Chiang Mai J. Sci. 2013; 40(1) 11

Chiang Mai J. Sci. 2013; 40(1) : 11-25http://it.science.cmu.ac.th/ejournal/Contributed Paper

Characteristics of Phosphate Solubilization byPhosphate-Solubilizing Bacteria Isolated fromAgricultural Chili Soil and Their Efficiency on theGrowth of Chili (Capsicum frutescens L. cv. Hua Rua)Worapon Surapat [a], Charida Pukahuta [a], Pongsak Rattanachaikunsopon [a],Tadanori Aimi [b], and Sophon Boonlue*[c][a] Department of Biological Sciences, Faculty of Science, Ubon Ratchathani University,

Ubon Ratchathani 34190, Thailand.[b] Department of Biochemistry and Biotechnology, Faculty of Agriculture, Tottori University,

Tottori 680-8553, Japan.[c] Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand.*Author for correspondence; e-mail: bsopho@kku.ac.th.

Received: 10 April 2012Accepted: 21 May 2012

ABSTRACTNo previous studies have been carried out on the isolation and characterization of

phosphate-solubilizing bacteria (PSB) in organic and conventionally grown chili (Capsicum frutescensL. cv. Hua Rua) in Ubon Ratchathani province, Thailand. This study aimed to isolate PSB fromrhizosphere soil of chili cultivated on organic and conventional farms. Characterization oforganic acid and ability of mineral phosphate solubilization were also investigated. The effectsof PSB on the growth of chili were observed in a pot trial. The most PSB (over 68%) couldbe obtained from organically managed soil. Ten isolates of PSB were selected based ontricalcium phosphate solubilizing ability, and further testing was carried out for characterizationof organic acids. The selected PSB exhibited phosphate-solubilizing ability ranging between126.36-488.55 μg/ml of phosphate. Additionally, the 16S rRNA gene and phylogenetic analysisshowed maximum similarity to Burkholderia ambifaria and B. tropica. HPLC analysis of organicacids produced from these isolates in a culture medium found eight different kinds of organicacids: acetic acid, citric acid, gluconic acid, lactic acid, succinic acid, propionic acid, and twounknown organic acids. Among the isolates in this study, multiple organic acids could beproduced from eight isolates, and a single organic acid was found from two isolates. Multipleorganic acids did not correspond with an ability to solubilize mineral phosphate. Isolate KS04was obtained from a conventional chili farm, and showed the greatest efficiency in promotionof chili growth cultivated in sterile organically managed soil; it significantly increased the growth,flowering and P uptake, compared to uninoculated plants. Thus, this isolate may be useful foruse as a bio-inoculant for chili production in organically managed soil.

Keywords: Burkholderia tropica KS04, Chili growth, Organic acid, Organically managed soil,Phosphate-solubilizing bacteria

12 Chiang Mai J. Sci. 2013; 40(1)

1. INTRODUCTIONChili (Capsicum frutescens L.) is an

economically important vegetable ofThailand that can be grown all yearround [1]. The biggest production of chili isin the northeast of Thailand [2], particularly inthe southern part of the northeast, namelyUbon Ratchathani and Sisaket provinces [3].Generally, agricultural practices of Thaifarmers for cultivation of chili have beenperformed using two systems: conventionaland organic. High chemical inputs, such assynthetic fertilizers and synthetic pesticides, aretypical in a conventional system, which ispracticed by most farmers. In an organicsystem, green manure, animal manures,liquid fertilizer (effective microorganism,called EM) and compost are applied. As aresult, the quality and price of chili productsis higher than those produced in aconventional system [4].

