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Characterization of chitinase secreted by Bacillus cereus strain CH2 and evaluation of its efficacy against Verticillium wilt of eggplant

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Abstract

A chitinase-secreting strain CH2 was one of 353 strains isolated from rhizosphere of eggplant. Based on 16S rDNA sequence alignment and several biochemical and physiological characteristics, the strain was identified to be of Bacillus cereus. On chitin–Ayers (CA) medium, the strain secreted chitinase. Evaluation of its activity, combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), showed it to be a 15.0-KD chitinase. On glass slides, germination of the fungal spores was effectively suppressed by the bacterial suspension, supernatant from the suspension, and 0.005% solution of chitinase extracted from the strain CH2. The optimum pH for chitinase was 7.1 and optimum temperature was 40°C. At that temperature, high-level chitinolytic activity was retained for 10 days. In greenhouse experiments, suspension of the cells of the CH2 strain reduced the severity of Verticillium wilt on eggplant by 69.69%, its supernatant by 54.04%, and the enzyme diluted to 0.01% strength by 53.13% in 14 days. Strain CH2 and its chitinase have good commercial potential in controlling Verticillium wilt.

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References

  • Abd-Allah EF (2001) Streptomyces plicatus as a model biocontrol agent. Folia Microbiol (Praha) 46(4):309–341

    Article  CAS  Google Scholar 

  • Backman PA, Brannen PM, Mahaffee WF (1994) Plant response and disease control following seed inoculation with Bacillus subtilis. Adelaide, Aust.: CSIRO, Div. Soils, pp 3–8

  • Balasubramanian R, Manocha MS (1992) Cytosolic and membrane-bound chitinase of two mucoraceous fungi:a comparative study. Can J Microbiol 38:331–338

    Article  PubMed  CAS  Google Scholar 

  • Berger F, Hong L, White D et al (1996) Effect of pathogen inoculum, antagonist density, and plant species on biological control of Phytophthora and Pythium damping-off by Bacillus subtilis Cot1 in high humidity fogging glasshouses. Phytopathology 86:429–433

    Article  Google Scholar 

  • Bhat RG, Subbarao KV (2001) Reaction of broccoli to isolates of Verticillium dahliae from various hosts. Plant Dis 85:141–146

    Article  Google Scholar 

  • Buchanan RE, Bergey DH et al (1984) Bergey’s manual of determinative bacteriology. 8th Chinese edn. Chinese Science Press

  • Chernin LS, Fuente L De la, Sobolev V et al (1997) Molecular cloning, structural analysis and expression in Escherichia coli of a chitinase gene from Enterobacter agglomerans. Appl Environ Microbiol 63(3):834–839

    PubMed  CAS  Google Scholar 

  • Cohen-Kupiec R, Chet I (1998) The molecular biology of chitin digestion. Curr Opin Biotechnol 9:270–277

    Article  PubMed  CAS  Google Scholar 

  • Colson-Hanks ES, Deverall BJ (2000) Effect of 2,6-dichloroisonicotinic acid, its formulation materials and benzothiadiazole on systemic resistance to Alternaria leaf spot in cotton. Plant Pathol 49:171–178

    Article  CAS  Google Scholar 

  • Conroy JJ, Green RJ, Ferris JM (1972) Interaction of Verticillium albo-atrum and the root lesion nematode, Pratylenchus penerrans, in tomato roots at controlled inoculum densities. Phytopathology 62:362–366

    Article  Google Scholar 

  • Fang ZD (1998) Method in plant pathology, 3rd edn Chinese Agricultural Press, Beijing

    Google Scholar 

  • Faulkner LR, Bolander WJ, Skotland CB (1970) Interaction of Verticillium dahliae and Pratylenchus minyus in Verticillium wilt of peppermint: Influence of the nematode as determined by a double root technique. Phytopathology 60:100–103

    Article  Google Scholar 

  • Felse PA, Panda T (1999) Regulation and cloning of microbial chitinase genes. Appl Microbiol Biotechnol 51:141–151

    Article  PubMed  CAS  Google Scholar 

  • Fernando WGD, Nakkeeran S, Zhang Y et al (2007) Biological control of Sclerotinia sclerotiorum (Lib.) de Bary by Pseudomonas and Bacillus species on canola petals. Crop Prot 26(2):100–107

    Article  Google Scholar 

  • Fitzell R, Evans G., Fahy PC (1980) Studies on the colonization of plant roots by Verticillium dahliae with use of immuno fluorescent staining. Aust J Bot 28:357–368

    Article  Google Scholar 

  • Flach J, Pilet PE, Jolles P (1992) What’s new in chitinase research? Experientia 48:701–716

