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Bacillus subtilis SQR 9 can control Fusarium wilt in cucumber by colonizing plant roots

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Abstract

Fusarium wilt is one of the major constraints on cucumber production worldwide. Several strategies have been used to control the causative pathogen, Fusarium oxysporum f. sp. cucumerinum J. H. Owen, including soil solarization, fungicide seed treatment and biological control. In this study, F. oxysporum f. sp. cucumerinum was successfully controlled by a newly isolated strain, Bacillus subtilis SQR 9, in vitro and in vivo. Greenhouse experiments were carried out to evaluate the effect of inoculation and solid fermentation of organic fertilizer with B. subtilis SQR 9, hereby defined as bio-organic fertilizer (BIO), on the control of Fusarium wilt. In comparison with the control, the wilt incidence was significantly reduced (49–61% reduction) by application of BIO. The rhizosphere population of F. oxysporum f. sp. cucumerinum, as detected both by selective plating and realtime PCR, was significantly lower in BIO-treated plants than the control. The localization of bacterial cells, pattern of colonization and survival of B. subtilis SQR 9 in the rhizsosphere of cucumber, was examined by fluorescent microscopy and explored following recovery of the green fluorescent protein (gfp)-labeled SQR 9 with the new gfp-marked shuttle vector pHAPII through selective plating. The preferential sites of the labeled strain were the differentiation and elongation zone, root hair and the lateral root junctions. The population of the strain was 106 cfu/g root in rhizoplane. These results indicate that the strain was able to survive well in the rhizosphere of cucumber, suppressed growth of F. oxysporum in the rhizosphere of cucumber and protected the host from the pathogen.

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References

  • Armstrong GM, Armstrong JK (1981) Formae speciales and races of Fusarium oxysporum causing wilt disease. In: Nelson PE, Toussoun TA, Cook RJ (eds) Fusarium: disease, biology, and taxonomy. Pennsylvania State University Press, University Park, pp 391–399

    Google Scholar 

  • Asaka O, Shoda M (1996) Biocontrol of Rhizoctonia solani damping off of tomato with Bacillus subtilis RB14. Appl Environ Microbiol 62:4081–4085

    PubMed  CAS  Google Scholar 

  • Bais HP, Fall R, Vivanco JM (2004) Biocontrol of Bacillus subtilis against infection of Arabidopsis root by Pseudomonas syringae is facilitated by biofilm formation and surfactin production. Plant Physiol 134:307–319

    Article  PubMed  CAS  Google Scholar 

  • Baysal Ö, Çalışkan M, Yeşilova Ö (2008) An inhibitory effect of a new Bacillus subtilis strain (EU07) against Fusarium oxysporum f. sp. radicis-lycopersici. Physiol Mol Plant Pathol 73:25–32

    Article  Google Scholar 

  • Becker JO, Schwinn FJ (1993) Control of soil-borne pathogens with living bacteria and fungi: status and outlook. Pestic Sci 37:355–363

    Article  Google Scholar 

  • Cazorla FM, Romero D, Pérez-García A, Lugtenberg BJJ, De Vicente A, Bloemberg G (2007) Isolation and characterization of antagonistic Bacillus subtilis strains from the avocado rhizoplane displaying biocontrol activity. J Appl Microbiol 103:1950–1959

    Article  PubMed  CAS  Google Scholar 

  • Chen L, Yang X, Raza W, Luo J, Zhang F, Shen Q (2011) Solid-state fermentation of agro-industrial wastes to produce bioorganic fertilizer for the biocontrol of Fusarium wilt of cucumber in continuously cropped soil. Bioresour Technol 102:3900–3910

    Article  PubMed  CAS  Google Scholar 

  • Chung S, Kong H, Buyer JS, Lakshman DK, Lydon J, Kim S, Roberts DP (2008) Isolation and partial characterization of Bacillus subtilis ME488 for suppression of soilborne pathogens of cucumber and pepper. Appl Microbiol Biotechnol 80:115–123