Inorganic fertilizer is an economical wayto supply enough nutrients to increase chiliproduction. Among the macronutrients usedfor chili production, phosphorus (P) is oneof the most essential for plant growth, thesecond being nitrogen. However, a largeamount of inorganic phosphate applied tothe soil as chemical fertilizer is immobilizedrapidly, causing unavailability and a limitationfor plant use [2, 5, 6]. Recent reports havedocumented that approximately 95-99% ofphosphorus in soil is present in the form ofinsoluble phosphates [7], but only 0.1% ofthe total phosphorus exists in plant-accessibleform [8]. This problem may be alleviated byreleasing P from an immobilized form to asoluble form through the activities ofsolubilization and mineralization by soilmicroorganisms [9].

Recently, plant growth-promotingrhizobacteria (PGPR) have become attractivefor use as soil inoculums due to theirimprovement of plant growth and yield by

various direct or indirect mechanisms [10, 11].Among PGPR, phosphate-solubilizingbacteria (PSB) are one of the most interestingmicroorganisms concerned with plantP nutrition. The principal mechanism formineral phosphate solubilization by thesebacteria is the production of low-molecular-weight organic acids, which have highpotential as cations bound to phosphate,as a result of their conversion into solubleforms [9]. PSB has the ability to dissolvetricalcium phosphate from an insolubleform into a soluble form, as has beenreported by many researchers [12, 13, 14, 15].Additionally, the use of PSB as inoculantssimultaneously increases P uptake by theplant, resulting in higher crop yields - as hasbeen documented in maize [16], greengram[17], sorghum [18], wheat, potato, bean [19]and tomato [20]. There have been few reportsconcerning growth promotion of chili byco-inoculation of arbuscular mycorrhizalfungi and PSB [21, 22]. However, there havebeen no reports on the isolation of PSBfrom organically managed soil; mostresults have been documented from otheragricultural and rangeland soils [12, 23, 24].

In order to investigate the potential ofPSB as inoculants for chili cultivation inorganically managed soil, the presentexperiment was designed to characterize thephosphate solubilization by PSB isolatedfrom conventional and organic soil and todetermine the effects of PSB on the growthand P uptake of chili (cv. Hua Rua) planted insterile, organically managed soil undergreenhouse condition.

2. MATERIALS AND METHODS2.1 Soil Sampling and Isolation of PSB

Soil samples were collected from oneorganic farm, Ratchathani Asok (15° 13’34.48" N, 104° 54’ 11.05" E), and from threeconventional chili farms, Ban Hua Rua

Chiang Mai J. Sci. 2013; 40(1) 13

(15° 22’ 4.82" N, 104° 49’ 49.74" E), BanKaset Nua (15° 18’ 9.97’’ N, 104° 89’ 84.91’’E)and Ban Pho Yai (15° 21’ 16.44’’ N, 104° 92’0.12’’ E), in Ubon Ratchathani province.Approximately 50 g of chili root zone soil(30 cm depth) was taken aseptically from thecentral areas (4 × 4 m) of three agriculturalchili plots at each of the four sites, withduplicates from each plot. Each soil samplewas thoroughly mixed and further enrichedon Pikovskaya’s medium, containing 0.5%tricalcium phosphate as a sole P source, at30° C for 24 h. Subsequently, PSB wereisolated by dilution plate technique onPikovskaya’s agar medium [25]. After 3 d ofincubation at 30° C, colonies with hollowclear zones were obtained, and thereafterpurified on Pikovskaya’s agar.

2.2 Morphological and BiochemicalCharacteristics

Gram staining is used to classify bacteriaon the basis of their forms, sizes, cellularmorphologies, and Gram reactions [26].Biochemical characterization of PSB isolateswas carried out according to Coenye et al.[27], Garrity et al. [28] and Reis et al. [19].Utilization of sugar and fermentation weredetermined by starch hydrolysis, gelatinhydrolysis and arginine hydrolysis, as well astests for oxidase, catalase, urease, nitratereduction, glucose, D-mannitol, lactose,dulcitol, maltose, fructose, D-raffinose, andsucrose.

