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Comparative efficiency of phosphate-solubilizing bacteria under greenhouseconditions for promoting growth andaloin-A content of Aloe barbadensisGupta Mamta a b , Praveen Rahi c , Vijaylata Pathania d , ArvindGulati c , Bikram Singh d , Ravinder Kumar Bhanwra e & RupinderTewari aa Department of Biotechnology , Panjab University , Chandigarh ,Indiab Department of Environment and Vocational Studies , PanjabUniversity , Chandigarh , Indiac Plant Pathology and Microbiology Laboratory , Institute ofHimalayan Bioresource and Technology , Palampur , Indiad Department of Natural Plant Products , Institute of HimalyanBioresource and Technology , Palampur , Indiae Department of Botany , Panjab University , Chandigarh , IndiaPublished online: 10 Aug 2011.

To cite this article: Gupta Mamta , Praveen Rahi , Vijaylata Pathania , Arvind Gulati ,Bikram Singh , Ravinder Kumar Bhanwra & Rupinder Tewari (2012) Comparative efficiency ofphosphate-solubilizing bacteria under greenhouse conditions for promoting growth and aloin-A content of Aloe barbadensis , Archives of Agronomy and Soil Science, 58:4, 437-449, DOI:10.1080/03650340.2010.522574

To link to this article: http://dx.doi.org/10.1080/03650340.2010.522574

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Comparative efficiency of phosphate-solubilizing bacteria under

greenhouse conditions for promoting growth and aloin-A content

of Aloe barbadensis

Gupta Mamtaa,b, Praveen Rahic, Vijaylata Pathaniad, Arvind Gulatic, BikramSinghd, Ravinder Kumar Bhanwrae and Rupinder Tewaria*

aDepartment of Biotechnology, Panjab University, Chandigarh, India; bDepartment ofEnvironment and Vocational Studies, Panjab University, Chandigarh, India; cPlant Pathologyand Microbiology Laboratory, Institute of Himalayan Bioresource and Technology, Palampur,

India; dDepartment of Natural Plant Products, Institute of Himalyan Bioresource andTechnology, Palampur, India; eDepartment of Botany, Panjab University, Chandigarh, India

(Received 25 May 2010; final version received 5 September 2010)

This study was conducted with Aloe barbadensis in order to investigate the efficacyof four phosphate-solubilizing bacteria (PSB), Pseudomonas synxantha 10223,Burkholderia gladioli 10242, Enterobacter hormaechei 10240 and Serratia marces-cens 10241 to solubilize Mussorie rock phosphate (MRP) and to evaluate its effectson growth, soluble P content andP uptake comparedwith control, i.e. uninoculatedplants. Pot experiments were conducted in a greenhouse, in soil supplemented withMRP. Each PSB treatment showed different effects on different plant growthparameters. The maximum increase in leaf length (23.7%), total number of leaves(33.33%) and dry rind weight (69.10%) was observed in plants treated with P.synxantha10223 comparedwith control.Whereas,maximum increase in root length(23.43%), fresh leaves weight (79.03%), dry gel weight (113.08%) and total gelvolume (112.10%), was observed in plants treated with S. marcescens 10241compared with uninoculated plants. Maximum increase in aloin-A content[114.92% (per g dry gel weight) and 322.32% (per plant dry gel weight)] wasobserved in plants treated with P. synxantha 10223 compared with control plants.Root colonization by inoculated PSB as estimated byRAPD technique showed thatall PSB were able to survive in the rhizosphere of Aloe plants.

Keywords: Aloe barbadensis; aloin-A; phosphate solubilizing bacteria; randomamplification of polymorphic DNA (RAPD); rhizosphere

Introduction

Phosphorus (P) is the secondmost essential macronutrient for plant growth. AvailableP is generally not sufficient to meet the crop requirement because most of the P isconverted into insoluble forms as phosphates of iron, aluminum and calcium(Altomare et al. 1999). Therefore, external chemical P fertilizers have to be applied,but unfortunately, excessive use of chemical P fertilizer has led to many environmentalproblems, such as eutrophication and soil salinity (Del Campillo et al. 1999).P-solubilizing microorganisms (PSM) are known to enhance the yield of crops by

