Advertisement

Purification and characterization of a potential antifungal protein from Bacillus subtilis E1R-J against Valsa mali

  • N. N. Wang
  • X. Yan
  • X. N. Gao
  • H. J. Niu
  • Z. S. Kang
  • L. L. Huang
Original Paper

Abstract

In order to identify the antagonistic substances produced by Bacillus subtilis E1R-J as candidate of biocontrol agents for controlling Apple Valsa Canker, hydrochloric acid precipitation, reverse phase chromatography, gel filtration, and ion exchange chromatography were used. The purified fraction EP-2 showed a single band in native-polyacrylamide gel electrophoresis (native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fraction EP-2 was eluted from native-PAGE and showed a clear inhibition zone against V. mali 03-8. These results prove that EP-2 is one of the most important antifungal substances produced by B. subtilis E1R-J in fermentation broth. SDS-PAGE and Nano-LC–ESI–MS/MS analysis results demonstrated that EP-2 was likely an antifungal peptide (trA0A086WXP9), with a relative molecular mass of 12.44 kDa and isoelectric point of 9.94. The examination of antagonistic mechanism under SEM and TEM showed that EP-2 appeared to inhibit Valsa mali 03-8 by causing hyphal swelling, distortion, abnormality and protoplasts extravasation. Inhibition spectrum results showed that antifungal protein EP-2 had significantly inhibition on sixteen kinds of plant pathogenic fungi. The stability test results showed that protein EP-2 was stable with antifungal activity at temperatures as high as 100 °C for 30 min and in pH values ranging from 1.0 to 8.0, or incubated with each 5 mM Cu2+, Zn2+, Mg2+, or K+. However, the antifungal activity was negatively affected by Proteinase K treatment.

Keywords

Endophytic Bacillus subtilis Apple Valsa Canker (AVC) Biocontrol agents (BCAs) Antifungal substances 

Notes

Acknowledgments

This study was supported by the Special Fund for Agro-Scientific Research in the Public Interest (No. 201203034), Science and Technology Innovation Research of Shaanxi (No. 2011KTZB02-02-02) and the National Natural Science Foundation of China (31101476). We are grateful to Dr. Ralf T. Voegele at Universität Hohenheim, Dr. Bing Liu (Jiangxi Agricultural University) and Prof. Heinrich Buchenauer (University Hohenheim) for comments and improvement of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

