Soil Biology & Biochemistry 37 (2005) 1970–1974 www.elsevier.com/locate/soilbio Short communication Isolation and characterization of phosphate solubilizing bacteria from the rhizosphere of crop plants of Korea Heekyung Chunga, Myoungsu Parka, Munusamy Madhaiyana, Sundaram Seshadria, Jaekyeong Songb, Hyunsuk Chob, Tongmin Saa,* a Department of Agricultural Chemistry, Chungbuk National University, 48, Gaeshing Dong, Heungduk Gu, Cheongju, Chungbuk 361-763, South Korea b Korean Agricultural Culture Collection (KACC), National Institute of Agricultural Biotechnology, Suwon 441-707, South Korea Received 6 April 2004; received in revised form 22 November 2004; accepted 21 February 2005Abstract Whole-cell fatty acids methyl ester (FAME) proﬁle and 16S rDNA sequence analysis were employed to isolate and identify the bacterialgroups that actively solubilized phosphates in vitro from rhizosphere soil of various crops of Korea. Out of several hundred colonies thatgrew on Pikovskaya’s medium 13 best isolates were selected based on the solubilization of insoluble phosphates in liquid culture and furthercharacterized and identiﬁed. They were clustered under the genera Enterobacter, Pantoea and Klebsiella and the sequences of threerepresentative strains were deposited in the GenBank nucleotide sequence data library under the accession numbers AY335552, AY335553,AY335554.q 2005 Published by Elsevier Ltd.Keywords: Phosphate solubilization; Pantoea agglomerans; Enterobacter aerogenes; Klebsiella sp. Microorganisms capable of producing a halo/clear zone Since the knowledge on the diversity of phosphatedue to solubilization of organic acids in the surrounding solubilizing bacteria (PSB) in Korean soils is lagging, anmedium (Singal et al., 1991) are selected as potential attempt to isolate and identify PSB through biochemical andphosphate solubilizers (Das, 1989) and are routinely molecular methods was made. The rhizosphere soil samplesscreened in the laboratory by a plate assay method collected and transferred under aseptic conditions were(Gerretson, 1948) using either Pikovskaya agar (Pikovskaya, stored in an ice pack at 4 8C in the laboratory. One milliliter1948) or Sperber agar (Sperber, 1958). Several reports on of the appropriate (10K5–10K7) dilutions of the soil samplesbacteria and fungi isolated from soil have evaluated their was plated on Pikovskaya’s medium (Pikovskaya, 1948) formineral phosphate solubilizing (MPS) activity with various P the isolation of PSB. The colonies distinguished bysources such as calcium phosphate tribasic [Ca3(PO4)2] producing halo zones, were identiﬁed and sub-cultured(Illmer and Schinner, 1995), iron phosphate (FePO4) (Jones (Table 1). As the plate assay is not considered a reliableet al., 1991) and aluminium phosphate (AlPO4) (Illmer et al., method in determining a strain as phosphate solubilizer1995). An increase in P availability to plants through the (Johri et al., 1999), the pure cultures were further screenedinoculation of PSBs has also been reported previously in pot in liquid medium containing Ca3(PO4)2, AlPO4 and FePO4experiments and under ﬁeld conditions (Banik and Dey, at a concentration of 5 g LK1 as insoluble P sources. The1981; Chabot et al., 1996; deFreitas et al., 1997; Zaidi et al., cultures supernatant obtained by centrifugation was passed2003). through a 0.45 mM Millipore ﬁlter (Sartorius) and the inorganic phosphate content of the culture ﬁltrate was determined by the molybdenum blue method (Murphy and * Corresponding author. Tel.: C82 43 261 2561; fax: C82 43 271 5921. Riley, 1962). Autoclaved medium served as a control for E-mail address: email@example.com (T. Sa). each set. All the isolates solubilized Ca3(PO4)2 to a greater0038-0717/$ - see front matter q 2005 Published by Elsevier Ltd. extent than AlPO4 and FePO4 with AlPO4 exhibiting poordoi:10.1016/j.soilbio.2005.02.025 solubilization (Table 2). Even the isolates that did not