2.3 Genomic DNA Isolation, 16S rRNASequencing and Phylogeny

Template DNA for 16S rRNA geneamplification was prepared by a methodmodified from Nilsson et al. [29]. To producethe fragments of the 16S rRNA gene,genomic DNA of PSB was amplified bypairs of a universal primer: UFUL (5’-GCCTAACACATGCAAGTCGA-3’) and

URUL (5’ CGTATTACCGCGGCTGCTGG-3’). The reaction conditions includedan initial denaturation of 5 min at 94° C,followed by 30 cycles of denaturation at94 ° C for 30 s, annealing at 55° C for 30 s,and extension at 72° C for 30 s. A finalextension at 72° C for 5 min was done at theend of the amplification. PCR productswere electrophoresed using 1.5% agarose,then stained with ethidium bromide (0.5 μg/ml), and visualized using a GelDoc-It®

(Upland, CA) UV transilluminator [30].Sequence data were compared visually

and aligned with available standardsequences of bacterial lineages from theGenBank of the National Center forBiotechnology Information (NCBI), usinga BLASTN analysis tool [31]. Mega4molecular software, version 4.0, was usedfor multiple sequence alignment andphylogenetic tree analysis. Multiple sequencealignment used the ClustalW softwarepackage. Phylogenetic trees were built usingneighbor-joining. Bootstrap analysis wasbased on 1,000 resamplings [32]. Distanceswere calculated according to the Kimura2-parameter [33]. The isolates were alsocompared with those present in the RibosomalDatabase Project (http://rdp.cme.msu.edu/), comprising reference profiles of closelytyped species strains.

2.4 Determination for Growth, PhosphateSolubilizing Abilities, pH and Analysisof Organic acid

PSB were inoculated into Pikovskaya’smedium containing 0.5% tricalciumphosphate, as described previously, by shakingat 160 rpm at 30° C for 72 h. To estimatebacterial growth yield, optical density (OD)of bacterial culture was determined at560 nm (OD560); then cell numberswere examined by standard plate countmethod [34], and later centrifuged for 5 min

14 Chiang Mai J. Sci. 2013; 40(1)

at 13,081 × g (10,000 rpm) at 4° C in aHettich Rotina 35R to remove bacterialbiomass. The supernatant was measured fortotal amount of released soluble phosphatecontents by the Molybdenum blue method[35]. The pH of the medium was alsorecorded from the cultured supernatant by apH meter (Sartorius PP-20, Edgewood, NY)in order to establish a relationship betweenthis parameter and the soluble phosphate [36].

For analysis of organic acids, culturedmedia were filtered though a 0.2 μm filter(GTBP; Millipore, Billerica, MA); 20 μl offiltrates were then injected into an HPLCcolumn (LC-20AD; Shimadzu, Kyoto,Japan) equipped with a SPD-M20A diodearray detector. Organic acid separation wascarried out on an Inertsil ODS-3 column(GL Sciences, Tokyo, Japan) with 0.1 MNH4H2PO4 in phosphoric acid (pH 2.6) asmobile phase. The retention time of eachsignal was recorded at a wavelength of210 nm.

2.5 Effects of PSB on growth of chili inpot experiment

PSB suspensions were prepared byculturing on NB and incubating on anorbital shaker at 160 rpm, 30° C, for 48 h.Then bacterial cells were harvested bycentrifugation for 5 min at 13,081 × g (10,000rpm), 4° C, in a Hettich Rotina 35Rcentrifuge, and washed with sterile distilledwater. The cell pellet was resuspended withsterile distilled water, and then further adjustedto be 1 at OD560 (2.0 × 108 CFU/ml).