    Article  PubMed  CAS  Google Scholar 

  • Compant S, Duffy B, Nowak J, Clément C, Barka EA (2005) Use of plant growth-promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects. Appl Environ Microbiol 71:4951–4959

    Article  PubMed  CAS  Google Scholar 

  • El-Hassan SA, Gowen SR (2006) Formulation and delivery of the bacterial antagonist Bacillus subtilis for management of Lentil vascular wilt caused by Fusarium oxysporum f. sp. lentis. J Phytopathol 154:148–155

    Article  Google Scholar 

  • Emmert EAB, Handelsman J (1999) Biocontrol of plant disease: a gram positive perspective. FEMS Microbiol Lett 171:1–9

    Article  PubMed  CAS  Google Scholar 

  • Erhart E, Burian KH, Stich K (1999) Suppression of Pythium ultimum by biowaste composts in relation to compost microbial biomass, activity and content of phenolic compounds. J Phytopathol 147:299–305

    CAS  Google Scholar 

  • Eva Z, Hans M (1986) Characterization of signals promoting gene expression on the Staphylococcus aureus plasmid pUB110 and development of a Gram-positive expression vector system. DNA 3:219–225

    Google Scholar 

  • Gamalero E, Lingua G, Capr FG, Fusconi A, Berta G, Lemanceau P (2004) Colonization pattern of primary tomato roots by Pseudomonas fluorescens A6RI characterized by dilution plating, flow cytometry, fluorescence, confocal and scanning electron microscopy. FEMS Microbiol Ecol 48:79–87

    Article  PubMed  CAS  Google Scholar 

  • Gao X, Jackson TA, Lambert KN, Li S (2004) Detection and quantification of Fusarium solani f. sp. glycines in soybean roots with real-time quantitative polymerase chain reaction. Plant Dis 88:1372–1380

    Article  CAS  Google Scholar 

  • Georgakopoulos DG, Fiddaman P, Leifert C, Malathrakis NE (2002) Biological control of cucumber and sugar beet damping-off caused by Pythium ultimum with bacterial and fungal antagonists. J Appl Microbiol 92:1078–1086

    Article  PubMed  CAS  Google Scholar 

  • Gorodecki B, Hadar Y (1990) Suppression of Rhizoctonia solani and Sclerotium rolfsii diseases in container media containing composted separated cattle manure and composted grape marc. Crop Prot 9:271–274

    Article  Google Scholar 

  • Haas D, Défago G (2005) Biological control of soil-borne pathogens by fluorescent Pseudomonads. Nat Rev Microbiol 3:307–319

    Article  PubMed  CAS  Google Scholar 

  • Haas D, Keel C (2003) Regulation of antibiotic production in root-colonizing Peudomonas spp. and relevance for biological control of plant disease. Annu Rev Phytopathol 41:117–153

    Article  PubMed  CAS  Google Scholar 

  • Hermansson A, Bäckman JSK, Svensson BH, Lindgren P (2004) Quantification of ammonia-oxidising bacteria in limed and non-limed acidic coniferous forest soil using real-time PCR. Soil Biol Biochem 36:1935–194

    Article  CAS  Google Scholar 

  • Hervás A, Landa B, Datnoff LE, Jiménez-Díaz RM (1998) Effects of commercial and indigenous microorganisms on Fusarium wilt development in chickpea. Biol Control 13:166–176

    Article  Google Scholar 

  • Hoitink HAJ, Fahy PC (1986) Basis for the control of soilborne plant pathogens with composts. Anu Rev Phytopathol 24:93–114

    Article  Google Scholar 

  • Isabella T, Simone G, Diego M, Carlo P, Enrica C, Matti K (2008) Generation and comparison of bioluminescent and fluorescent Bacillus licheniformis. Curr Microbiol 57:245–250

    Article  Google Scholar 

  • Jansson JK (2003) Marker reporter genes illuminating tools environmental microbiologists. Curr Opin Microbiol 6:310–316

