The Korean Journal of Microbiology, Vol., No., September 006, p. -9 Copyright 006, The Microbiological Society of Korea w ³ ƒ ƒ y ³ p»x 1 Á y 1,, * w œw w w 1/6*(# $» w» w w ³ ƒ ƒ y ³ «m l w. ³ w e mw, Pseudomonas fluorescens RAF15. P. fluorescens RAF15 q ³ Botrytis cinerea» ³ Rhizoctonia solani w. Ca l ƒ w» w glucose 1.5%, urea 0.005%, MgCl Á6H O 0.%, MgSO Á7H O 0.01%, CaCl ÁH O 0.01%, NaCl 0.05%, 0 o C» ph 7.0, 5 z, 86 mg/l ƒ. ƒ y» w ph w ƒ. P. fluorescens RAF15 10-5 o C, 1-% ph.0-11.0 ƒ w. ³ CaHPO, Ca hydroxyapatite ƒƒ 971-111 mg/l, 791-908 mg/l 8 mg/l ƒ w. ù FePO AlPO, ƒ ƒƒ 18 mg/l, 5 mg/l. Key words ý antifungal activity, biofertilizer, phosphate-solubilizing bacteria, Pseudomonas fluorescens RAF15 ú yw ù yw w m y, y y w t w w ƒ y ƒw jš. yw ù yw ù w w ƒ š, p m k y w w ù w ƒ w w ƒ w ü w š (1-. 10% w w,», w œ w»., m œ z œ soil solution ü ww w ƒ û š ù z w» (., e m, š e ww e (5. m w, m w ƒ w š, w ƒ w. ƒ g w t y w w ƒ. w x y t w ƒ š. w w y w w *To whom correspondence should be addressed. Tel: 055-50-550, Fax: 055-50-55 E-mail: shjoo@pusan.ac.kr y d m ƒ y k m k p w y w w w w š w. ¾ w yw w, yw w ü x» š. yw w w» w ¼w wš p ³ wš w wš (6, 7. p, ³ w w ³ y ƒ ƒ š, Bacillus spp. Pseudomonas spp. w w ³ y ƒ š (8, 9. w ³ w w ³ y ƒ š, m w» xk ƒ ƒ y k ³ w» ƒ š» w» wš w.» ww w z, ƒ y mw. w ³ ƒ ƒ y ³ ƒ y ³ m ƒ y w š Pikovskaya (PVK w plate assay method w (10. û ƒ
Ki-Hyun Park and Hong-Joo Son Kor. J. Microbiol m «m w, m xk w z, PVK agar plate w 0 o C 7 w. g n y j g ƒ y ³ 1 w. ³ ƒƒ 1% NaCl w PVK broth w 0 o C, 00 rpm, 5 z k w. w ³ w ƒ d w w ³ š w ƒ y k ³ w. w ³ ƒ ƒ y ³ w» w t ³ Botrytis cinerea, Rhizoctonia solani, Fusarium oxysporum, Phythium ultimum, Alternaria porri PDA plate e w ƒ j w ³ x³ w. ƒ y PVK glucose 1%, (NH SO 0.05%, NaCl 0.0%, KCl 0.0%, MgSO Á 7H O 0.01%, MnSO Á7H O 0.05%, FeSO Á7H O 0.05 % yeast extract 0.05% (ph 7.0, Ca 0.5% ƒw (10.» NBRIY [glucose 1%, (NH SO 0.05%, NaCl 0.0 %, MgSO Á7H O 0.01%, KCl 0.0%, MnSO Á7H O 0.05%, FeSO Á 7H O 0.05%, ph 7.0](11, NBRIP [glucose 1%, (NH SO 0.01%, MgSO Á7H O 0.05%, MgCl Á6H O 0.5%, ph 7](11 Son [glucose 1%, NH NO 0.1%, MgSO Á7H O 0.0%, CaCl ÁH O 0.06%, ph 7.0](1, Ca 0.5% ƒƒ ƒw. x³ w» w xkw, p w p Biochemical tests for identification of medical bacteria (1 w mw, Cowan and Steel's Manual for the identification of medical bacteria (1 Bergey's manual of determinative bacteriology (15 w w. API 0NE kit (biomerieux, France mw w. ƒ w m x³ ƒ w e» w ƒ k,»,,» ph ³, ƒ ph d w, p w w 00 rpm 5 z k w. ƒ m ƒ w w» w ƒ (CaHPO, Ca, FePO, AlPO, hydoxyapatite ƒƒ ƒ x³ w 0 o C, 00 rpm 5 w z, ƒ ph d w. 1N HCl 1 : 1 yww ƒ y k z, spectrophotometer w 660 nm Ÿ d w ³ w (16. ƒ 17,18 g, 0 w ³ w vanadomolybdophosphoric acid colorimetric method (17 w, ³ w ƒ w x w. x z w, ùkü ƒ s³. š w ³ ƒ ƒ y ³ û, «m l Ca ƒ w PVK plate 1 cm n y w ³ w z, PVK broth 00 mg/l ƒ w ³ w. ³ 0 o C 7 e w ƒ ³ w w ³ z mw, q w Botrytis