A pot experiment was carried out in agreenhouse employing randomized completeblock design with four replications and11 treatments. The treatments consisted ofone control (non-inoculated with PSB)and 10 PSB species. All treatments wereconducted in pots (12-in diameter) containing8 kg of soil. The soil used for preparation

of the seedling and pot experiment wasobtained from a plot that had been organicallymanaged for more than five years(Ratchathani Asok). This organic soil wasamended by adding home made compost(5,555.56 kg/ha), coarse rice bran (5,555.56kg/ha), soybean press cake (2,777.78 kg/ha),sesame press cake (2,777.78 kg/ha) andrice husk ash (5,555.56 kg/ha). It had asilt/loam texture, pH 4.6, 1.3% organicmatter, 0.08% total N, 66.8 ppm total P,37.5 ppm available P (Bray II Method [37]),and 78.3 ppm extractable K (1 N NH4OAc[38]). Soil was sterilized by fumigation with60g/m2 of dazomet (3,5-dimethyl-1,3,5-thiadiazinane-2-thione). Chili (cv. Hua Rua)seedlings were prepared from sterile(10% v/v, NaOCl solution, for 10 min) seedsplanted in small plastic pots (top diameter,7 cm) filled with fumigated soil. After 21 d,individual chili seedlings were transplantedinto individual plastic pots (12-in diameter)and then inoculated with 5 ml of PSB cellsuspension (2.0 × 108 CFU/ml). The plantswere watered with sterile distilled waterfor 90 d. Five ml of PSB cell suspension(2.0 × 108 CFU/ml) was reinoculated ontothe plant root zone by injection with asterilized syringe at every 2 weeks ofcultivation. At the end of the study,plant growth parameters - such as shootheight, stem diameter, fresh weight ofshoots and roots, dry weight of shoots androots, number of flowers per plant, and Puptake - were determined.

2.6 Statistical analysisData were analyzed using SPSS for

Windows software, version 11.5.0 (SPSS,Chicago, IL). All data were subjected toanalysis of variance. Comparisons of meanswere made by Duncan’s multiple range test(P≤0.05).

Chiang Mai J. Sci. 2013; 40(1) 15

3. RESULTS3.1 Isolation of PSB and PreliminaryScreening for Phosphate Solubilization

PSB were isolated from rhizospheresoil of chili cultivated at four sites fromagricultural farms in Ubon Ratchathaniprovince, Thailand. Fifty isolates of PSBwere obtained (Figure 1). A large amount ofPSB isolated strains were obtained fromthe organic farm at Ratchathani Asok(34 isolates). PSB isolates at conventionalchili plots were found to be fewer than at theorganic chili farm: at Ban Hua Rua (7 isolates),Ban Kaset Nua (6 isolates), and Ban Pho Yai(3 isolates). In order to screen for highphosphate-solubilizing activity of PSBstrains for a further pot experiment, all PSBstrains were cultured on Pikovskaya’s

medium containing 0.5% tricalciumphosphate. Ten isolates - namely, RA01,RA02, RA12, RA28, RA32, HR06, HR07,KS01, KS04 and PI01 - exhibited highability of phosphate solubilization forreleasing of soluble phosphorus fromtricalcium phosphate (Figure 1).

3.2 Morphological and BiochemicalCharacterization of Bacteria

The morphological characteristics ofPSB were Gram negative, rod-shaped andnon-spore forming. All isolates were positivefor nitrate reductase, citrate, oxidase, catalase,hydrolysis of glucose, D-mannitol, andfructose tests, but negative for a urease test.RA01, RA02, RA12 and KS01 presentedpositive results for gelatin hydrolysis,

Figure 1. Mineral solubilizing activity of PSB isolated from organic and conventionalchili farms. Organic farm: RA, Ratchathani Asok; Conventional farm: HR, Ban HuaRua; KS, Ban Kaset Nua; P, Ban Pho Yai.

16 Chiang Mai J. Sci. 2013; 40(1)

arginine hydrolysis, dulcitol, D-raffinoseand sucrose; whereas isolates RA28, RA32,HR06, HR07, KS04 and PI01 showednegative results for gelatin and argininehydrolysis. In addition, four isolates(RA32, HR06, HR07 and KS04) presentedpositive results for a lactose test, whileother isolates exhibited negative results.

