Journal of Plant Pathology

, Volume 100, Issue 2, pp 179–190 | Cite as

Characterization of antagonistic microorganisms against Aspergillus spp. from grapevine leaf and berry surfaces

  • Kazem Kasfi
  • Parissa Taheri
  • Behrooz Jafarpour
  • Saeed Tarighi
Original Article


This study aimed at controlling the common fungi causing postharvest Aspergillus rot of cv. Thompson seedless table grape (Vitis vinifera L.) via application of epiphytic biocontrol agents. Antagonistic yeasts and bacteria were isolated from the epiphytic flora associated with grape berries and leaves from five vineyards in Iran. A total of 130 yeast and bacterial isolates from grapevine surfaces were screened for antagonism against Aspergillus flavus, A. niger and A. ochraceus, the main species responsible for the accumulation of aflatoxin and ochratoxin A in grape berries. Seven yeast and bacterial isolates were selected based on their inhibitory effects on Aspergillus spp. and assayed by an in vitro nutritional competition test for their antagonistic capability. These isolates showed obvious antifungal activity against three different species of Aspergillus. Five yeast isolates were identified based on ITS region sequences as Candida membranifasciens (isolates Ka15 and Kh69) and Meyerozyma guilliermondii (Ka21, Kh59 and Kh60). Two bacterial isolates were identified based on the 16S rRNA gene sequences as Bacillus sp. (Ka3 and A10). Finally, the effect of antagonistic isolates on inoculated grape berries for their ability to inhibit infection by Aspergillus spp. was also investigated. All isolates showed antagonistic properties against the pathogens assayed at 25 °C and significantly reduced the disease progress on grape berries. Our data demonstrated that application of antagonistic microorganisms could be a promising alternative to fungicide treatments for controlling postharvest diseases of grapevine.


Aspergillus spp. Biocontrol agents Epiphytic flora Grapevine Mold 



We thank Ferdowsi University of Mashhad, Iran, for financial support of this research with project number 3/39326 approved on 1/12/2015.