*Corresponding author. Email: rupinder@pu.ac.in

Archives of Agronomy and Soil Science

Vol. 58, No. 4, April 2012, 437–449

ISSN 0365-0340 print/ISSN 1476-3567 online

� 2012 Taylor & Francis

http://dx.doi.org/10.1080/03650340.2010.522574

http://www.tandfonline.com

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converting insoluble soil P to soluble P through the release of organic acids (oxalic acid,gluconic acid and ketogluconic acid, etc.), chelation and ion exchange (Omar 1998;Narula et al. 2000; Whitelaw 2000) and thereby make it available to plants. The role ofphosphate-solubilizing bacteria (PSB) in enhancing the production rate of food andfodder crops is well documented (Selvakumar et al. 2008; Kumar et al. 2009). Atpresent, more attention is being paid towards the potential of PSB to promote thegrowth of medicinal plants (Jaleel et al. 2007; Kaymak et al. 2008) because these areimportant for pharmaceuticals, neutraceuticals and cosmetics. Organic cultivation ofmedicinal plants is becoming an opportunity sector for global trade and commerce.Aloe barbadensis is an importantmedicinal plant, andbelongs to the familyLiliaceae. Ithas highly valuable ingredients that exhibit interesting biological functions and istherefore in great demand in the pharmaceutical, food and cosmetic industries. Aloebarbadensis contains aloin-A (barbaloin; Figure 1), one of the major phenoliccompounds in 68 species ofAloe investigated (Groom andReynolds 1987). Some otherphenolic compounds present are aloesin, isoaloeresin D and aloeresin E (Okamuraet al. 1996). Phenolic compounds have an important role in the treatment of tumors,diabetes, ulcer and cancer (Ishii et al. 1990).

There are limited reports on the effect of PSMon the growthofAloe vera (Tawarayaet al. 2007; Pandey and Banik 2009). Attempts have been made in the past to explorethe potential of PSB toutilize rockphosphate because there exist substantial deposits oflow-grade rock phosphate, which may partly meet crop demands for P; one suchdeposit is Mussorie rock phosphate (MRP) (Illmer and Schinner 1992; Sharma et al.2009). In this study, the potential of PSB toutilizeMRPwas evaluated and the effects ofMSP on plant-growth parameters, available P content in soil, P uptake in plants andaloin-A content in dry gel of leaves of A. barbadensis were studied.

Materials and methods

Microorganisms

Four PSB isolated from the rhizosphere of A. barbadensis were used asbioinoculants. Serial dilutions (1:10) of rhizospheric soil were made in phosphate

Figure 1. Structure of aloin–A.

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buffered saline (pH 7.2) and spread on Pikovskaya’s (PVK) agar (Pikovskaya 1948),amended with 0.5% tricalcium phosphate. Bacterial isolates showing a zone of Psolubilization on PVK agar were marked as PSB. PSB were identified asPseudomonas synxantha 10223, Burkholderia gladioli 10242, Enterobacter hormaechei10240 and Serratia marcescens 10241 based on morphological, biochemical andmolecular techniques (Mamta et al. 2010, personal information).

MRP solubilization assay

P-solubilization by PSB was estimated by inoculating pure culture in 100 ml ofmodified PVK broth having the following composition (g l71): 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 (Pikovskaya 1948). Cultureswere allowed to grow at 308C on a rotary shaker (130 rpm). MRP was obtainedfrom M/S Pyrites, Phosphates and Chemicals Ltd (Noida, India) as a 100-mesh sizepowder. The MRP contained following components (%): CaO 38.50, P2O5 21.20, F2.30, CO3

2� 13.80, Na2O 0.17, MgO 5.60, K2O 0.25, Al2O3 0.73, SiO2 6.60, Fe2O3

4.41, sulfide–sulfur 4.00, organic-C 1.14, chlorides 0.015, SO4–S 0.10, neutralammonium citrate (C6H14N2O7) 2.20 (Mittal et al. 2008). Cultures were removed atregular intervals of 24 h, for 7 days and centrifuged. The pH of the supernatant wasmeasured using a pH meter (HPG G-2001, India) and the soluble P content wasestimated colorimetrically (Jenway 6305 spectrophotometer, UK) using thechlorostannous reduced molybdophosphoric acid blue method (Jackson 1973).Final values were calculated with the help of a standard curve obtained using 0 to2 mg l71 KH2PO4.