H. Chung et al. / Soil Biology & Biochemistry 37 (2005) 1970–1974 1971Table 1Location, soil series, crops and colony morphology of PSB isolatesLocation Soil series Crops (scientiﬁc name) Isolates Colony morphologyGae Sin Dong Yesan Spring onion (Allium ﬁstulosum L.) HK 11-1 White, slender Yesan Pepper (Capsicum annuum L.) HK 14-1 White, circular Yesan Spring onion HK 17-1 White, circular Yesan Sesame (Sesamum indicum L.) HK 18-3 White, circularGang Seo Dong Sangju Sesame HK 20-1 White, circular Sangju Pepper HK 23-2 White, circular Sangju Pepper HK 24 White, circularHyeong Dong Ri Sangju Spring onion HK 34-1 White, circular Sangju Spring onion HK 34-2 White, circularSek Pan Ri Sachon Rice (Oryza sativa L.) HK 52-1 White, circularJeung Pyung Sachon Rice HK 68-1 Yellow, circular Sachon Rice HK 68-3 White, circularBong Yang Up Sachon Rice HK 69 White, circularperform well in plate assays exhibited signiﬁcant phos- DNA (16S rDNA). fD1 (5 0 -AGAGTTTGATCCTGGCT-phates solubilization in the liquid cultures. These isolates CAG-3 0 ) and rP2 (3 0 -ACGGCTACCTTGTTACGACTT-5 0 )presumably identiﬁed as PSBs further characterized by a primers (Weisburg et al., 1991) were used. A GeneAmpseries of biochemical reactions as per the Bergey’s Manual PCR System (Perkin–Elmer Co., Norwalk, CT) with Taqof Systemic Bacteriology (Holt et al., 1994) were Gram- DNA polymerase (Promega Co., Southampton, England)negative rods with positive for catalase activity and negative was used for PCR (Park et al., in press). The sequencing wasfor oxidase activity, H2S production, gelatin, starch and performed using Big-Dye Terminator Cycle Sequencinglipid hydrolysis (Table 3). and an ABI Prism 310 Genetic Analyzer (Tokyo, Japan). The isolates were identiﬁed based on whole-cell cellular The phylogenetic tree for the data sets was inferred by thefatty acids, derivatized to methyl esters, i.e. FAMEs and neighbor-joining method using the neighbor-joining pro-analyzed by gas chromatography (GC) using the MIDI gram, MEGA version 2.0 (Kumar et al., 1993).system (MIDI, Newark, DE). The analysis was performed The GC-FAME analysis placed most of the isolatesusing the Sherlock Microbial Identiﬁcation system TSBA under Enterobacter sp., Klebsiella sp., and Pantoea sp. that4.0 software and library general system software version are grouped under one family, Enterobacteriaceae. Results4.1. Qualitative and quantitative differences in the fatty acid of 16S rDNA identiﬁcation agreed with that of GC-FAMEproﬁles were used to compute the distance for each strain for ﬁve PSBs at the genus level in Enterobacter andrelative to the strains in the library (Sasser, 1990a,b; Sasser Klebsialla (Table 4). Isolation of bacteria belonging to theand Wichman, 1991). Genomic DNA was extracted by the family Enterobacteriaceae from various soils, and their MPSphenol/chloroform method (Sambrook et al., 1989) and activities has also been reported earlier (Kim et al., 1997,ampliﬁed using PCR ampliﬁcation of the 16S ribosomal 1998; Vassilev et al., 1997, 1999; Remus et al., 2000).Table 2Solubilization of inorganic phosphates by the PSB isolates in liquid culturesIsolates Liquid culture (mg P mLK1) Ca3(PO4)2 AlPO4 FePO4HK 11-1 96.2G5.9 8.0G5.0 18.8G6.3HK 14-1 127.2G2.1 13.8G1.3 22.9G1.7HK 17-1 113.7G3.3 4.3G0.9 4.5G0.9HK 18-3 121.7G2.7 3.7G0.7 10.2G1.1HK 20-1 142.1G2.1 5.1G1.6 24.4G1.1HK 23-2 114.3G8.4 9.9G0.9 18.8G4.3HK 24 136.4G1.1 5.7G0.6 19.3G1.0HK 34-1 138.6G5.7 5.9G1.1 27.2G3.7HK 34-2 126.9G2.2 10.7G1.3 12.6G1.0HK 52-1 107.7G2.1 6.3G0.4 15.5G6.4HK 68-1 119.2G5.3 8.7G0.4 20.7G4.6HK 68-3 107.5G2.2 7.2G0.6 49.1G2.2HK 69 113.0G5.7 4.1G1.7 49.5G4.7Liquid cultures were assayed after seven days. Ca3(PO4)2, tricalcium phosphate; AlPO4, aluminium phosphate; FePO4, ferric phosphate.
