lowest colonization was by E. aerogenes 10208 in unamended (3.8 × 107 CFU g−1) and MRP amended soil (4.1 × 107 CFU g−1). Only B. gladioli 10216 and S. marcescens 10238 were reisolated and only at the lowest dilution tested in the plants treated with mixed PSB inoculum (Table 4).
Table 4

Rhizosphere colonization by PSB in Stevia rebaudiana at 12th week after inoculation

PSB isolate

CFU × 108 g−1 soil

(Individual PSB treatment)

CFU × 108 g−1 soil

(PSB consortium treatment)

S + B. gladioli (MTCC 10216)

26.0 ± 4.0

1.2 ± 2.5

S + B. gladioli (MTCC 10217)

5.0 ± 2.0

ND

S + E. aerogenes (MTCC 10208)

3.8 ± 9.1

ND

S + S. marcescens (MTCC 10238)

17.0 ± 5.6

0.7 ± 1.5

S + MRP + B. gladioli (MTCC 10216)

30.0 ± 2.0

1.7 ± 4.2

S + MRP + B. gladioli (MTCC 10217)

5.6 ± 4.2

ND

S + MRP + E. aerogenes (MTCC 10208)

4.1 ± 3.6

ND

S + MRP + S. marcescens (MTCC 10238)

21.0 ± 2.0

1.0 ± 2.0

Values are mean of three replications (mean ± SD), S uninoculated soil, MRP Mussoorie rock phosphate

ND not detected

Discussion

Stevia rebaudiana as a natural sweetener is in demand particularly by the persons suffering from diabetes and obesity due to antihypertensive and antihyperglycemic attributes (Melis et al. 2009). Integrated application of microbial inoculants with agrotechnologies is required for improving biomass productivity and biochemical constituents in the plant. This study demonstrates the beneficial effect of mixed inoculations of PSB on plant biomass generation in Stevia (Table 1). The significantly higher levels of available P in inoculated plants and supporting soil indicated that the enhanced growth was probably due to the greater availability of P resulting from solubilization of the phosphate source by the mixed PSB treatments (Ekin 2010). The individual inoculant strains were initially shown to have the capability to solubilize MRP in liquid medium (Fig. 1). B. gladioli 10216 showed the minimum phosphate solubilization in vitro, but resulted in higher shoot biomass production of plants grown in MRP amended soil. The results support previous observations that rate of in vitro phosphate solubilization has no simple relationship with in vivo plant growth (de Freitas et al. 1997; Mittal et al. 2008). The reason may be the production of plant growth promoting metabolites (indole acetic acid and siderophores) besides phosphate solubilization (Mamta et al. 2010). Out of the four PSB, two (B. gladioli 10216 and 10217) produced IAA as well as siderophore, whereas E. aerogenes 10208 produced only IAA and S. marcescens 10238 only siderophore. However, in present report no direct relationship could be seen between in vitro production of IAA and siderophore and in vivo plant growth.

This is the first report showing the influence of PSB along with MRP on Stevia growth. The higher plant growth with the combined treatments of PSB compared with their individual treatments is similar to the higher growth reported for rice with combined inoculations of Azospirillumlipoferum, Bacillusmegaterium and Pseudomonasfluorescens over the corresponding individual applications (Raja et al. 2006).

The PSB individually or collectively showed a significant increase in P content of soils (Table 2), which would be expected to increase its uptake in plants (Sárdi et al. 2009). A direct positive relationship could be established between the liberation of phosphate from MRP in soil by individual inoculant strains and plant growth. All PSB liberated higher amounts of phosphate from MRP in pure cultures and also showed higher increase in total shoot biomass in plants grown in soil amended with MRP as compared to the control plants.