Evaluation of PSB through plant growth experiments

Soil and plants

Soil was characterized for various parameters such as pH, available N (Subbiah andAsija 1956), available P (Olsen et al. 1954), available K (Muhr et al. 1965), total Caand Mg (Cheng and Bray 1951), and total organic carbon (Walkley and Black 1934).The soil (pH, 7.8; available N, 46.875 mg kg71; available P, 5 mg kg71; available K,14.866 mg kg71; total Ca, 0.6 mmol 100 g71; total Mg, 0.12 mmol 100 g71; totalorganic carbon, 0.10%) was thoroughly mixed and passed through a 2-mm sieve toremove large particulate matter and allowed to dry in sunlight for 7 days. Tissue-cultured plantlets were obtained from Haryali Biotech (Zirakpur, India).

Cultivation conditions

Ethanol-disinfected pots were filled with 4.0 kg of soil. Roots of tissue-culturedplantlets were sterilized by dipping in 2% NaOCl solution for 10 min and thenwashed three times with distilled water. Root dip treatment with the desired culturehaving 108 CFU ml71 was performed. Experiments were performed in a completelyrandomized block design. Plantlets were planted (one per pot) in five different sets ofthree replications each: (1) soil þ MRP (300 mg kg71 soil); (2) soil þ MRP þ P.synxantha 10223; (3) soil þ MRP þ B. gladioli 10242; (4) soil þ MRP þ E.hormaechei 10240 and (5) soil þ MRP þ S. marcescens 10241.

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Soil and plant analysis for P content

Soil samples were analyzed for available P content using the colorimetric sodiumbicarbonate-extractable P method (Olsen et al. 1954). P uptake in Aloe plants wasdetermined by using the vanadomolybdo phosphoric yellow color method (Koeingand Johnson 1942).

Analysis of aloin-A

The gel part of the leaves of A. barbadensis was freeze dried. The 0.5 g freeze-dried sample was extracted with 10 ml methanol for 24 h at 48C. The suspensionobtained was centrifuged at 4000 g for 10 min at 48C. The supernatant was subjectedto HPLC (WATER Corp., Milford, MA, USA; Lichrocart1 C-18 column,EMPOWER software, flow rate of 0.8 ml min71 and pressure 800 psi, 290 nmwavelength, photodiode array detector). The mobile phase used was 0.1% acetic acidand acetonitrile (60:40). Aloin-A (Sigma Chemicals) was used as a standard.

Analysis of root colonization by PSB in the plant rhizosphere

The inoculated PSB in each treatment were analyzed for their root colonization abilityon the basis of random amplification of polymorphic DNA (RAPD) banding patternanalysis. The rhizosphere soil adhering to the roots of harvested plants was separatedby gentle tapping and stored in sterilized Petri plates at 48C. Multiple dilutions (1.0 gsoil in 10 ml phosphate buffer saline, pH 7.2) of each replicated soil sample were spreadon PVK agar and incubated at 308C for 5 days. Isolates showing P-solubilization weremarked and counted (number of isolates showing differentmorphology and number ofisolates showing similar morphology). The genomic DNA of PSB was isolated usingthe protocol suggested by Himedia (MB505 HiPurATM Bacterial and Yeast GenomicDNA Purification Spin Kit). Amplification of DNA samples was carried out in athermocycler (My cyclerTM, BioRad, USA) using primer OPA-04, 50-AATCGGGCTG-30 (Betancor et al. 2004). The thermocycler conditions involved: denaturation,948C for 40 s; annealing, 558C, 1 min; extension, 728C for 2 min; and final extension at728C for 10 min. Thermocycler was primed for 36 cycles of amplification. The PCRproducts were separated in 1.5% agarose gels run at 100 V. Gels were stained withethidium bromide (0.5 mgml71), photographed on aGelDoc (BioradUniversal HoodII, BioRad, USA). The similarity of band patterns was quantified by simple matching(Apostol et al. 1993). PSB isolates having a similar banding pattern to the inoculatedPSB were counted at 1076 dilution factor and PSB isolates were expressed as log CFUg71 of soil using the mathematical formula:

Log CFUg�1soil ¼ Log Number of colonies counted=dilution factorð Þ � 10a � 10b

afor converting per 100 ml into per 1.0 ml, bfor converting per 1.0 ml into per 10 mlor per g of soil.