1972 H. Chung et al. / Soil Biology & Biochemistry 37 (2005) 1970–1974Table 3Biochemical characteristics of the PSB isolatesBiochemical reactions PSB isolates HK HK HK HK HK HK HK HK HK HK HK HK HK 11-1 14-1 17-1 18-3 20-1 23-2 24 34-1 34-2 52-1 68-1 68-3 69Gram staining K K K K K K K K K K K K KCatalase C C C C C C C C C C C C COxidase K K K K K K K K K K K K KIMViC test Indole production K K K K K K K K K K K K K Methyl red K K K K K K K K K K K K K Voges-Proskauer C C C C C C C C C C C C C Citrate (Simmons) C C C C C C C C C C C C CLysine decarboxylase K K K C K C K K K K K C CArginine dihydrolase C C C C C K C C K C C K KOrnithine decarboxylase C C C C C K K C K C C K KCarbon source utilization Sucrose C C C C C C C C C C C C C Fructose C C C C C C C C C C C C C Glucose C C C C C C C C C C C C C Glycerol C C C C C C C C C C C C C Maltose C C C C C C C C C C C C C Mannitol C C C C C C C C C C C C C Inositol C C C C C C C C C C C C C Dulicitol K K K K C C C K C K C C K Lactose K K K K C C C K C K C C C Melibiose C C C C C C K C C C C C C D-Rafﬁnose C C C C C C C C C C C C C Sorbitol C C C C C C C C C C C C CH2S production K K K K K K K K K K K K KGelatin hydrolysis K K K K K K K K K K K K KStarch hydrolysis K K K K K K K K K K K K KLipid hydrolysis K K K K K K K K K K K K KC, tested positive/utilized as substrate; K, tested negative/not utilized as substrate.Further, it has been reported that multiple strains of closely nucleotide sequence data library under the followingrelated bacteria to dominate the rhizosphere soils of paddy accession numbers: AY335552 (HK 14-1, P. agglomerans),(Chin et al., 1999). The phylogenetic positions of the four AY335553 (HK 34-2, Klebsiella sp.) and AY335554best performing strains: Enterobacter aerogenes (HK 201, (HK 20-1, E. aerogenes).HK 34-1), Pantoea agglomerans (HK 14-1), Klebsiella sp. Application of bacterial inoculants as biofertilizers has(HK 34-2) is presented in Fig. 1. The sequences of three been reported to result in improved plant growth andrepresentative strains were deposited in the GenBank increased yield (Bashan and Holguin, 1998; Vessey, 2003).Table 4Identiﬁcation of PSB isolates by GC-FAME and 16S rDNA sequencing from rhizosphere soil samplesIsolate GC-FAME identiﬁcation Similarity (%) 16S rDNA identiﬁcation Identity (%)HK 11-1 Enterobacter cancerogenus 64.9 Pantoea sp. 98HK 14-1 Enterobacter cloacae 82.7 Pantoea agglomerans 99HK 17-1 Enterobacter cloacae 82.9 Pantoea sp. 96HK 18-3 Kluyvera ascorbata 37.6 Enterobacter cloacae 99HK 20-1 Klebsiella pneumoniae 14.6 Enterobacter aerogenes 99HK 23-2 Klebsiella pneumoniae 14.6 Klebsiella sp. 98HK 24 Kluyvera ascorbata 60.6 Enterobacter cloacae 99HK 34-1 Kluyvera ascorbata 5.1 Enterobacter sp. 95HK 34-2 Klebsiella pneumoniae 92.1 Klebsiella sp. 98HK 52-1 Klebsiella planticola 83.1 Enterobacter cloacae 99HK 68-1 Enterobacter cancerogenus 7.6 Enterobacter aerogenes 99HK 68-3 Klebsiella pneumoniae 43.8 Klebsiella sp. 99HK 69 Klebsiella pneumoniae 89.1 Klebsiella sp. 98