cinerea» w Rhizoctonia solani ³ w RAF15 x³ w. RAF15 ƒ w ³ Fig. 1. ³ ƒ w ³ ƒ œ ƒ j q. x³ w e Table 1. ³ m x» ³, yw p Cowan and Steel's Manual for the identification of medical bacteria (1 Bergey's manual of determinative bacteriology (15 w mw, Pseudomonas sw ƒ ƒ. w, API 0NE kit (biomerieux, France w k z w, Pseudomonas fluorescens 99.7% w ùkû. x³ RAF15 Pseudomonas fluorescens RAF15 w. ƒ w ƒ y. ³ w» w» w x š ƒ w ƒ w, Table. NBRIP ƒ w ƒ ùkû ù ³ ƒ û. NBRIY, Son ³ w PVK ³ ù ƒ û. x ƒ ƒ phƒ ƒ û. PVK NBRIY
인산 가용화세균의 특성 Vol., No. 5 Biochemical parameters showing biological activities of isolate RAF15. Clear zone indicates solubilization of insoluble phosphate around colony of isolate RAF15 on agar plate containing 0.5% Ca(PO (left. Antifungal activity of isolate RAF 15 against phytopathogenic Botrytis cinerea (middle and Rhizoctonia solani (right. Fig. 1. Taxonomical characteristics of isolated strain RAF15 Contents Characteristics Morphological characteristics Cell shape Rod Gram stain Spore formation Motility Cultural characteristics Colony shape Circular, undulate, convex Colony surface Smooth Colony color White Biochemical characteristics Growth in air Growth anaerobically Acid from glucose Oxidase Catalase O/F test Oxidation Yellow pigmentation Arginine dihydrolase Nitrate reduction Indole production Urease β-glucosidase β-galactosidase Gelatin hydrolysis Assimilation : glucose, arabinose, mannose, mannitol, gluconate, N-acetyl-glucosamine, malate Assimilation : maltose, caprate, adipate, citrate Table 1. 의 경우, 각각 배양 일 후 가용성 인산의 생성량이 감소하기 시작하였으며, 이에 비례하여 균체 생육도는 증가하였다. 이것은 생성된 가용성 인산이 실험균주의 생육에 이용되고, 이에 따라 균체 생육도가 증가한 것으로 추정되나 차후에 좀 더 자세한 실 험이 필요한 것으로 판단된다. 본 실험에서는 생성된 균체당 가 용성 인산 생성능이 가장 우수한 NBRIP 배지를 기본배지로 선 정하여 이후의 실험을 진행하였다. 탄소원이 가용성 인산 생성능에 미치는 영향을 조사하기 위하 여 각종 탄소원을 1%씩 첨가하여 0 C, 00 rpm에서 5일간 배 양한 결과는 Table 에서 보는 바와 같다. Glucose, galactose, mannitol로부터 각각 6, 16, 99 mg/l의 가용성 인산이 생성 되었으며, lactose, sucrose, maltose, sorbitol 등으로부터는 가용성 인산이 전혀 생산되지 않았다. 또한 가용성 인산 생성에 탄소원 은 필수적인 것으로 나타났으며, 가용성 인산 생성은 배지의 ph 감소와 밀접한 관련이 있는 것으로 추정되었다. 일반적으로 불용 성 인산이 가용성 인산으로 전환되는 이유는 미생물이 생산한 각종 유기산에 의한 배양액의 ph 감소가 주원인으로 보고되어 있다(17. 따라서 앞으로 본 균주가 생성하는 유기산의 종류와 농도에 대한 실험이 뒤따라야 할 것으로 판단된다. 가용성 인산 생성에 최적인 glucose의 농도를 조사한 결과, glucose의 농도 증 가에 따라 가용성 인산의 생성량도 증가하여 1.5%에서 785 mg/ L의 가용성 인산이 생성되었으며, 그 이상의 농도에서는 일정하 였다. ph는.1-. 범위를 유지하였다(Fig.. 항진균능이 없는 Pantoea agglomerans R-는 % glucose로부터 750 mg/l의 가 용성 인산을 생성할 수 있는 것으로 보고되어 있다(1. 따라서 본 실험균주가 기질 이용면에서 P. agglomerans R-보다 우수 Soluble phosphate production and cell growth in different media for the selection of a basic medium Soluble P (mg/l Growth (A660 Culture time (day Medium 1 5 1 5 1 PVK 151 9 0 99 0.85 1.5 1.51 1.9 1.95.5 NBRIP 169 09 91 598 0.19 0.75 0.67 0.6 0.657.6 NBRIY 1 58 16 8 0.6 1.05 1.555..658.9 Son 111 77 16 0.916 1.165 1.5 1.08 0.85.7 o Table. Final ph.....7. 5.. 5..1 6.9. 5 5.6.1 6..
