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![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 2005
Abstract
Whole-cell fatty acids methyl ester (FAME) profile and 16S rDNA sequence analysis were employed to isolate and identify the bacterial
groups that actively solubilized phosphates in vitro from rhizosphere soil of various crops of Korea. Out of several hundred colonies that
grew on Pikovskaya’s medium 13 best isolates were selected based on the solubilization of insoluble phosphates in liquid culture and further
characterized and identified. They were clustered under the genera Enterobacter, Pantoea and Klebsiella and the sequences of three
representative 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 phosphate
due to solubilization of organic acids in the surrounding solubilizing bacteria (PSB) in Korean soils is lagging, an
medium (Singal et al., 1991) are selected as potential attempt to isolate and identify PSB through biochemical and
phosphate solubilizers (Das, 1989) and are routinely molecular methods was made. The rhizosphere soil samples
screened 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 milliliter
1948) or Sperber agar (Sperber, 1958). Several reports on of the appropriate (10K5–10K7) dilutions of the soil samples
bacteria and fungi isolated from soil have evaluated their was plated on Pikovskaya’s medium (Pikovskaya, 1948) for
mineral phosphate solubilizing (MPS) activity with various P the isolation of PSB. The colonies distinguished by
sources 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 reliable
et al., 1991) and aluminium phosphate (AlPO4) (Illmer et al., method in determining a strain as phosphate solubilizer
1995). An increase in P availability to plants through the (Johri et al., 1999), the pure cultures were further screened
inoculation of PSBs has also been reported previously in pot in liquid medium containing Ca3(PO4)2, AlPO4 and FePO4
experiments and under field conditions (Banik and Dey, at a concentration of 5 g LK1 as insoluble P sources. The
1981; Chabot et al., 1996; deFreitas et al., 1997; Zaidi et al., cultures supernatant obtained by centrifugation was passed
2003). 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: tomsa@chungbuk.ac.kr (T. Sa). each set. All the isolates solubilized Ca3(PO4)2 to a greater
0038-0717/$ - see front matter q 2005 Published by Elsevier Ltd. extent than AlPO4 and FePO4 with AlPO4 exhibiting poor
doi:10.1016/j.soilbio.2005.02.025 solubilization (Table 2). Even the isolates that did not](https://image.slidesharecdn.com/9-120510163259-phpapp01/85/9-1-320.jpg)
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- 1. 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 2005 Abstract Whole-cell fatty acids methyl ester (FAME) profile and 16S rDNA sequence analysis were employed to isolate and identify the bacterial groups that actively solubilized phosphates in vitro from rhizosphere soil of various crops of Korea. Out of several hundred colonies that grew on Pikovskaya’s medium 13 best isolates were selected based on the solubilization of insoluble phosphates in liquid culture and further characterized and identified. They were clustered under the genera Enterobacter, Pantoea and Klebsiella and the sequences of three representative 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 phosphate due to solubilization of organic acids in the surrounding solubilizing bacteria (PSB) in Korean soils is lagging, an medium (Singal et al., 1991) are selected as potential attempt to isolate and identify PSB through biochemical and phosphate solubilizers (Das, 1989) and are routinely molecular methods was made. The rhizosphere soil samples screened 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 milliliter 1948) or Sperber agar (Sperber, 1958). Several reports on of the appropriate (10K5–10K7) dilutions of the soil samples bacteria and fungi isolated from soil have evaluated their was plated on Pikovskaya’s medium (Pikovskaya, 1948) for mineral phosphate solubilizing (MPS) activity with various P the isolation of PSB. The colonies distinguished by sources 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 reliable et al., 1991) and aluminium phosphate (AlPO4) (Illmer et al., method in determining a strain as phosphate solubilizer 1995). An increase in P availability to plants through the (Johri et al., 1999), the pure cultures were further screened inoculation of PSBs has also been reported previously in pot in liquid medium containing Ca3(PO4)2, AlPO4 and FePO4 experiments and under field conditions (Banik and Dey, at a concentration of 5 g LK1 as insoluble P sources. The 1981; Chabot et al., 1996; deFreitas et al., 1997; Zaidi et al., cultures supernatant obtained by centrifugation was passed 2003). 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: tomsa@chungbuk.ac.kr (T. Sa). each set. All the isolates solubilized Ca3(PO4)2 to a greater 0038-0717/$ - see front matter q 2005 Published by Elsevier Ltd. extent than AlPO4 and FePO4 with AlPO4 exhibiting poor doi:10.1016/j.soilbio.2005.02.025 solubilization (Table 2). Even the isolates that did not