Statistical analysis

Statistical analysis was conducted with a one-way analysis of variance (ANOVA)using SPSS software, version 30. Comparison of means was performed by LSD testat p � 0.05.

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Results

PSB isolates and their potential for MRP solubilization

Four PSB (P. synxntha 10223, B. gladioli 10242, E. hormaechei 10240 and S.marcescens 10241) were isolated from the rhizospheric soils of A. barbadensis plantsby serial dilutions (1:10) and spreading on PVK agar amended with tricalciumphosphate. Because the objective of this experiment was to solubilize the MRP byPSB isolates, the four PSB isolates showing zones of tricalcium phosphatesolubilization on solid PVK agar were further selected for quantitative estimationof MRP solubilization in a liquid medium. MRP solubilization by various PSBisolates over 7 days varied from 1.33 to 54.44 mg ml71 (Figure 2). The maximum Psolubilization was shown by E. hormaechei 10240 (54.4 mg ml71 on the third day)followed by B. gladioli 10242 (29.6 mg ml71 on the first day), S. marcescens10241(28.8 mg ml71 on the seventh day) and P. synxantha 10223 (15.6 mg ml71 onthe third day). With the increase in P solubilization rate, the pH of the medium wasseen to decrease (Figure 2). A negative correlation of 70.35, 70.79, 70.81 and70.70 was observed between P solubilization and the pH of medium inoculated withP. synxntha 10223, B. gladioli 10242, E. hormaechei 10240 and S. marcescens 10241,respectively.

Figure 2. Mussorie rock phosphate solubilization by phosphate-solubilizing bacteria isolatesin a liquid medium: (a) Pseudomonas synxantha 10223, (b) Burkholderia gladioli 10242, (c)Enterobacter hormaechei 10240, (d) Serratia marcescens 10241. Error bars represent standarddeviation.

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Plant growth promotion by PSB

Increase in leaf length, root length and total number of leaves

The PSB inoculations showed stimulatory effects on the growth of A. barbadensiscompared with uninoculated plants (Table 1). A significant increase (15.25–23.70%)in leaf length was found in all the PSB treatments but the maximum stimulatoryeffect was shown by single treatment with P. synxantha 10223 (23.70%). In case ofroot length, a significant increase (15.35–23.43%) was observed in each PSB-treatedplant compared with control plants, whereas, in case of total number of leaves inplants treated with P. synxantha 10223, a significant increase of 33.33% wasobserved compared with control plants.

Increase in dry gel weight and dry rind weight

PSB-inoculated plants showed a significant increase in dry gel weight (64.15–113.08%) and dry rind weight (25.43–69.10%) compared with control plants.Inoculation with S. marcescens 10241 and P. synxantha 10223 showed greaterincrease in dry gel weight (102.78–113.08%) and dry rind weight (67.77–69.10%)compared with control plants. Whereas treatment with B. gladioli 10242 and E.hormaechei 10240 showed an increase of 64.15–73.24% in dry gel weight and 25.43–30.35% in dry rind weight compared with control plants (Table 1).

Increase in fresh leaf weight and total gel volume

PSB-inoculated plants showed a significant increase in fresh leaf weight (32.71–79.03%) and total gel volume (68.65–112.10%) compared with control plants.Treatment with S. marcescens 10241 showed the maximum increase in fresh leafweight (79.08%) and total gel volume (112.103%) compared with control plants(Table 1).