6 Ki-Hyun Park and Hong-Joo Son Kor. J. Microbiol Table. Effect of different carbon sources on Ca solubilization by Pseudomonas fluorescens RAF15 Carbon source (1% Soluble P (mg/l Growth (A 660 Final ph None 0 0.0 7.0 Glucose 6 0.61.1 Fructose 5 0.67 6.0 Galactose 16 0.7. Lactose 0 0.07 6.9 Sucrose 0 0.06 6.8 Maltose 0 0.09 6.8 Sorbitol 0 0.05 6.9 Mannitol 99 0.518 5. Glycerol 8 0.917 5.9 Table. Effect of different nitrogen sources on Ca solubilization by Pseudomonas fluorescens RAF15 Nitrogen source (0.01% Soluble P (mg/l Growth (A 660 Final ph None 81 0.165.5 (NH SO 80 0.67. NHCl 808 0.771. NH NO 80 0.95.1 KNO 79 0.79. NaNO 770 0.57. Urea 80 1.016.1 Beef extract 69 0.59.5 Casamino acid 70 0.1. Corn steep liquor 8 0.9.5 Polypeptone 5 0.19.5 Tryptone 516 0.08.5 Yeast extract 60 0.6. w q. ƒ e w w» w ƒ 0.01% ƒw 0 o C, 00 rpm 5 w Table.»» ƒ ³ ùkü, ƒƒ ƒ v.» w» ³ ƒ û p w. ƒ ƒ ³ ùkü urea w z, w 0.005%¾ ƒ ƒw ù w w» w 0.0% ƒ (10-1 mg/l. ù ³ urea ƒ ƒw 0.5% (A 660 = 5.7. w 0.0% ph 8.1-8.8 ùkü š, ƒ y k (Fig.. wr, P. agglomerans R- ammonium nitrate ƒ w, nitrite ƒ x w (1. Fig.. Effect of glucose concentrations on Ca solubilization by Pseudomonas fluorescens RAF15. ø, cell growth; ù, soluble phosphate; ÿ, final ph. Fig.. Effect of urea concentrations on Ca solubilization by Pseudomonas fluorescens RAF15. ø, cell growth; ù, soluble phosphate; ÿ, final ph.» MgCl Á6H O MgSO Á7H O, ƒƒ 0.% 0.01% ƒ ƒ, KCl ƒ w e (.» sw» ƒ z w, 0.01% CaCl ÁH O 0.05% NaCl ƒ ƒ g (. P. fluorescens RAF15» ph w Table 5. 5 z, 0 C ƒ o ƒ ùkü ù 10-5 C o, 1-1 ¾ ƒ (705-9 mg/l ùkü. ³» 10 o C w ƒ w q. 0 C ƒ ³ o ùkù. wr, ³» ph.0-11.0 ƒ (701-86 mg/lw, ph 1 ƒ x w. 10-5 o C ph.0-11.0 y ƒ w ³
Vol., No. ƒ y ³ p 7 Table 5. Effect of temperature and initial ph on Ca (PO solubilization by Pseudomonas fluorescens RAF15 Temperature ( o C Time (day a Soluble P (mg/l Growth (A 660 Initial ph Time (day* Soluble P (mg/l Growth (A 660 10 1 705 0.97 5 701 0.71 15 1 896 0.977 5 81 0.759 0 1 905 0.998 5 80 0.85 5 1 9 1.091 5 5 8 0.787 0 5 858 0.98 5.5 5 8 0.787 5 5 1 0.68 6 5 86 0.78 0 5 0 0.011 6.5 5 89 0.776 7 5 86 0.975 7.5 5 89 0.7 8 5 86 0.77 9 5 8 0.75 10 5 8 0.77 11 5 81 0.708 1 5 0 0.1 a indicates maximal soluble P production and cell growth. P. agglomerans R-ƒ (1. ù P. agglomerans R-» y 69-15 mg/l ƒ w x³ ƒ q. x³ ƒ» ƒ y { y ƒ š, z ³ w. y ƒ y w glucose 1.5%, urea 0.005%, MgCl Á6H O 0.%, MgSO Á 7H O 0.01%, CaCl ÁH O 0.01%, NaCl 0.05%, 0 o C» ph 7.0. w ƒ w m ww ƒ x, m w (19. m w ƒ w w w w Fig.. ³ KCl (1-% NaCl (1-% ƒƒ 89-610 mg/l -588 mg/l ƒ w, 1% CaCl ÁH O Fig.. Soluble phosphate production by Pseudomonas fluorescens RAF15 in the medium containing high concentration of salts. ø, KCl; ù, NaCl; ÿ, CaCl ÁH O. 100 mg/l ƒ w. ³ ry ü m ƒ w š q. Table 6. Changes in soluble phosphate and final ph mediated by Pseudomonas fluorescens RAF15 in optimal culture media containing various insoluble phosphates Phosphate source Concentration (% Soluble P (mg/l Final ph Phosphate source Concentration (% Soluble P (mg/l Final ph Ca (PO 0.5 861. CaHPO 0.5 971.0 1.0 908. 1.0 108.0 1.5 888. 1.5 111.0.0 880..0 1091.1.0 791..0 996.1 AlPO 0.5 5.1 Hydroxyapatite 1.0 8.1 FePO 0.5 18.0