3.3 Identification and PhylogeneticAnalysis of Bacteria

Identification of PSB isolates based on16S rRNA gene sequence and a phylogenetic

tree are presented in Figure 2. Thesesequence data have been submitted to theDDBJ/EMBL/GenBank databases underaccession number AB673035-AB673045.All isolates displayed close homology tothe genus Burkholderia. Our isolates weredivided into two phylogenetic clades. Thefirst clade (PI01, RA28, RA32, KS04,HR06 and HR07) was maximally closelyrelated to Burkholderia tropica Ppe8T. Inaddition, two clusters were subdividedwithin this clade, as cluster I (PI01 and RA28) and cluster II (RA32, KS04, HR06 and

Figure 2. Phylogenetic analysis based on 16S rRNA sequence, which was generated basedon pairwise nucleotide distance of the Kimura 2-parameter using the neighbor-joining methodincluded in the MEGA4 software package. The bar indicates a difference of 1 nucleotide per100. The number beside the node is the statistical bootstrap value. In brackets are the GenBankaccession numbers of the16S rRNA genes.

Chiang Mai J. Sci. 2013; 40(1) 17

HR07). 16S rRNA gene sequence analysisof RA28 and PI01 in cluster Ishowed 99.1% similarity to Burkholderiatropica Ppe8T. However, members of clusterII - HR07, HR06, KS04 and RA32 presented98.5%, 98.6%, 98.8% and 98.5% similaritiesto Burkholderia tropica Ppe8T, respectively.The second clade (RA02, RA12, KS01 andRA01) was closely related to Burkholderiaambifaria, with a genetic distance farther fromother strains and completely separate fromthe other clade. The isolates in this group(RA02, RA12, KS01 and RA01) presented99.6%, 99.7%, 99.3% and 99.7% similaritiesto Burkholderia ambifaria AMMDT, respectively.From these results, we identified our isolatesto be B. tropica and B. ambifaria.

3.4 Phosphate Solubilization, Growth,pH and Production of Organic acid

Ten isolates with high phosphate-solubilizing activity PSB were investigatedfor phosphate solubilization during a 72 hperiod by monitoring growth, pH, available

phosphorus and organic acid production inthe culture medium (Table 1). Growth rate interms of cell numbers measured by standardplate count methods was not significantlydifferent among observed isolates (1.86 × 108

to 2.06 × 108 CFU/ml); whereas pH in theculture medium declined (to 3.31-5.11) froman initial pH of 6.60 after incubation for72 h. In addition, isolate HR07 was able toproduce significantly higher acid than otherisolates (except for RA28, RA32 and PI01),which was detected from a pH drop(pH 3.31). In consideration of solublephosphorus released from mineral-solubilizingactivity of PSB, the result showed thatbacterial isolates RA02, RA12, HR06, HR07,KS01 and KS04 had significantly higherpotential for solubilization of tricalciumphosphate than the other isolates. Amongthese strains, KS04 was proven to be the best(499.85 μg/ml).

An analysis of organic acid productionby HPLC is presented in the chromatogramin Figure 3. The results revealed that 8

Table 1. Bacterial cell growth, pH, soluble-P concentration and types of organic acid observedafter 72 hrs incubationa.

Note: aIn a column, means followed by a common letter are not significantly different byDMRT0.05 bG, gluconic acid; L, lactic acid; C, citric acid; A, acetic acid; S, succinic acid; P, propionicacid; U1, unknown acid 1; and U2, unknown acid 2.