- 2. H. Chung etal. / Soil Biology & Biochemistry 37 (2005) 1970–1974 1971 Table 1 Location, soil series, crops and colony morphology of PSB isolates Location Soil series Crops (scientific name) Isolates Colony morphology Gae 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, circular Gang Seo Dong Sangju Sesame HK 20-1 White, circular Sangju Pepper HK 23-2 White, circular Sangju Pepper HK 24 White, circular Hyeong Dong Ri Sangju Spring onion HK 34-1 White, circular Sangju Spring onion HK 34-2 White, circular Sek Pan Ri Sachon Rice (Oryza sativa L.) HK 52-1 White, circular Jeung Pyung Sachon Rice HK 68-1 Yellow, circular Sachon Rice HK 68-3 White, circular Bong Yang Up Sachon Rice HK 69 White, circular perform 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 GeneAmp series of biochemical reactions as per the Bergey’s Manual PCR System (Perkin–Elmer Co., Norwalk, CT) with Taq of 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 was for oxidase activity, H2S production, gelatin, starch and performed using Big-Dye Terminator Cycle Sequencing lipid 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 the fatty 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 isolates using the Sherlock Microbial Identification system TSBA under Enterobacter sp., Klebsiella sp., and Pantoea sp. that 4.0 software and library general system software version are grouped under one family, Enterobacteriaceae. Results 4.1. Qualitative and quantitative differences in the fatty acid of 16S rDNA identification agreed with that of GC-FAME profiles were used to compute the distance for each strain for five PSBs at the genus level in Enterobacter and relative to the strains in the library (Sasser, 1990a,b; Sasser Klebsialla (Table 4). Isolation of bacteria belonging to the and Wichman, 1991). Genomic DNA was extracted by the family Enterobacteriaceae from various soils, and their MPS phenol/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 2 Solubilization of inorganic phosphates by the PSB isolates in liquid cultures Isolates Liquid culture (mg P mLK1) Ca3(PO4)2 AlPO4 FePO4 HK 11-1 96.2G5.9 8.0G5.0 18.8G6.3 HK 14-1 127.2G2.1 13.8G1.3 22.9G1.7 HK 17-1 113.7G3.3 4.3G0.9 4.5G0.9 HK 18-3 121.7G2.7 3.7G0.7 10.2G1.1 HK 20-1 142.1G2.1 5.1G1.6 24.4G1.1 HK 23-2 114.3G8.4 9.9G0.9 18.8G4.3 HK 24 136.4G1.1 5.7G0.6 19.3G1.0 HK 34-1 138.6G5.7 5.9G1.1 27.2G3.7 HK 34-2 126.9G2.2 10.7G1.3 12.6G1.0 HK 52-1 107.7G2.1 6.3G0.4 15.5G6.4 HK 68-1 119.2G5.3 8.7G0.4 20.7G4.6 HK 68-3 107.5G2.2 7.2G0.6 49.1G2.2 HK 69 113.0G5.7 4.1G1.7 49.5G4.7 Liquid cultures were assayed after seven days. Ca3(PO4)2, tricalcium phosphate; AlPO4, aluminium phosphate; FePO4, ferric phosphate.
- 3. 1972 H. Chung et al. / Soil Biology & Biochemistry 37 (2005) 1970–1974 Table 3 Biochemical characteristics of the PSB isolates Biochemical 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 69 Gram staining K K K K K K K K K K K K K Catalase C C C C C C C C C C C C C Oxidase K K K K K K K K K K K K K IMViC 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 C Lysine decarboxylase K K K C K C K K K K K C C Arginine dihydrolase C C C C C K C C K C C K K Ornithine decarboxylase C C C C C K K C K C C K K Carbon 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 C H2S production K K K K K K K K K K K K K Gelatin hydrolysis K K K K K K K K K K K K K Starch hydrolysis K K K K K K K K K K K K K Lipid hydrolysis K K K K K K K K K K K K K C, 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 following related 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 AY335554 best 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 and representative strains were deposited in the GenBank increased yield (Bashan and Holguin, 1998; Vessey, 2003). Table 4 Identification of PSB isolates by GC-FAME and 16S rDNA sequencing from rhizosphere soil samples Isolate GC-FAME identification Similarity (%) 16S rDNA identification Identity (%) HK 11-1 Enterobacter cancerogenus 64.9 Pantoea sp. 98 HK 14-1 Enterobacter cloacae 82.7 Pantoea agglomerans 99 HK 17-1 Enterobacter cloacae 82.9 Pantoea sp. 96 HK 18-3 Kluyvera ascorbata 37.6 Enterobacter cloacae 99 HK 20-1 Klebsiella pneumoniae 14.6 Enterobacter aerogenes 99 HK 23-2 Klebsiella pneumoniae 14.6 Klebsiella sp. 98 HK 24 Kluyvera ascorbata 60.6 Enterobacter cloacae 99 HK 34-1 Kluyvera ascorbata 5.1 Enterobacter sp. 95 HK 34-2 Klebsiella pneumoniae 92.1 Klebsiella sp. 98 HK 52-1 Klebsiella planticola 83.1 Enterobacter cloacae 99 HK 68-1 Enterobacter cancerogenus 7.6 Enterobacter aerogenes 99 HK 68-3 Klebsiella pneumoniae 43.8 Klebsiella sp. 99 HK 69 Klebsiella pneumoniae 89.1 Klebsiella sp. 98
- 4. H. Chung etal. / Soil Biology & Biochemistry 37 (2005) 1970–1974 1973 Fig. 1. Phylogenetic tree showing the relationships among the PSB isolates and between representatives of other related taxa. The tree was constructed by using the MEGA2 after aligning the sequences with Megalign and generating evolutionary distance matrix inferred by the neighbor-joining method using Kimura parameter 2. The numbers at the nodes indicate the levels of bootstrap support based on data for 1000 replicates; values inferred greater than 50% are only presented. 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 growth in 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 maize depth studies based on the plant growth promoting activities and lettuce by phosphate solubilizing Rhizobium leguminosarium of 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. Indian Acknowledgements 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 phosphate Korea 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 soluble References 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.
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