8 Ki-Hyun Park and Hong-Joo Son Kor. J. Microbiol ƒ ƒ ƒ w» w CaHPO, Ca, FePO, AlPO hydroxyapatite ƒƒ 0.5-.0% ƒw w Table 6. CaHPO, Ca hydroxyapatite, ƒ 971-111 mg/l, 791-908 mg/l 8 mg/l ƒ, ph.0-.. ù FePO AlPO, phƒ ƒƒ.0,.1 ù ƒ ƒƒ 18 mg/l, 5 mg/l. Penicillium radicum CaHPO, FePO, AlPO l ƒƒ 176, 10, 0 mg/l ƒ w (0, Rahnella aquatilis hydroxyapatite l 0 mg/l ƒ w š š (1. ³ { w ƒ ƒ š. wr, calcium phosphate activew w iron aluminum phosphate ƒ w (, ³ w ùkü. w w ( w,. š x 1. Shoda, M. 000. Bacterial control of plant diseases. J. Biosci. Bioeng. 89, 515-51.. Sholberg, P.L., A. Marchi, and J. Bechard. 1995. Biocontrol of postharvest diseases of apple using Bacillus spp. isolated from stored apples. Can. J. Microbiol. 1, 7-5.. Subhash, C.V., K.L. Jagdish, and K.T. Anil. 001. Evaluation of plant growth promoting and colonization ability of endophytic diazotrophs from deep water rice. J. Biotechnol. 91, 17-11.. Narsian, V. and H.H. Patel. 000. Aspergillus aculeatus as a rock phosphate solubilizer. Soil Biol. Biochem., 559-565. 5. Vassilev, N., M.T. Baca, M. Vassileva, I. Franco, and R. Azcon. 1995. Rock phosphate solubilization by Aspergillus niger grown on sugar-beet waste medium. Appl. Microbiol. Biotechnol., 56-59. 6. Kobayashi, D.Y. and N.E.H. El-Bararad. 1996. Selection of bacterial antagonists using enrichment cultures for the control of summer patch disease in Kentucky Bluegrass. Curr. Microbiol., 106-111. 7. Chaurasia, B., A. Pandey, L.M.S. Palni, P. Trivedi, B. Kumar, and N. Colvin. 005. Diffusible and volatile compounds produced by an antagonistic Bacillus subtilis strain cause structural deformations in pathogenic fungi in vitro. Microbiol. Res. 160, 75-81. 8. Patel, V.J., S.R. Tendulkar, and B.B. Chattoo. 00. Bioprocess development for the production of an antifungal molecules by Bacillus licheniformis BC98. J. Biosci. Bioeng. 98, 1-5. 9. Nautiyal, C.S. 1997. Selection of chickpea-rhizosphere-competent Pseudomonas fluorescens NBRI10 antagonistic to Fusarium oxysporum f. sp. ciceri, Rhizoctonia bataticola and Phythium sp. Curr. Microbiol. 5, 5-58. 10. Gupta, R., R. Singal, A. Shankar, R.C. Kuhad, and R.K. Saxena. 199. A modified plate assay for screening phosphate solubilizing microorganisms. J. Gen. Appl. Microbiol. 0, 55-60. 11. Nautiyal, C.S. 1999. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol. Lett. 170, 65-70. 1. Son, H.J., G.T. Park, M.S. Cha, and M.S. Heo. 006. Solubilization of insoluble inorganic phosphates by a novel salt- and ph-tolerant Pantoea agglomerans R- isolated from soybean rhizosphere. Biores. Technol. 97, 0-10. 1. Macfaddin, J.F. 1980. Biochemical Tests for Identification of Medical Bacteria. The Williams and Wilkins Co., Baltimore. 1. Barrow, G.I. and R.K.A. Felthanm. 199. Cowan and Steel's Manual for the identification of medical bacteria. Cambridge University Press, New York. 15. Holt, J.G., N.R. Krieg, P.H.A. Sneath, J.T. Staley, and S.T. Williams. 199. Bergey's Manual of Determinative Bacteriology, 9th ed. The Williams and Wilkins Co., Baltimore. 