References

  1. Abe K, Kotoda N, Kato H, Soejima J (2007) Resistance sources to Valsa canker (Valsa ceratosperma) in a germplasm collection of diverse Malus species. Plant Breed 126:449–453. doi: 10.1111/j.1439-0523.2007.01379.x CrossRefGoogle Scholar
  2. Baindara P, Mandal SM, Chawla N, Singh PK, Pinnaka AK, Korpole S (2013) Characterization of two antimicrobial peptides produced by a halo tolerant Bacillus subtilis strain SK.DU.4 isolated from a rhizosphere soil sample. AMB Express 3:2. http://www.amb-express.com/content/3/1/2
  3. Berry C, Fernando WGD, Loewen PC, Kievit TR (2010) Lipopeptides are essential for Pseudomonas sp. DF41 biocontrol of Sclerotinia sclerotiorum. Biol Control 55:211–218. doi: 10.1016/j.biocontrol.2010.09.011 CrossRefGoogle Scholar
  4. Chen Y, Yan F, Chai YR, Liu HX, Kolter R, Losick R, Guo JH (2012) Biocontrol of tomato wilt disease by Bacillus subtilis isolates from natural environments depends on conserved genes mediating biofilm formation. Environ Microbiol 15:848–864. doi: 10.1111/j.1462-2920.2012.02860.x CrossRefGoogle Scholar
  5. Deng ZS, Zhao LF, Zhang WW, Ji YL, Wei GH (2009) Isolation of endophytic fungi from Ginkgo biloba L. and their antagonism on the Valsa mali Mayabe et Yamada. Acta Bot Boreali-Occident Sin 29:0608–0613 (in Chinese)Google Scholar
  6. Emrick D, Ravichandran A, Gosai J, Lu S, Gordon DM, Smith L (2013) The antifungal occidiofungin triggers an apoptotic mechanism of cell death in yeast. J Nat Prod 76:829–838. doi: 10.1021/np300678e CrossRefGoogle Scholar
  7. Fiddaman PJ, Rossall S (1993) The production of antifungal volatiles by Bacillus subtilis. J Appl Bacteriol 74:119–126. doi: 10.1111/j.1365-2672.1993.tb03004.x CrossRefGoogle Scholar
  8. Gao KX, Liu XG, Guo RF, Gao BJ, Zhu TB (2002) Mycoparasitism of Trichoderma spp. on five plant pathogenic fungi. J Shandong Agricult Univ 33:37–42 (in Chinese) Google Scholar
  9. Gao XN, Han QM, Chen YF, Qin HQ, Huang LL, Kang ZS (2014) Biological control of oilseed rape Sclerotinia stem rot by Bacillus subtilis strain Em7. Biocontrol Sci Technol 24:39–52. doi: 10.1080/09583157.2013.844223 CrossRefGoogle Scholar
  10. Gopinathan S (2013) Detection of FUR1 gene in 5-flucytosine resistant Candida isolates in vaginal candidiasis patients. J Clin Diagn Res 7(11):2452–2455. doi: 10.7860/JCDR/2013/6160.3574 Google Scholar
  11. Huang BQ, Huang LL, Kang ZS, Qiao HP (2009) Purification and characterization of an extracellular antifungal protein from wheat endophytic Bacillus subtilis strain E1R2j. Acta Agricult Boreali-Occident Sin 18(6):285–290Google Scholar
  12. Kang ZS (1995) Ultrastructure of plant pathogenic fungi. China Science and Technology Press, Beijing, pp 9–10 (in Chinese) Google Scholar
  13. Kloepper JW, Ryu CM, Zhang S (2004) Induced systemic resistance and promotion of plant growth by Bacillus spp. Phytopathology 94:1259–1266. doi: 10.1094/PHYTO.2004.94.11.1259 CrossRefGoogle Scholar
  14. Kumar A, Prakash A, Johri BN (2011) Bacillus as PGPR in crop ecosystem. In: Maheshwari DK (ed) Bacteria in agrobiology: crop ecosystems. Springer, New York, pp 37–59. doi: 10.1007/978-3-642-18357-7 CrossRefGoogle Scholar
  15. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacterio phage T4. Nature 227:680–685. doi: 10.1038/227680a0 CrossRefGoogle Scholar
  16. Li H, Zhao J, Feng H, Huang LL, Kang ZS (2013) Biological control of wheat stripe rust by an endophytic Bacillus subtilis strain E1R-j in greenhouse and field trials. Crop Prot 43:201–206. doi: 10.1016/j.cropro.2012.09.008 CrossRefGoogle Scholar
  17. Li ZP, Gao XN, Fan DY, Yan X, Kang ZS, Huang LL (2015) Saccharothrix yanglingensis strain Hhs.015 is a promising biocontrol agent on Apple Valsa Canker. Plant Dis. doi: 10.1094/PDIS-02-15-0190-RE Google Scholar
  18. Liu B, Qiao HP, Huang LL (2009) Biological control of take-all in wheat by endophytic Bacillus subtilis E1R-j and potential mode of action. Biol Control 49:277–285. doi: 10.1016/j.biocontrol.2009.02.007 CrossRefGoogle Scholar
  19. Liu B, Huang LL, Buchenauer H, Kang ZS (2010) Isolation and partial characterization of an antifungal protein from the endophytic Bacillus subtilis strain EDR4. Pest Biochem Physiol 98:305–311. doi: 10.1016/j.pestbp.2010.07.001 CrossRefGoogle Scholar
  20. Onishi J, Meinz M, Thompson J, Curotto J, Dreikorn S (2000) Discovery of novel antifungal (1, 3)-beta-D-glucan synthase inhibitors. Antimicrob Agents Chemother 44:368–377. doi: 10.1128/AAC.44.2.368-377.2000 CrossRefGoogle Scholar