Influence of PSB on aloin-A content of Aloe barbadensis

PSB treatments showed significant increase in the aloin-A content in dry gel of A.barbadensis on the basis of both ‘per g’ and ‘per plant’ dry gel weight comparedwith control plants (Table 2). The maximum increase in aloin-A content(114.92% per g dry gel weight; 322.32% per plant dry gel weight) was shownby P. synxantha 10223-treated plants compared with control plants. The otherPSB-treated plants showed an increase in aloin-A content of 66.23–37.71% and246.70–30.24% on the basis of ‘per g and ‘per plant’ dry gel weight, respectively,compared with control plants.

Effect of PSB on available P content in soil

The available P content of soil was greater in PSB-inoculated pots compared withuninoculated pots (Table 3). The soil of S. marcescens 10241 inoculated pots wasfound to have maximum amount of available P (6.57 mg kg71) and showed anincrease of 268.33% compared with control plants. The soil of other PSB-treatedpots showed an increase of 118.33–237.50% in available P content compared withcontrol plants.

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Table

1.

Effectofphosphate-solubilizingbacteria

inoculationonplantgrowth

ofAloebarbadensisgrownin

MRP-amended

soil.

Treatm

ent

Leaflength

(cm)

Rootlength

(cm)

Totalno.

ofleaves

Dry

gel

weight

(gplant7

1)

Dry

rindweight

(gplant7

1)

Fresh

leafweight

(gplant7

1)

Totalgel

volume

(mlplant7

1)

Sþ

MRP(control)

30.8

+0.24(a)

9.9

+0.56(a)

6+

0.70(a)

0.45+

0.01(a)

1.5

+0.02(a)

131.67+

1.14(a)

100.8

+5.40(a)

Sþ

MRPþ

Pseudomonas

synxantha(M

TCC

10223)

38.1

+0.69(c)

12.02+

0.52(b)

8+

0.70(b)

0.91+

0.04(c)

2.54+

0.07(c)

220.45+

5.89(c)

185.8

+7.01(b)

Sþ

MRPþ

Burkholderia

gladioli(M

TCC

10242)

35.52+

0.53(b)

11.46+

0.27(b)

6.4

+0.54(a)

0.78+

0.01(b)

1.95+

0.08(b)

182.10+

4.90(b)

175.2

+4.15(b)

Sþ

MRPþ

Enterobacter

horm

aechei

(MTCC

10240)

35.5

+0.77(b)

11.42+

0.30(b)

6.6

+0.54(a

b)

0.74+

0.02(b)

1.88+

0.05(b)

174.75+

1.56(b)

170+

4.85(b)

Sþ

MRPþ

Serratia

marcescens

(MTCC

10241)

37.38+

0.55(c)

12.22+

0.21(b)

7+

0.70(a

b)

0.96+

0.01(c)

2.52+

0.05(c)

235.74+

6.01(c)

213.8

+15.75(c)

Note:Values

given

are

themeanofthreereplications.Meanvalues

(mean+

S.D

)followed

bythesameletter

donotdiffer

significantlybyLSD

atp�

0.05.MRP,Mussorierock

phosphate;S,

unamended

soil.

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Effect of PSB on P uptake in plants

With the increase in available P content in soil, an increase in P uptake by the Aloeplants was observed (Table 3). The maximum increase (118.05%) in P uptake wasshown by plants treated with S. marcescens 10241 followed by P. synxantha 10223(111.75%) compared with control plants. However, plants treated with B. gladioli10242 (61.52%) and E. hormaechei 10240 (45.56%) showed less increase in P uptakethan plants treated with S. marcescens 10241 and P. synxantha 10223 compared withuninoculated plants.

Soil analysis for inoculated strains by RAPD

The rhizosphere soil was tested for the survivability of inoculated PSB isolates usinga RAPD technique with primer OPA-04 (Figure 3). It was found that all PSB wereable to survive in the rhizosphere of Aloe plants. Some unknown PSB were alsofound to survive in the rhizosphere of Aloe plants along with inoculated PSB, but thenumber of inoculated PSB on Pikovskaya’s agar plate was very high compared withunknown PSB isolates. Among individual PSB treatments, maximal colonizationwas observed with P. synxantha 10223 (9.72 log CFU g71 soil) followed by S.