The increased plant growth resulted in an increase in the yield of ST and R-A due solely to an increase in leaf biomass, as no significant increase was observed in the concentrations of steviol glycosides on unit mass basis. The highest increment in ST and R-A contents was obtained in MRP amended soil inoculated with the combined application of strains which also induced the highest biomass production (Table 2). The results on efficacy of combined microbial treatments are in agreement with the earlier report on the highest stevioside content when the plants were treated with the combined application of biofertilizers (Bacillus megaterium, Azospirillum sp. and VA mycorrhiza) compared with the corresponding individual applications (Das and Dang 2010).

The rhizoshere colonization of host plant by PSB is an important factor for plant growth (Lugtenberg et al. 2001). All PSB strains were able to survive in the rhizosphere of Stevia plants with the maximum rhizosphere colonization shown by B. gladioli 10216 in the soil with and without MRP. In plants inoculated with the mixture of PSB, only B. gladioli 10216 and S. marcescens 10238 were detected in the rhizosphere after 12 weeks. The other two strains were not present above the limit of detection (10–100 CFU g−1 soil). This is probably due to the greater competitive ability of inoculants B. gladioli 10216 and S. marcescens 10238 in the rhizosphere. There was no direct relationship between rhizosphere colonization data and plant growth as both E. aerogenes 10208 and S. marcescens 10238 showed the same good increase in total shoot biomass in plants grown in soil+MRP, even though rhizosphere colonization by E. aerogenes was relatively poor and that by S. marcescens was good. The reason may be production of the different plant growth promoting metabolites by these PSB strains (Mamta et al. 2010).

The PSB strains grown under uniform culture conditions showed distinctive patterns for utilization of carbon sources and whole-cell fatty acids methyl esters. Carbon-source utilization pattern of B. gladioli 10216 and 10217 corroborated well with those described for 15 strains of B. gladioli (Gillis et al. 1995). Strains E. aerogenes 10208 and S. marcescens 10238 showed resemblance to E. aerogenes strain R11-2 and S. marcescens strain 90–166, respectively (Rascoe et al. 2003; Wang et al. 2010). B. gladioli 10216 and S. marcescens 10238 were able to utilize more carbon sources than B. gladioli 10217 could explain their superior rhizosphere colonizing ability. The utilization of wide range of carbon sources has been considered the main factor for successful colonization and advantageous in acquiring dominance in the rhizosphere (Lugtenberg et al. 2001).

Polyphasic characterization by 16S rRNA gene sequencing (Mamta et al. 2010), carbon-source utilization and cellular fatty acid analysis confirmed that strains 10216 and 10217 belong to B. gladioli, 10208 to E. aerogenes and 10238 to S. marcescens. Cellular fatty acid profiles of B. gladioli 10216 and 10217 showed the presence of 16:0, 16:1 w7c and 18:1 w7c as the major fatty acids which were similar with those described for B. gladioli (Coenye et al. 2001). S. marcescens 10238 with 14:00, 16:1 iso I/14:0 3OH, 16:00, 16:1w7c, 17:0 cyclo, and 18:1 w7c as the major fatty acids was related to S. marcescens (Li et al. 2011). The presence of major fatty acids 16:00, 16:1 iso I/14:0 3OH, 16:1 w7c and 18:1w7c in E. aerogenes 10208 corresponded to the expected fatty acid composition of E. aerogenes (Wang et al. 2010).

In conclusion, we have found that inoculation with Burkholderia gladioli strains 10216 and 10217, Enterobacter aerogenes 10208 and Serratia marcescens 10238 effectively utilized MRP resulting in enhanced shoot biomass and consequently higher yield of ST and R-A contents of Stevia rebaudiana. Treatment with single inoculum strain was less effective than treatment with a mixture of strains. The potential of PSB along with MRP application needs to be studied further under field conditions for commercial cultivation of the plant.

Acknowledgments

Thanks are due to University Grant Commission, New Delhi, India for the financial support. Thanks are also due to the Director, Institute of Himalayan Bioresource Technology (CSIR), Palampur-176061, Himachal Pradesh, India for providing facilities for BIOLOG and FAME analyses under the Project “Exploitation of India’s Rich Microbial Wealth” (NWP006).

Copyright information

© Springer Science+Business Media B.V. 2011