Isolates OD560 Cell density(log CFU/ml)

pH ofmedium

Soluble-P(μg/ml)

Type of organic acidb

RA01RA02RA12RA28RA32HR06HR07KS01KS04PI01CV%

1.120a1.288a1.236a1.153a1.228a1.219a1.181a1.155a1.199a1.232a16.4

1.86 × 108a2.14 × 108a2.06 × 108a1.92 × 108a2.04 × 108a2.03 × 108a1.96 × 108a1.92 × 108a2.00 × 108a2.05 × 108a

41.34

4.25bc5.01d5.11d3.76ab3.53ab4.77cd3.31a

4.86cd4.05b3.99ab19.95

294.26b479.51de403.44cd129.28a

335.15bc448.44de427.54de433.81de499.85e133.60a98.76

G L A C S U1 U2 G L A C P U2G L A C P U2A S U1AA U1A SG L C P U2AG A C P U2

18 Chiang Mai J. Sci. 2013; 40(1)

different kinds of organic acid - lactic acid,acetic acid, citric acid, succinic acid, propionicacid, gluconic acid, and 2 unknownacids - could be produced from all PSBisolates. Eight isolates (RA01, RA02, RA12,RA28, HR06, HR07, KS01 and PI01) showedthe presence of multiple organic acids(as shown in Table 2 and Figure 3). Onlytwo isolates (RA32 and KS04) couldproduce only one organic acid (acetic acid).In addition, acetic acid could be producedfrom all observed isolates except KS01.The retention times of the unknown organic

acids were 2.6 and 20.4 min for unknown1 and unknown 2, respectively.

3.5 Effects of PSB on the Growth of Chiliin a Pot Experiment

The effects of PSB on the growth ofchili in a pot trial demonstrated that chiliinoculated with PSB isolate KS04 showedthe best growth performance. All plantgrowth parameters including height, freshweight, dry weight of shoots and roots andnumber of flowerings found in plantsinoculated with KS04 were significantly

Figure 3. HPLC chromatogram of organic acids produced from PSB cultured on Pikovskaya’smedium for 72 h. G, gluconic acid; L, lactic acid; C, citric acid; A, acetic acid; S, succinic acid;P, propionic acid; U1, unknown acid 1; and U2, unknown acid 2.

Chiang Mai J. Sci. 2013; 40(1) 19

Table 2. Growth parameters and phosphorus uptake in chili inoculated with ten differentPSB isolates cultivated on sterile organically managed soila.

higher than in uninoculated plants(control). Chili inoculated with RA12,HR07, KS01, KS04 and PI01 showedsignificantly higher P uptake than thecontrol (uninoculated plant). Among them,the highest P uptake was evident in theplant inoculated with KS04. Furthermore,the height and P uptake of chili inoculatedwith KS04 was significantly higher thanplants inoculated with the other PSBisolates.

4. DISCUSSIONPSB is a phosphate-solubilizing

microorganism which can be routinelyscreened by a plate assay method usingPikovskaya medium. The bacteria willgrow on this medium and form a clearzone around the colony [25, 39]. Thesebacteria can convert tricalcium phosphate inthe medium from insoluble to soluble forms[40]. A total of 50 PSB isolates were screened

by the halo zone presented around a colonyin Pikovskaya’s medium which were obtainedfrom organically managed soil (RatchathaniAsok), higher than at conventional chilifarms (Ban Hua Rua, Ban Kaset Nua andBan Pho Yai). Juottonen et al. [41] reportedthat environmental conditions and farmmanagement practices had a considerableimpact on soil microorganisms. Thenumbers of microorganisms found in eachsoil sample were different because thecomposition and function of the microbialcommunity was shown to vary according toland-use type, plant species, temperature,nutrients, oxygen availability, pollutants andother environmental variables. Our resultscorresponded with findings by Chen et al.[12] which reported that several isolates ofPSB could be isolated from fertile soil inTaichung, Taiwan. Kumar et al. [42] alsofound different numbers of PSB isolates invarious agricultural farms in eastern Uttar

Note: aIn a column, means followed by a common letter are not significantly different byDMRT0.05

Treatments

Shoot

Height(cm)

Diameter(cm)

Freshweight(g/pot)

Dryweight(g/pot)

RootFreshweight(g/pot)