16. Rodriguez, H., T. Gonzalez, and G. Selman. 000. Expression of a mineral phosphate solubilizing gene from Erwinia herbicola in two rhizobacterial strains. J. Biotechnol. 8, 155-161. 17. Clesscerl, L.S., A.E. Greenberg, and A.D. Eaton. 1998. Standard methods for the examination of water and wastewater, 0th ed.apha-awwa-wef. Washington, D.C. 18. Nautiyal, C.S., S. Bhadauria, P. Kumar, H. Lal, R. Mondal, and D. Verma. 000. Stress induced phosphate solubilization in bacteria isolated from alkaline soils. FEMS Microbiol. Lett. 18, 91-96. 19. Van Elas, J.D., J.T. Trevors, and E.M.H. Wellington. 1997. Modern soil microbiology. Marcel Dekker, Inc., New York. 0. Whitelaw, M.A., T.J. Harden, and K.R. Helyar. 1999. Phosphate solubilization in solution culture by the soil fungus Penicillium radicum. Soil Biol. Biochem. 1, 655-665. 1. Kim, K.Y., D. Jordan, and H.B. Krishnan. 1997. Rahnella aquatilis, a bacterium isolated from soybean rhizosphere, can solubilize hydroxyapatite. FEMS Microbiol. Lett. 15, 7-77.. Illmer, P., A. Barbato, and F. Schinner. 1995. Solubilization of hardly-soluble AlPO with P-solubilizing microorganisms. Soil Biol. Biochem. 7, 65-70. (ReceivedGAugust 5, 006/Accepted September 1, 006
Vol., No. ƒ y ³ p 9 ABSTRACT : Isolation and Characterization of Insoluble Phosphate-Solubilizing Bacteria with Antifungal Activity Ki-Hyun Park 1 and Hong-Joo Son 1,, * ( 1 Department of Biotechnology, Miryang National University, Miryang 67-706, Korea, School of Applied Life Science and Joint Research Center of Pusan National University-Fraunhofer Institute, Pusan National University, Miryang 67-706, Korea To develop multifunctional microbial inoculant, an insoluble phosphate-solubilizing bacterium with antifungal activity was isolated from plant rhizospheric soil. On the basis of its morphological, cultural and physiological characteristics and Biolog analysis, this bacterium was identified as Pseudomonas fluorescens RAF15. P. fluorescens RAF15 showed antifungal activities against phytopathogenic fungi Botrytis cinerea and Rhizoctonia solani. The optimal medium composition and cultural conditions for the solubilization of insoluble phosphate by P. fluorescens RAF15 were 1.5% of glucose, 0.005% of urea, 0.% MgCl 6H O, 0.01% of MgSO 7H O, 0.01% of CaCl H O, and 0.05% of NaCl along with initial ph 7.0 at 0 o C. The soluble phosphate production under optimum condition was 86 mg/l after 5 days of cultivation. The solubilization of insoluble phosphates was associated with a drop in the ph of the culture medium. P. fluorescens RAF15 showed resistance against different environmental stresses like 10-5 o C temperature, 1-% salt concentration and ph -11 range. The strain produced soluble phosphate to the culture broth with the concentrations of 971-111 mg/l against CaHPO, 791-908 mg/l against Ca, and 8 mg/l against hydroxyapatite, respectively. However, the strain produced soluble phosphate to the culture broth with the concentrations of 15 mg/l against FePO, and 5 mg/l against AlPO, respectively.