    Article  PubMed  CAS  Google Scholar 

  • Garland JL, Mills AL (1991) Classification and characterization of heterotrophic microbial communities on the basis of patterns of community level: sole-carbon-source-utilization. Appl Envion Microb 57:2351–2359

    CAS  Google Scholar 

  • Gerik JS, Huisman OC (1988) Study of fieldgrown cotton roots infected with Verticillium dahliae using an immunoenzymatic staining technique. Phytopathology 78:1174–1178

    Article  Google Scholar 

  • Graham LS, Sticklen MB (1994) Plant chitinases. Can J Bot 72:1057–1083

    Article  CAS  Google Scholar 

  • Halebian S, Harris B, Finegold SM et al (1981) Rapid methods that aid in distinguishing Gram-positive from Gram-negative anaerobic bacteria. J Clin Microbiol 13:444–448

    PubMed  CAS  Google Scholar 

  • Heimbrook ME, Wang WL (1989) Campbell G1 staining bacterial flagella easily. J Clin Microbiol 27(11):2612–2615

    PubMed  CAS  Google Scholar 

  • Helistö P, Aktuganov G, Galimzianova N et al (2001) Lytic enzyme complex of an antagonistic Bacillus sp. X-b: isolation and purification of components. J Chromatogr B 758:197–205

    Article  Google Scholar 

  • Hoell IA, Klemsdal SS, Vaaje-Kolatad G et al (2005) Overexpression and characterization of a novel chitinase from Trichoderma atovirid strain P1. Biochim et Biophysica Acta 1748:180–190

    CAS  Google Scholar 

  • Huang JL, Li HL, Yuan HX (2006) Effect of organic amendments on Verticillium wilt of cotton. Crop Prot 25(11):1167–1173

    Article  Google Scholar 

  • Huang CJ, Wang TK, Chung SC et al (2005) Identification of an antifungal chitinase from a potential biocontrol agent, Bacillus cereus 28–9. J Biochem Mol Biol 38(1):82–88

    PubMed  CAS  Google Scholar 

  • Jiang ZQ, Guo YH, Li SM et al (2006) Evaluation of biocontrol efficiency of different Bacillus preparation and field application methods against Phytophthora blight of bell pepper. Biol Control 36:216–223

    Article  Google Scholar 

  • Karasuda S, Tanaka S, Ksjihara H et al (2003) Plant chitinase as a possible biocontrol agent for use instead of chemical fungicides. Biosci Biotechnol Biochem 67(1):221–224

    Article  PubMed  CAS  Google Scholar 

  • Korsten L, De Villiers EE, Weimer FC et al (1993) A review of biological control of postharvest diseases of subtropical fruits. In: Champ BR, Highley E, Johnson GI (eds) Postharvest handling of tropical fruits. In: Proceedings of an international conference held at Chiang Mai, Thailand. Watson, Ferguson and Company, pp 172–185

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Lee YS, Park IH, Yoo JS et al (2007) Cloning, purification, and characterization of chitinase from Bacillus sp. DAU101. Bioresour Technol 98:2734–2741

    Article  PubMed  CAS  Google Scholar 

  • Levin AG., Lavee S, Tsror L (2003) Epidemiology of Verticillium dahliae on olive (cv. Picual) and its effect on yield under saline conditions. Plant Pathol 52:212–218

    Article  Google Scholar 

  • Li DC, Chen S, Jing LU (2005) Purification and partial characterization of two chitinases from the mycoparasitic fungus Talaromyces flavus. Mycopathologia 159:223–229

    Article  CAS  Google Scholar 

  • Mathivannan N, Kabilan V, Murugesan K (1998) Purification, characterization, and antifungal activity of chitinase from Fusarium chlamydosporum, a mycoparasite to groundnut rust, Puccinia arachidis. Can J Microbiol 44:646–652

    Article  Google Scholar 

  • Mercado-Blanco J, Rodríguez-Jurado D, Hervás A et al (2004) Suppression of verticillium wilt in olive planting stocks by root-associated fluorescent Pseudomonas spp. Biol Control 30:474–486

    Article  Google Scholar 

  • Millis AAS, Platt HW, Hurta RAR (2004) Effect of salt compounds on mycelial growth, sporulation and spore germination of various potato pathogens. Postharvest Biol Tec 34:341–350

    Article  CAS  Google Scholar 

  • Nannipieri P, Klug MT, Reddy CA (1984) Microbial biomass and activity measurement in soils: ecological significance. American Society for Microbiology, Washington

