Abstract
Antifungal metabolites produced by Bacillus amyloliquefaciens AG1, previously isolated from wood of grapevine with “esca syndrome”, were studied. The crude protein extract (CPE) obtained from culture supernatant fluid by precipitation with ammonium sulfate was assayed against many grapevine fungal pathogens. B. amyloliquefaciens strain AG1 showed a broad spectrum of antifungal activity, inhibiting mycelial growth in vitro of all tested fungal microorganisms. The metabolites contained in CPE were heat stable and remained active over a wide pH range (2–10). Their activity was not affected by proteolytic or glycolytic enzymes. Tricine-SDS-polyacrylamide gel electrophoresis revealed a single band within the range of 2,510–3,480 Da, that showed inhibitory activity when used in the antifungal assay. Mass spectrometry analysis of this band allowed the substances involved in antibiosis to be identified as two tryptic peptides that correspond to the N-terminal sequence of subtilisin BPN’. These results suggest a potential role of B. amyloliquefaciens AG1 as a biocontrol agent.
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References
Alfonzo A, Ventorino V, Torta L, Burruano S, Moschetti G (2008) In vitro antagonism of a grapevine endophytic Bacillus subtilis strain towards “esca” fungi. Integrated Protection in Viticulture IOBC/wprs Bulletin 36:19–24
Alfonzo A, Conigliaro G, Torta L, Burruano S, Moschetti G (2009) Antagonism of Bacillus subtilis strain AG1 against vine wood fungal pathogens. Phytopathol Mediterr 48:155–158
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Baumgartner K, Warnock AE (2006) A soil inoculant inhibits Armillaria mellea in vitro and improves productivity of grapevines with root disease. Plant Dis 90:439–444
Bhunia AK, Johnson MC, Ray B (1987) Direct detection of an antimicrobial peptide of Pediococcus acidilactici in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Ind Microbiol 2:319–322
Burruano S, Mondello V, Conigliaro G, Alfonzo A, Spagnolo A, Mugnai L (2008) Grapevine decline in Italy caused by Lasiodiplodia theobromae. Phytopathol Mediterr 47:132–136
Esikova TZ, Temirov YV, Sokolov SL, Alakhov YB (2002) Secondary antimicrobial metabolites produced by thermophilic Bacillus spp. strains VK2 and VK21. Appl Biochem Microbiol 38(3):226–231
Gupta R, Beg QK, Lorenz P (2002) Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 59:15–32
Hiradate S, Yoshida S, Sugie H, Yada H, Fujii Y (2002) Mulberry anthracnose antagonists (iturins) produced by Bacillus amyloliquefaciens RC-2. Phytochemistry 61:693–698
Mondello V, Conigliaro G, Alfonzo A, Ferraro V, Torta L, Burruano S (2008) Preliminary studies on “esca” disease in Sicilian vineyards. IOBC/wprs Bullettin 36:181–187
Munimbazi C, Bullerman LB (1998) Isolation and partial characterization of antifungal metabolites of Bacillus pumilus. J Appl Microbiol 84(6):959–968
Nair JR, Singh G, Sekar V (2002) Isolation and characterization of a novel Bacillus strain from coffee phyllosphere showing antifungal activity. J Appl Microbiol 93(5):772–780
Niu Q, Huang X, Zhang L, Lian L, Li Y, Li J, Yang J, Zhang K (2007) Functional identification of the gene bace16 from nematophagous bacterium Bacillus nematocida. Appl Microbiol Biot 75(1):141–148
Paul B, Girard I, Bhatnagar T, Bouchet P (1997) Suppression of Botrytis cinerea causing grey mould disease of grape vine (Vitis vinifera) and its pectinolytic activities by a soil bacterium. Microbiol Res 152:413–420
Pinchuk IV, Bressollier P, Sorokulova IB, Verneuil B, Urdaci MC (2002) Amicoumacin antibiotic production and genetic diversity of Bacillus subtilis strains isolated from different habitats. Res Microbiol 153:269–276
Rao MB, Tanksale AM, Ghatge MS, Deshpande VV (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 62:597–635
Romero D, de Vicente A, Rakotoaly RH, Dufour SE, Veening JW, Arrebola E, Cazorla FM, Kuipers OP, Paquot M, Pérez-Garcìa A (2007) The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca. Mol Plant Microbe Interact 20:430–440
Schägger H, V Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166(2):368–379
Schimdt C, Lorenz D, Wolf GA (2001) Biological control of the grapevine dieback fungus Eutypa lata I: screening of bacterial antagonists. J Phytopathol 149:427–435
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599
Thompson JD, Higgins DG, Gibson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Villani F, Salzano G, Sorrentino E, Pepe O, Marino P, Coppola S (1993) Enterocin 226 NWC, a bacteriocin produced by Enterococcus faecalis 226, active against Listeria monocytogenes. J Appl Bacteriol 74:380–387
Villani F, Pepe O, Mauriello G, Salzano G, Moschetti G, Coppola S (1994) Antimicrobial activity of Staphylococcus xylosus from Italian sausages against Listeria monocytogenes. Lett Appl Microbiol 18:159–161
Wang SL, Shih IL, Liang TW, Wang CH (2002) Purification and characterization of two antifungal chitinases extracellularly produced by Bacillus amyloliquefaciens V656 in a shrimp and crab shell powder medium. J Agric Food Chem 50:2241–2248
Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703
Wells JA, Ferrari E, Henner DJ, Estell DA, Chen EY (1983) Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis. Nucleic Acids Res 11:7911–7925
Wichitra L, Punpen H, Samerchai C (2008) Growth inhibitory properties of Bacillus subtilis strains and their metabolites against the green mold pathogen (Penicillium digitatum Sacc.) of citrus fruit. Postharvest Biol Technol 48:113–121
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The author thank Dr. Luca Settani, Department DEMETRA, University of Palermo, for his help in this study.
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Alfonzo, A., Lo Piccolo, S., Conigliaro, G. et al. Antifungal peptides produced by Bacillus amyloliquefaciens AG1 active against grapevine fungal pathogens. Ann Microbiol 62, 1593–1599 (2012). https://doi.org/10.1007/s13213-011-0415-2
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DOI: https://doi.org/10.1007/s13213-011-0415-2