Dryweight(g/pot)

No. offlowerings

per pot

P uptake(mg/pot)

ControlRA01RA02RA12RA28RA32HR06HR07KS01KS04PI01CV%

23.88a32.50b29.75b42.00b29.50b43.00b33.50b42.50b41.50b49.00c39.00b35.7

0. 26a0.30ab0.26a

0.32ab0.26a0.40b0.33ab0.40b0.37ab0.41b0.35ab26.5

2.72a5.18a4.23a

8.45ab4.57ab9.17ab6.88ab10.81ab9.36ab14.50b8.32ab73.4

0.38a0.82a0.65a

1.39ab0.58ab1.52ab1.09ab1.70ab1.72ab2.66b1.38ab91.3

2.40ab2.02ab2.72bc2.00ab2.27abc1.61ab2.40abc1.76ab1.20a3.25c

1.97ab92.8

0.06a0.28a0.26a

0.53ab0.54ab0.61ab0.26a

0.70ab0.54ab1.17b0.51ab84.9

1.00ab4.50cd0.00a

1.50ab3.00bc1.50ab2.75bc4.25cd1.50ab5.50d0.25a

190.28

0.002a0.006abc0.005ab0.014bc0.006abc0.011abc0.006abc0.017bc0.017bc0.028d

0.015bc 84.85

20 Chiang Mai J. Sci. 2013; 40(1)

Pradesh, India.Based on the ability of solubilization of

tricalcium phosphate, PSB isolated fromconventional farm had higher solubilizingactivity than those isolated from organicfarm. This finding was similar to the reportof Bhakta et al. [43] who found thatphosphate solubilizing activity of bacteriaisolated from sediment soil exposed tovarious doses of chemical fertilizerincreased correlating to the amount offertilizer in the soil.

In addition, the general characteristicsof the isolates in this study correspondedwith the genus Burkholderia as described byLinu et al. [44]: Gram negative, rod-shaped,motile, non-sporing, and containing catalaseand citrate. Analysis of 16S rRNA andthe phylogeny of PSB found that allisolates belonged to Burkholderia spp.(Figure 2), which are completely separatedfrom Enterobacter sp. by 100 bootstrap values.In addition, all isolates in this study weremaximally related to Burkholderia tropica andB. ambifaria. Our results contrasted with thereport of Kumar et al. [42], which could onlyisolate genera belonging to members ofEnterobacteriaceae (Enterobacter spp. andExiguobacterium spp.) from agricultural farms.However, our results corresponded with thefindings of Balandreau et al. [45], whichdocumented that Burkholderia was a commonplant-associated bacterium which could befound naturally in wet soil and decayingplants [46].

Generally, available phosphorus in soilcan be increased by low-molecular-massorganic acids produced from PSB [12].The results of this study demonstratedthat various types of organic acids wereproduced by PSB. This was a significantcharacteristic of these bacteria, including adrop in pH and soluble phosphorus whichwas obtained after culturing in mineral

phosphate for a few days. The results inTable 1 and Figure 3. demonstrate that mostPSB isolates (8 isolates) produced multipleorganic acids, while a few (2 isolates)produced only one type of organic acid(acetic acid). However, multiple organicacid producers did not show phosphate-solubilizing activity or a pH drop greaterthan those from one organic acid producer.This result contrasting with the findings ofChen et al. [12], who reported that acombination of multiple organic acids couldaccomplish mineral solubilization andpH drop better than a single organic acid.In addition, our results revealed that aceticacid could be produced from all isolatesin this study, except isolate KS01. Thisindicated that acetic acid may be a majoracid concerned with the solubilizationactivity of PSB [47]. However, thephosphate solubilization efficiency maydepend on other factors such as substrate,medium, temperature, time, and othermechanisms in addition to organic acidproduction. However, the acidification ofculture supernatants seemed to be generallythe main mechanism for phosphatesolubilization [48]. When they releasedorganic acid, the medium pH dropped toa low level rapidly. Valverde et al. [49]reported that Burkholderia spp. producedgluconic acid, acetic acid and citric acid,and showed that these bacterial genera hadhigh efficiency in solubilizing insolublephosphate. Other organic acids could beproduced by them as well, such as butyric acid,lactic acid, succinic acid, malic acid, glycolicacid, fumaric acid [50], propionic acid,and unknown organic acids [51].