    Article  PubMed  CAS  Google Scholar 

  • Jørgensen C, Leser TD (2007) Estimating amplification efficiency improves multiplex real-time PCR quantification of Bacillus licheniformis and Bacillus subtilis spores in animal feed. J Microbiol Methods 68:588–595

    Article  PubMed  Google Scholar 

  • Kokalis-Burelle N, Vavrina CS, Rosskopf EN, Shelby RA (2002) Field evaluation of plant growth-promoting rhizobacteria amended transplant mixes and soil solarization for tomato and pepper production in Florida. Plant Soil 238:257–266

    Article  CAS  Google Scholar 

  • Komada H (1975) Development of a selective medium for qualitative isolation of Fusarium oxysporum from natural soil. Rev Plant Protec Res 8:114–125

    Google Scholar 

  • Leifert C, Li H, Chidburee S, Hampson S, Workman S, Sigee D, Epton HAS, Harbour A (1995) Antibiotic production and biocontrol activity by Bacillus subtilis CL27 and Bacillus pumilus CL45. J Appl Bacteriol 78:97–108

    PubMed  CAS  Google Scholar 

  • Lievens B, Claes L, Vakalounakis DJ, Vanachter ACRC, Thomma BPHJ (2007) A robust identification and detection assay to discriminate the cucumber pathogens Fusarium oxysporum f. sp. cucumerinum and f. sp. radicis-cucumerinum. Environ Microbiol 9:2145–2161

    Article  PubMed  CAS  Google Scholar 

  • Ling N, Xue C, Huang Q, Yang X, Xu Y, Shen Q (2010) Development of a mode of application of bioorganic fertilizer for improving the biocontrol efficacy to Fusarium wilt. Biocontrol 55:673–683

    Article  Google Scholar 

  • Liu X, Zhao H, Chen S (2006) Colonization of maize and rice plants by strain Bacillus megaterium C4. Curr Microbiol 52:186–190

    Article  PubMed  CAS  Google Scholar 

  • Lugtenberg B, Kamilova F (2009) Plant-growth-promoting rhizobacteria. Anu Rev Microbiol 63:541–556

    Article  CAS  Google Scholar 

  • Luo J, Ran W, Hu J, Yang X, Xu Y, Shen Q (2010) Application of bio-organic fertilizer significantly affected fungal diversity of soils. Soil Sci Soc Am J 74:2039–2048

    Article  CAS  Google Scholar 

  • McKeen CD, Rielly CC, Pusey PL (1986) Production and partial characterization of antifungal substances antagonistic to Monilinia fructicola from Bacillus subtilis. Phytopathology 76:136–139

    Article  CAS  Google Scholar 

  • Mckenzie T, Hoshino T, Tanaka T, Sueoka N (1986) The nucleotide sequence of pUB110: some salient features in relation to replication and its regulation. Plasmid 15:93–103

    Article  PubMed  CAS  Google Scholar 

  • Minuto A, Spadaro D, Garibaldi A, Gullino ML (2006) Control of soilborne pathogens of tomato using a commercial formulation of Streptomyces griseoviridis and solarization. Crop Prot 25:468–475

    Article  Google Scholar 

  • Moyne AL, Clevel TE, Tuzun S (2001) Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D. FEMS Microbiol Lett 234:43–49

    Article  Google Scholar 

  • Poonguzhali S, Madhaiyanm M, Yim W, Kim K, Sa T (2008) Colonization pattern of plant root and leaf surfaces visualized by use of green-fluorescent-marked strain of Methylobacterium suomiense and its persistence in rhizosphere. Appl Microbiol Biotechnol 78:1033–1043

    Article  PubMed  CAS  Google Scholar 

  • Rampach GS, Kloepper JW (1998) Mixtures of plant growth-promoting rhizobacteria enhance biological control of multiple cucumber pathogen. Phytopathology 88:1158–1164

    Article  Google Scholar 

  • Rasmussen R (2001) Quantification on the Lightcycler. In: Meuer S, Wittwer C, Nakagawara K (eds) Rapid cycle real-time PCR: methods and applications. Springer, Berlin, pp 129–144