    Google Scholar 

  • Naosekpam SAA, Rajni V, Shanmugam V (2006) Extracellular chitinase of fluorescent Pseudomonads antifungal to Fusarium oxysporum f. sp. dianthi causing carnation wilt. Curr Microbiol 52:310–316

    Article  CAS  Google Scholar 

  • Parameswaran B, Tünde P, Viviana N et al (2004) Production and purification of extracellular chitinases from Penicillium aculeatum NRRL 2129 under solid-state fermentation. Enzyme Microb Technol 36:880–887

    Google Scholar 

  • Paulitz TC, Belanger RR (2001) Biological control in greenhouse systems. Annu Rev Phytopathol 39:103–133

    Article  PubMed  CAS  Google Scholar 

  • Perry JW, Evert RF (1983) The effect of colonization by Verticillium dahliae on the root tips of Russet Burbank potatoes. Can J Botany 61:3422–3429

    Article  Google Scholar 

  • Pleban S, Chermin L, Chet I (1997) Chitinolytic activity of an endophytic of Bacillus cereus. Lett Appl Microbiol 25:284–288

    Article  PubMed  CAS  Google Scholar 

  • Schaad NW (1988) Laboratory guide for identification of plant pathogenic bacteria. American Phytopatholgical Society Press, Minnesota, pp 1–16

    Google Scholar 

  • Shternshis MV, Beljaev AA, Shpatova TV et al (2002) Field testing of BACTICIDE, PHYTOVERM and CHITINASE for control of the raspberry midge blight in Siberia. BioControl 47:697–706

    Article  CAS  Google Scholar 

  • Sietsma JH, Wessels JGH (1979) Evidence for covalent linkages between chitin and β-glucan in a fungal cell wall. J Gen Microbiol 114:99–108

    CAS  Google Scholar 

  • Stabb EV, Johnson L, Handelsman J (1994) Zwittermycin A-producing strains of Bacillus cereus from diverse soils. Appl Environ Microbiol 60:4404–4412

    PubMed  CAS  Google Scholar 

  • Storey GW, Evans K (1987) Interactions between Globodera pallida juveniles, Verticillium dahliae and three potato cultivars, with descriptions of associated histopathologies. Plant Pathol 36:192–200

    Article  Google Scholar 

  • Tang WH (1994) Yield-increasing bacteria (YIB) and biocontrol of sheath blight of rice. Adelaide, Aust.: CSIRO. Div. Soils, pp 267–273

  • Thanassoulopoulos CC, Biris DA, Tjamos EC (1979) Survey of Verticillium wilt of olive trees in Greece. Plant Dis Reptr 63:936–940

    Google Scholar 

  • Tronsmo A, Harman GE (1993) Detection and quantification of N-Acetyl-β-d-glucosaminidase, chitiobiosidase, and endochitinase in solutions and on gels. Anal Biochem 208:4–79

    Article  Google Scholar 

  • Watanabe T, Oysnsgi W, Suzushi K et al (1990) Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation. J Bacteriol 172:74–79

    Google Scholar 

  • Wei LH, Ding GC, Guo JH et al (2006) Effect of biocontrol preparation GJ23 against root-knot nematodes in cucumber. J Jiangsu Agric Sci 5:46–48

    Google Scholar 

  • Weller DM (1988) Biological control of soilborne plant pathogens in the rhizosphere with bacteria. Annu Rev Phvtopathol 26:379–407

    Article  Google Scholar 

  • Wersberg WG, Brans SM, Pelletier DA et al (1991) 16S Ribosomal DNA amplification for phytogenetic study. J Bacteriol 173(2):697–703

    Google Scholar 

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Acknowledgments

This research was supported by a grant-in-aid for science research from the Chinese 863 high-tech Program (2002AA244041), Sino-Germany Cooperation Project on Agricultural Science and Technology (2004-Z17) and Program for New Century Excellent Talents in University (NCET-06-0492). We thank David Cushley to modify the manuscript.

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  1. Department of Plant Pathology, Nanjing Agricultural University, Nanjing, 210095, China

    Jian-Gang Li, Zhi-Qiang Jiang, Liu-Ping Xu, Fei-Fei Sun & Jian-Hua Guo

  2. Nantong Entry-Exit Inspection and Quarantine Bureau, Jiangsu, 226005, China

    Zhi-Qiang Jiang

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  1. Jian-Gang Li
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Correspondence to Jian-Hua Guo.

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Li, JG., Jiang, ZQ., Xu, LP. et al. Characterization of chitinase secreted by Bacillus cereus strain CH2 and evaluation of its efficacy against Verticillium wilt of eggplant. BioControl 53, 931–944 (2008). https://doi.org/10.1007/s10526-007-9144-7

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