Table 2. Effect of phosphate-solubilizing bacteria on aloin-A content of Aloe barbadensisplants grown in MRP-amended soil.

TreatmentAloin-A content(mg g71 dry gel)

Aloin-A content(mg plant71 dry gel)

S þ MRP (control) 3.8 + 0.1(a) 1.75 + 0.02 (a)S þ MRP þ Pseudomonas synxantha

(MTCC 10223)8.16 + 0.15 (d) 7.42 + 0.24 (c)

S þ MRP þ Burkholdia gladioli (MTCC 10242) 5.23 + 0.25 (c) 4.04 + 0.23 (b)S þ MRP þ Enterobacter hormaechei

(MTCC 10240)5.6 + 0.1 (bc) 4.10 + 0.16 (b)

S þ MRP þ Serratia marcescens (MTCC 10241) 6.31 + 0.16 (b) 6.09 + 0.16 (c)

Note: Values given are the mean of three replications. Mean values (mean + SD) follwoed by the sameletter do not differ significantly by LSD at p � 0.05. MRP, Mussorie rock phosphate; S, unamended soil.

Table 3. Effect of phosphate-solubilizing bacteria on available P content as well as P uptakein Aloe barbadensis plants in MRP-amended soil.

Treatment

AvailableP content

in soil (mg kg71)

Total P uptake(gel þ outer rind)

(mg plant71)

S þ MRP (control) 1.78 + 0.08 (a) 5.19 + 0.47 (a)S þ MRP þ Pseudomonas synxantha (MTCC 10223) 6.02 + 0.19 (b) 10.99 + 1.38 (b)S þ MRP þ Burkholdia gladioli (MTCC 10242) 5.01 + 0.13 (c) 8.38 + 1.2 (ab)S þ MRP þ Enterobacter hormaechei (MTCC 10240) 3.89 + 0.14 (d) 7.55 + 1.04 (ab)S þ MRP þ Serratia marcescens (MTCC 10241) 6.57 + 0.22 (e) 11.31 + 1.43 (bc)

Note: Values given are the means of three replications. Mean values (mean + S.D) followed by the sameletter do not differ significantly by LSD at p � 0.05. MRP, Mussorie rock phosphate; S, unamended soil.

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marcescens 10241 (9.67 log CFU g71 soil), B. gladioli 10242 (8.68 log CFU g71 soil)and E. hormaechei 10240 (8.60 log CFU g71 soil).

Discussion

Our studies were aimed at testing the efficiency of selected strains of P-solubilizingbacteria (P. synxantha 10223, S. marcescens 10241, B. gladioli 10242 and E.hormaechei 10240) to utilize MRP and its effects on plant growth of A. barbadensis.There are limited reports on the influence of microbial biofertilizers on Aloe vera; forexample, Tawaraya et al. (2007) reported enhancement in the growth of Aloe plantsby the use of arbuscular mycorrhizae (Glomus clarum and Gigaspora decipiens),whereas Pandey and Banik (2009) reported the influence of Glomus mossae andAzotobacter sp. on Aloe vera. Our report demonstrates for the first time the influenceof bacteria P. synxantha, B. gladioli, E. hormaechei and S. marcescens on Aloe plants.However, these microbes have been used as biofertlizers for other plants, forexample, P. synxantha for enhancing bud growth of Pinus sylvestris (Pirttila et al.2004), B. gladioli for plant growth promotion of Cucumis sativus (Raupach andKloepper 1998); E. hormaechei for increasing wheat yield (Egamberdieva et al. 2008)and S. marcescens for increased biomass production of Cucurbita pepo (Selvakumaret al. 2008).

Figure 3. Genotypic profiling through RAPD analysis of phosphate-solubilizing bacteria(PSB), isolated from the rhizosphere of Aloe barbadensis plants treated with Pseudomonassynxantha 10223 (a), Burkholderia gladioli 10242 (b), Enterobacter hormaechei 10240 (c), andSerratia marcescens 10241 (d). Lane L represents a 1 kb DNA ladder; circled lane numbersrepresent inoculated PSB isolates in their respective treatments and noncircled lanes representthe unknown PSB, isolated along with inoculated PSB. Analysis of each PSB was performed inthree replicates.