  21. Pane C, Villecco D, Campnile F, Zaccardelli M (2012) Novel strains of Bacillus, isolated from compost and compost amended soils, as biological control agents against soil-borne phytopathogenic fungi. Biocontrol Sci Technol 22:1373–1388. doi: 10.1080/09583157.2012.729143 CrossRefGoogle Scholar
  22. Qiao HP, Huang LL, Kang ZS (2006) Endophytic bacteria isolated from wheat and their antifungal activities to soil-borne disease pathogens. Chin J Appl Ecol 17:690–694Google Scholar
  23. Rao Q, Guo W, Chen X (2015) Identification and characterization of an antifungal protein, AFAFPR9, produced by marine-derived Aspergillus fumigatus R9. J Microbiol Biotechnol 25:620–628. doi: 10.4014/jmb.1409.09071 Google Scholar
  24. Ren JJ, Shi GL, Wang XQ, Liu JG, Wang YN (2013) Identification and characterization of a novel Bacillus subtilis strain with potent antagonistic activity of a flagellin-like protein. World J Microbiol Biotechnol 29:2343–2352. doi: 10.1007/s11274-013-1401-6 CrossRefGoogle Scholar
  25. Sathishkumar R, Ananthan G, Raghunathan C (2015) Production and characterization of haloalkaline protease from ascidian-associated Virgibacillus halodenitrificans RSK CAS1 using marine wastes. Ann Microbiol 65:1481–1493. doi: 10.1007/s13213-014-0987-8 CrossRefGoogle Scholar
  26. Senol M, Nadaroglu H, Dikbas N, Kotan R (2014) Purification of chitinase enzymes from Bacillus subtilis bacteria TV-125, investigation of kinetic properties and antifungal activity against Fusarium culmorum. Ann Clin Microbiol Antimicrob 13:35. doi: 10.1186/s12941-014-0035-3 CrossRefGoogle Scholar
  27. Skouri Gargouri H, Gargouri A (2008) First isolation of a novel thermostable antifungal peptide secreted by Aspergillus clavatus. Peptides 29:1871–1877. doi: 10.1016/j.peptides.2008.07.005 CrossRefGoogle Scholar
  28. Slimene IB, Tabbene O, Gharbi D, Mnasri B, Schmitter JM, Urdaci MC, Limam F (2015) Isolation of a chitinolytic Bacillus licheniformis S213 strain exerting a biological control against phoma medicaginis infection. Appl Biochem Biotechnol 175:3494–3506. doi: 10.1007/s12010-015-1520-7 CrossRefGoogle Scholar
  29. Stein T (2005) Bacillus subtilis antibiotics: structures, synthesis and specific functions. Mol Microbiol 56:845–857. doi: 10.1111/j.1365-2958.2005.04587.x CrossRefGoogle Scholar
  30. van der Weerden NL, Bleackley MR, Anderson MA (2013) Properties and mechanisms of action of naturally occurring antifungal peptides. Cell Mol Life Sci 70:3545–3570. doi: 10.1007/s00018-013-1260-1 CrossRefGoogle Scholar
  31. Wang XL, Wei JL, Huang LL, Kang ZS (2011) Re-evaluation of pathogens causing Valsa canker on apple in China. Mycologia 103:317–324. doi: 10.3852/09-165 CrossRefGoogle Scholar
  32. Xie D, Peng J, Wang J, Hu J, Wang Y (1998) Purification and properties of antifungal protein X98III from Bacillus subtilis. Act Microbiol Sin 38:13–19 (in Chinese) Google Scholar
  33. Xin YF, Shang JJ (2005) Biocontrol trials of Chaetomium spirale ND35 against apple canker. J For Res 16:121–124. Article id:1007-662X(2005)02-0121-04Google Scholar
  34. Yang J, Ji JY, Kang ZS, Huang LL (2012) Optimization of fermentation conditions and purification of antifungal lipopeptide produced by Bacillus subtilis E1R-J. Act Agricult Boreal-Occident Sin 21:54–60 (in Chinese) Google Scholar
  35. Yang LR, Quan X, Xue BG, Goodwin PH, Lu SB, Wang JH, Wei D, Wu C (2015) Isolation and identification of Bacillus subtilis strain YB-05 and its antifungal substances showing antagonism against Gaeumannomyces graminis var. tritici. Biol Control 85:52–58. doi: 10.1016/j.biocontrol.2014.12.010 CrossRefGoogle Scholar
  36. Zhan LR, Zhang KC, Ran LX, Shi YP (2008) Isolation and identification of the antagonistic actinomycetes against Valsa mali. Hebei J For Orchard Res 23:182–186 (in Chinese) Google Scholar
  37. Zhang JX, Gu YB, Chi FM, Ji ZR, Wu JY, Dong QL, Zhou ZS (2015) Bacillus amyloliquefaciens GB1 can effectively control Apple Valsa Canker. Biol Control 88:1–7. doi: 10.1016/j.biocontrol.2015.04.022 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • N. N. Wang
    • 1
  • X. Yan
    • 1
  • X. N. Gao
    • 1
  • H. J. Niu
    • 1
  • Z. S. Kang
    • 1
  • L. L. Huang
    • 1
  1. 1.State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant ProtectionNorthwest A&F UniversityYanglingPeople’s Republic of China

Personalised recommendations