    Google Scholar 

  • Raviv M, Oka Y, Katan J, Hadar Y, Yogev A, Medina S, Krasnovsky A, Ziadna H (2005) High-nitrogen compost as a medium for organic container-grown crops. Bioresour Technol 96:419–427

    Article  PubMed  CAS  Google Scholar 

  • Romero D, De Vicente A, Rakotoaly RV, Dufour SE, Veening JW, Arrebola E, Cazorla FM, Kuipers OP (2007) The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis towards Podosphaera fusca. Mol Plant Microb Interact 20:430–440

    Article  CAS  Google Scholar 

  • Rosado A, Duarte G, Seldin L (1994) Optimization of electroporation procedure to transform B. polymyxa SCE2 and other nitrogen-fixing Bacillus. J Microbiol Methods 19:1–11

    Article  CAS  Google Scholar 

  • Sambrook J, Fitsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory

  • Sneath PHA (1986) Endospore-forming gram-positive rods and cocci. In: Sneath PHA (ed) Bergey's manual of systematic bacteriology, vol. 2. Williams and Wilkins, Baltimore, pp 1104–1138, ISBN 0–683–07893–3

    Google Scholar 

  • Sun X, Chen Y, Wu C, Yang G, Guo B, Shen D (2006) Functional evaluation of a novel constitutive promoter F1 of Bacillus pumilus, as a rice epiphytic strain, and construction of an efficient expression an secretion system under the control of F1. Biotechnol Lett 28:979–985

    Article  PubMed  CAS  Google Scholar 

  • Timmusk S, Grantcharova N, Wagner EG (2005) Paenibacillus polymyxa invades plant roots and forms biofilms. Appl Environ Microbiol 71:7292–7300

    Article  PubMed  CAS  Google Scholar 

  • Turner JT, Backman PA (1991) Factors relating to peanut yield increases after seed treatment with Bacillus subtilis. Plant Dis 75:347–353

    Article  Google Scholar 

  • Waseem R, Yang XM, Wu HS, Wang Y, Xu YC, Shen QR (2009) Isolation and characterisation of fusaricidin-type compound-producing strain of Paenibacillus polymyxa SQR-21 active against Fusarium oxysporum f.sp. nevium. Eur J Plant Pathol 125:471–481

    Article  Google Scholar 

  • Whelan JA, Russell NB, Whelan MA (2003) A method for the absolute quantification of cDNA using real-time PCR. J Immunol Methods 278:261–269

    Article  PubMed  CAS  Google Scholar 

  • Wu HS, Yang X, Fan J, Miao W, Ling N, Xu Y, Huang Q, Shen Q (2009) Suppression of Fusarium wilt of watermelon by a bio-organic fertilizer containing combinations of antagonistic microorganisms. Biocontrol 54:287–300

    Article  Google Scholar 

  • Zhang X, Cui Z, Hong Q, Li S (2005) High-level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800. Appl Environ Microbiol 7:4101–4103

    Article  Google Scholar 

  • Zhang S, Waseem R, Yang X, Hu J, Huang Q, Xu Y, Liu X, Ran W, Shen Q (2008) Control of Fusarium wilt disease of cucumber plants with the application of a bioorganic fertilizer. Biol Fertil Soils 44:1073–1080

    Article  Google Scholar 

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Acknowledgments

This research was financially supported by Chinese Ministry of Science and Technology (2007CB109304 and 2009120) and Science and Technology Bureau of Jiangsu Province (BE2009672 and BA2008027). We would like to thank Dr. Hongsheng Wu from Nanjing University of Information Science and Technology, Nanjing, China, for his kind correction of the manuscript.

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Correspondence to Qirong Shen.

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Cao, Y., Zhang, Z., Ling, N. et al. Bacillus subtilis SQR 9 can control Fusarium wilt in cucumber by colonizing plant roots. Biol Fertil Soils 47, 495–506 (2011). https://doi.org/10.1007/s00374-011-0556-2

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