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The PSB isolates were tested in vitro for MRP solubilization. All PSB isolatesshowed the potential to solubilize MRP in liquid medium, although the degree ofsolubilization varied. A negative correlation of 70.35 to 70.81 was found betweenP solubilization and the pH of the medium. Our results are in agreement with Chenet al. (2006), who also reported a negative correlation (70.80) between Psolubilization and the pH of the medium. Some other reports also support ourresults (Illmer and Schinner 1992; Seshadri et al. 2000).

The isolates E. hormaechei 10240 and B. gladioli 10242 showed the greater MRPsolubilization than P. synxatha 10223 and S. marcescens 10241 under laboratoryconditions, but showed a lesser increase in plant growth during pot experiment,therefore it is suggested that in vitro values of P-solubilizing activities may not correlatewith production levels under in vivo conditions (de Freitas et al. 1997; Mittal et al.2008). The reasons for the variation between in vitro and in vivo results might beassociated with the production of several other secondary metabolites like indole-3-acetic acid (Patten and Glick 2002), siderophore (Koo and Cho 2009), gibberellins(Bottini et al. 2004) and cytokinins (Ortiz-Castro et al. 2008) by PSB in addition to Psolubilization. All PSB showed the potential to enhance various growth parameters incombination with MRP. Our results are in agreement with Sharma et al. (2009), whoreported that MRP along with PSB inoculation showed efficient increase inproductivity and phosphorus balance in rice-rapeseed-mungbean cropping system.

In this study, treatment with P. synxantha 10223 and S. marcescens 10241showed greater potential for enhancing plant growth and aloin-A production than B.gladioli 10242 and E. hormaechei 10240. The reasons for this might be associatedwith differences in P absorption by the plants inoculated with these PSB (Raj et al.1981; Piccini and Azcon 1987). In comparison with control plants, plants treatedwith P. synxantha 10223 and S. marcescens 10241 showed a greater increase in Puptake (111.75–118.05%) than plants treated with B. gladioli 10242 and E.hormaechei 10240 (45.56–61.52%).

The significantly lower plant growth in control plants than in PSB-inoculatedplants indicated that the native PSB did not contribute directly towards the plantgrowth through P solubilization because of their low number compared withinoculated PSB isolates. The increase in the uptake of P by plants in the treatmentswith PSB inoculations indicated the solubilization of insoluble phosphates by PSBby excreting organic acids and chelating substances (Omar 1998). Isolates, P.synxantha 10223 and S. marcescens 10241 showed the greater potential for enhancingplant growth than B. gladioli 10242 and E. hormaechei 10240 and likewise alsoshowed better P uptake in plants.

Other reasons for greater plant growth and aloin-A production by P. synxantha10223 and S. marcescens 10241 might be associated with root colonization abilitiesbecause it is reported that bacterial isolates that promote plant growth are also rapidcolonizers of the root system (deWeger et al. 1995; Hoflich et al. 1995).Maximum rootcolonization was shown by P. synxantha 10223 (9.72 log CFU g71 soil) followed by S.marcescens 10241 (9.67 log CFU g71 soil) and likewise maximum plant growth andaloin-A productions was observed in plants treated with these PSB.

Conclusions

According to this investigation it can be concluded that the PSB inoculationseffectively utilized the MRP and resulted in enhanced plant growth and aloin-A

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content in A. barbadensis. The effects of P. synxantha 10223 and S. marcescens 10241were more pronounced than B. gladioli 10242 and E. hormaechei 10240. Our studywas conducted under greenhouse conditions but the potential of PSB along withother rock phosphate application needs to be studied further under field conditions.

Acknowledgements

The authors would like to express their gratitude to Union Grant Commission, New Delhi,India for financial support and Haryali Biotech, Zirakpur (Punjab) for soil sample collectionand providing tissue culture plantlets. We are also thankful to Dr Vani Mittal and Mr OnkarBal for their useful suggestions and Mr Navtej Singh (Micrographer, SAIF, PanjabUniversity, Chandigarh) for timely help.

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