H. Chung et al. / Soil Biology & Biochemistry 37 (2005) 1970–1974 1973Fig. 1. Phylogenetic tree showing the relationships among the PSB isolates and between representatives of other related taxa. The tree was constructed by usingthe MEGA2 after aligning the sequences with Megalign and generating evolutionary distance matrix inferred by the neighbor-joining method using Kimuraparameter 2. The numbers at the nodes indicate the levels of bootstrap support based on data for 1000 replicates; values inferred greater than 50% are onlypresented. The scale bar indicates 0.005 substitutions per nucleotide position.Though no direct correlation could be established between Bashan, Y., Holguin, G., 1998. Proposal for the division of plant growthin vitro solubilization of P, plant P accumulation and promoting rhizobacteria into two classiﬁcations: biocontrol-PGPB (plant growth promoting bacteria) and PGPB. Soil Biol. Biochem. 30,available soil P, the results of this study make these isolates 1225–1228.attractive as phosphate solubilizers. It requires further in- Chabot, R., Antoun, H., Cescas, M.P., 1996. Growth promotion of maizedepth studies based on the plant growth promoting activities and lettuce by phosphate solubilizing Rhizobium leguminosariumof these isolates under pot culture as well as ﬁeld conditions biovar phaseoli. Plant Soil 184, 311–321.before they are recommended as biofertilizers. Chin, K.J., Hahn, D., Hengstamann, U., Leisack, W., Janssen, P.H., 1999. Characterization and identiﬁcation of numerically abundant culturable bacteria from the anoxic bulk soil of rice paddy microcosms. Appl. Environ. Microbiol. 65, 5042–5049. Das, A.C., 1989. Utilization of insoluble phosphates by soil fungi. J. IndianAcknowledgements Soc. Soil Sci. 58, 1208–1211. deFreitas, J.R., Banerjee, N.R., Germida, J.J., 1997. Phosphate solubilizing The authors feel grateful to the Agriculture Research and rhizobacteria enhance the growth and yield but not phosphorus uptake of canola (Brassica napus L.). Biol. Fertil. Soils 24, 358–364.Promotion Center, Ministry of Agriculture and Forestry, Gerretson, F.C., 1948. The inﬂuence of microorganisms on the phosphateKorea for the support rendered by them. Sundaram Seshadri intake by the plant. Plant Soil 1, 51–81.also acknowledges the Korea Science and Engineering Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T., Williams, S.T., 1994.Foundation for the ﬁnancial assistance through Brain pool Bergey’s Manual of Determinative Bacteriology, ninth ed. Williams &fellowship. Wilkins, Baltimore, MD. Illmer, P., Schinner, F., 1995. Solubilization of inorganic calcium phosphates-solubilization mechanisms. Soil Biol. Biochem. 27, 257– 263. Illmer, P., Barbato, A., Schinner, F., 1995. Solubilization of hardly solubleReferences AlPO4 with P-solubilizing microorganisms. Soil Biol. Biochem. 27, 265–270.Banik, S., Dey, B.K., 1981. Phosphate solubilizing microorganisms of a Johri, J.K., Surange, S., Nautiyal, C.S., 1999. Occurrence of salt, pH, and lateritic soil: III. Effect of inoculation of some tricalcium phosphate temperature-tolerant, phosphate-solubilizing bacteria in alkaline soils. solubilizing microorganisms on available phosphorus content of Curr. Microbiol. 39, 89–93. rhizosphere soils of rice (Oryza sativa L. cv IR 20) plants and their Jones, D., Smith, B.F.L., Wilson, M.J., Goodman, B.A., 1991. Phosphate uptake of phosphorus. Zentralblatt. Fur Bakteriologie. 136, 493–501. solubilizing fungi in a Scottish upland soil. Mycol. Res. 95, 1090–1903.