The effects of PSB on the growthof chili in a pot trial found that KS04significantly increased shoot height and Puptake, to a greater extent than the otherPSB isolates observed in this study.

Chiang Mai J. Sci. 2013; 40(1) 21

Furthermore, KS04 was the best isolate forsignificantly enhancing all plant growthparameters (higher than uninoculatedplants), including fresh and dry weight ofshoots, fresh and dry weight of roots,number of flowerings per plant, and Puptake (Table 3). Regarding the ability ofmineral phosphate solubilization and typesof organic acids of KS04 (Table 2), wefound that this isolate had high efficiencyfor solubilization of tricalcium phosphate,and produced only acetic acid. This indicatedthat acetic acid may be related to themechanism of PSB in promoting chiligrowth. Generally, soil pH was inducedto decline to pH 4.0-5.0 by 99% pure aceticacid. At these pH values, phosphorus in thesoil could be combined with aluminumor ferrous metals and converted intohydroxyapatites (insoluble phosphorus) [52].The subsequent activity of acidification byorganic acids produced from PSB was highunder these conditions.

This study showed that KS04significantly enhanced P uptake and growthof chili. Our findings correspond to resultsreported by Afzal and Bano [53] whoreported that wheat (Triticum aestivum)inoculated with PSB (Pseudomonas sp. strain54RB) significantly increased root andshoot weight, plant height, spike length,grain yield and P uptake higher than thecontrol. Similar results were also found incowpea (Vigna unguiculata (L.) Walp.) whichrevealed the enhancement of nodulation,root and shoot biomass, straw and grainyield and P and N uptake of plants inoculatedwith Gluconacetobacter sp. and Burkholderia sp.[44]. The growth parameters and P uptakeof chili plants in the present study suggestedthat bacterial isolate KS04 had the highestpotential for promoting chili growth, as aresult of its highest potential for enhancingP uptake of the plant. In addition, KS04

showed the strongest activity forsolubilization of insoluble tricalciumphosphate as well. Thus, this indicated thatP-solubilization was the main mechanismresponsible for positive growth response.Based on the results found in present study,we concluded that KS04 had the greatpotential for use as soil inoculants fororganic chili production in Thailand.However, before using of this bacterialisolates as a biofertilizer, its effect on thegrowth of chili in the actual organic chilicropping should be investigated.

5. CONCLUSIONSThe results of this study represented

that the number of PSB obtained fromorganic chili farms was higher than fromconventional chili farms. A high ability oforganic acid producer was identified usingconventional biochemical characteristics ofBurkholderia genera. The results of 16S rRNAgene and phylogenetic analysis of theseisolates showed a close relationship toBurkholderia ambifaria and B. tropica. Eightdifferent kinds of organic acid weredetected from the culture medium byHPLC analysis after 72 h. The multipleorganic acids and the single one which werefound did not correspond with the abilityof mineral phosphate solubilization andpH drop. Chili inoculated with KS04 whichwas planted in organic soil showedsignificantly higher values for all plantgrowth parameters and P uptake thanuninoculated plants (control). These findingssuggest the potential of PSB isolate KS04for use as a bio-inoculant for production ofchili by organic farming in Thailand.

ACKNOWLEDGMENTSThis research work was kindly

supported by grants from the Commissionon Higher Education of Thailand, the

22 Chiang Mai J. Sci. 2013; 40(1)

Royal Thai government, the ThailandResearch Fund (TRF), and MicrobialResources and Applications group.

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