1974 H. Chung et al. / Soil Biology & Biochemistry 37 (2005) 1970–1974Kim, K.Y., McDonald, G.A., Jordan, D., 1997. Solubilization of Sasser, M., 1990b. Identiﬁcation of bacteria through fatty acid analysis. In: hydroxyapatite by Enterobacter agglomerans and cloned Escherichia Klement, Z., Rudolph, K., Sands, D. (Eds.), Methods in Phytobacter- coli in culture medium. Biol. Fertil. Soils 24, 347–352. iology. Akademiai Kiado, Budapest, Hungary, pp. 199–204.Kim, K.Y., Jordan, D., McDonald, G.A., 1998. Enterobacter agglomerans, Sasser, M., Wichman, M.D., 1991. Identiﬁcation of microorganisms phosphate solubilizing bacteria, and microbial activity in soil: effect of through use of gas chromatography and high-performance liquid carbon source. Soil Biol. Biochem. 30, 995–1003. chromatography. In: Balows, A., Hausler Jr., W.J., Herrman, K.L.,Kumar, S., Tamura, K., Nei, M., 1993. MEGA: Molecular Evolutionary Isenberg, H.D., Shadomy, H.J. (Eds.), Manual of Clinical Micro- Genetics Analysis, Version 1.0. The Pennsylvania State University, biology, ﬁfth ed. American Society for Microbiology, Washington, DC. University Park, PA. Singal, R., Gupta, R., Kuhad, R.C., Saxena, R.K., 1991. Solubilization ofMurphy, J., Riley, J.P., 1962. A modiﬁed single solution method for the inorganic phosphates by a Basidiomycetous fungus Cuathus. Indian determination of phosphate in natural waters. Anal. Chim. Acta 27, J. Microbiol. 31, 397–401. 31–36. Sperber, J.I., 1958. The incidence of apatite dissolving organisms producing organic acids. Aust. J. Agric. Res. 9, 778–781.Park, M.S., Kim, C.W., Yang, J.C., Lee, H.S., Sa, T.M., in press. Isolation Vassilev, N., Marcia, T., Vassileva, M., Azcon, R., Barea, J.M., 1997. Rock and characterization of diazotrophic growth promoting bacteria from phosphate solubilization by immobilized cells of Enterobacter sp. in rhizosphere of agricultural crops of Korea. Microbiol. Res. (in press). fermentation and soil conditions. Bioresour. Technol. 61, 29–32.Pikovskaya, R.I., 1948. Mobilization of phosphorus in soil in connection Vassileva, M., Azcon, R., Barea, J.M., Vassilev, N., 1999. Effect of with vital activity of some microbial species. Microbiologiya 17, 362– encapsulated cells of Enterobacter sp. on plant growth and phosphate 370. uptake. Bioresour. Technol. 67, 229–232.Remus, R., Ruppel, S., Jacob, H.J., Chrlotte, H.B., Merbach, W., 2000. Vessey, J.K., 2003. Plant growth promoting rhizobacteria as biofertilizers. Colonization behaviour of two enterobacterial strains on cereals. Biol. Plant Soil 255, 571–586. Fertil. Soils 30, 550–557. Weisburg, W.G., Barns, S.M., Pelletier, D.A., Lane, D.J., 1991. 16SSambrook, J., Fritsch, E.E., Maniatis, T., 1989. Molecular Cloning: A ribosomal DNA ampliﬁcation for phylogenetic study. J. Bacteriol. 173, Laboratory Manual. Cold Spring Harbour Laboratory Press, Cold 697–703. Spring Harbor, New York. Zaidi, A., Khan, M.S., Amil, M.D., 2003. Interactive effect of rhizotrophicSasser, M., 1990a. Technical Note 102. Tracking a Strain Using the microorganisms on yield and nutrient uptake of chickpea (Cicer Microbial Identiﬁcation System. MIS, Newark, DE. arietinum L.). Eur. J. Agron. 19, 15–21.