  1. Abrunhosa L, Paterson RRM, Venancio A (2010) Biodegradation of ochratoxin a for food and feed decontamination. Toxins 2:1078–1099. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Allam N.G., El-Shanshoury A.R., Emara H.A., Mohamed A.Z., 2008. Fungi, toxigenic fungi, mycotoxins, pathogenic bacteria and heavy metal levels in some medicinal and food herbs. Proceedings. 5 th International Conference of Biological Science (Botany), Tanta (Egypt): 29–39Google Scholar
  3. Allam NG, El-Shanshoury AR, Emara HA, Zaky AZ (2012) Decontamination of ochratoxin a producing Aspergillus niger and ochratoxin a in medicinal plants by gamma irradiation and essential oils. Journal of International Environmental Application and. Science 7:161–169Google Scholar
  4. Arras G (1996) Mode of action of an isolate of Candida famata in biological control of Penicillium digitatum in orange fruit. Postharvest Biol Technol 8:191–198CrossRefGoogle Scholar
  5. Arrebola E, Sivakumar D, Korsten L (2010) Effect of volatile compounds produced by Bacillus strains on postharvest decay in citrus. Biol Control 53:122–128CrossRefGoogle Scholar
  6. Bae S, Fleet GH, Heard GM (2004) Occurrence and significance of Bacillus thuringiensis on wine grapes. Int J Food Microbiol 94:301–312CrossRefPubMedGoogle Scholar
  7. Batta YA (2000) Alternaria leaf spot disease on fig trees: varietal susceptibility and effect of some fungicides and Trichoderma. The Islamic University of Gaza. Journal 8:83–97Google Scholar
  8. Batta YA (2001) Effect of fungicides and antagonistic microorganisms on the black fruit spot disease on persimmon., Dirasat. Agric Sci 28:165–171Google Scholar
  9. Batta YA (2004a) Postharvest biological control of apple gray mold by Trichoderma harzianum Rifai formulated in an invert emulsion. Crop Prot 23:19–26CrossRefGoogle Scholar
  10. Batta YA (2004b) Effect of treatment with Trichoderma harzianum Rifai formulated in invert emulsion on postharvest decay of apple blue mold. Int J Food Microbiol 96:281–288CrossRefPubMedGoogle Scholar
  11. Benitez T, Rincon AM, Limon MC, Codon AC (2004) Biocontrol mechanisms of Trichoderma strains. Int Microbiol 7:249–260PubMedGoogle Scholar
  12. Bleve G, Grieco F, Cozzi G, Logrieco A, Visconti A (2006) Isolation of epiphytic yeasts with potential for biocontrol of Aspergillus carbonarius and A. niger on grape. Int J Food Microbiol 108:204–209CrossRefPubMedGoogle Scholar
  13. Castoria R, de Curtis F, Lima G, Caputo L, Pacifico S, de Cicco V (2001) Aureobasidium pullulans (LS 30), an antagonist of postharvest pathogens of fruits: study on its mode of action. Postharvest Biol Technol 22:7–17CrossRefGoogle Scholar
  14. Chalutz E, Ben Arie R, Droby S, Cohen L, Weiss B, Wilson CL (1988) Yeasts as biocontrol agents of postharvest diseases of fruits. Phytoparasitica 16:69–75Google Scholar
  15. Chen H, Xiao X, Wang J, Wu LJ, Zheng ZM, Yu ZL (2008) Antagonistic effects of volatiles generated by Bacillus subtilis on spore germination and hyphal growth of the plant pathogen, Botrytis cinerea. Biotechnol Lett 30:919–923CrossRefPubMedGoogle Scholar
  16. Csutak O, Vassu T, Sarbu I, Stoica I, Cornea P (2013) Antagonistic activity of three newly isolated yeast strains from the surface of fruits. Food Technol Biotechnol 51:70–77Google Scholar
  17. Diguta CF, Rousseaux S, Weidmann S, Bretin N, Vincent B, Guilloux-Benatier M, Alexandre H (2010) Development of a qPCR assay for specific quantification of Botrytis cinerea on grapes. FEMS Microbiol Lett 313:81–87CrossRefPubMedGoogle Scholar
  18. Drik R (2000) Specific PCR primers to identify arbuscular mycorrhizal fungi within colonized roots. Mycorrhiza 10:73–80CrossRefGoogle Scholar
  19. Droby S, Chalutz E, Wilson CL, Wisniewski M (1989) Characterization of the biocontrol activity of Debaromyces hansenii in the control of Penicillium digitatum on grapefruit. Can J Microbiol 35:794–800CrossRefGoogle Scholar
  20. Droby S, Chalutz E, Wilson CL, Wisniewski ME (1992) Biological control of postharvest diseases: a promising alternative to the use of synthetic fungicides. Phytoparasitica 20:S149–S153CrossRefGoogle Scholar
  21. Droby S, Wisniewski ME, Cohen L, Weiss B, Touitou D, Eilam Y, Chalutz E (1997) Influence of CaCl2 on Penicillium digitatum, grapefruit peel tissue, and biocontrol activity of Pichia guilliermondii. Phytopathology 87:310–315CrossRefPubMedGoogle Scholar
  22. Droby S., Wilson C.L., Wisniewski M., El Ghaouth A., 2000. Biologically based technology for the control of postharvest diseases of fruits and vegetables. In: Wilson C.L., Droby S. (eds). Microbial food contamination, pp 187–205. CRC Press, Boca RatonGoogle Scholar
  23. Eckert JW, Ogawa JM (1988) The chemical control of postharvest diseases: deciduous fruit, berries, vegetables and root tuber crops. Annu Rev Phytopathol 26:433–469CrossRefGoogle Scholar
  24. Elad Y, Kirshner B (1993) Survival in the phylloplane of an introduced biocontrol agent (Trichoderma harzianum) and populations of the plant pathogen Botrytis cinerea as modified by biotic conditions. Phytoparasitica 21:303–313CrossRefGoogle Scholar
  25. El-Ghaouth A, Wilson CL, Wisniewski M (1998) Ultrastructural and cytochemical aspects of the biological control of Botrytis cinerea by Candida saitoana in apple fruit. Phytopathology 88:282–291CrossRefPubMedGoogle Scholar
  26. Filonow AB, Vishniac HS, Anderson JA, Janisiewicz WJ (1996) Biological control of Botrytis cinerea in apple by yeasts from various habitats and their putative mechanism of antagonism. Biol Control 7:212–220CrossRefGoogle Scholar
  27. Foldes T, Banhegyi I, Herpai Z, Varga L, Szigeti J (2000) Isolation of Bacillus strains from the rhizosphere of cereals and in vitro screening for antagonism against phytopathogenic, food-borne pathogenic and spoilage microorganisms. J Appl Microbiol 89:840–846CrossRefPubMedGoogle Scholar
  28. Gabriolotto C, Monchiero M, Negre M, Spadaro D, Gullino ML (2009) Effectiveness of control strategies against Botrytis cinerea in vineyard and evaluation of the residual fungicide concentrations. J Environ Sci Health B 44:389–396CrossRefPubMedGoogle Scholar
  29. Gachomo EW, Kotchoni SO (2008) The use of Trichoderma harzianum and T. viride as potential biocontrol agents against peanut microflora and their effectiveness in reducing aflatoxin contamination of infected kernels. Biotechnology 7:439–447CrossRefGoogle Scholar
  30. Harman GE, Howell Ch R, Viterbo A, Chet I, Matteo L (2004) Trichoderma species opportunistic, a virulent plant symbionts. Nat Rev Microbiol 2:43–56CrossRefPubMedGoogle Scholar
  31. Janisiewicz WJ, Roitman J (1988) Biological control of blue mold and gray mold on apple and pear with Pseudomonas cepacia. Postharvest Pathology and Mycotoxins 78:1697–1700Google Scholar
  32. Jiang C, Shi J, Liu Y, Zhu C (2014) Inhibition of Aspergillus carbonarius and fungal contamination in table grapes using Bacillus subtilis. Food Control 35:41–48CrossRefGoogle Scholar
  33. Karabulut OA, Smilanic JL, Gabler FM, Mansour M, Droby S (2003) Near-harvest applications of Metschnikowia fructicola, ethanol, and sodium bicarbonate to control postharvest diseases of grape in Central California. Plant Dis 87:1384–1389CrossRefGoogle Scholar
  34. Kim DS, Cook RJ, Weller DM (1997) Bacillus sp. L324-92 for biological control of three root diseases of wheat grown with reduced tillage. Phytopathology 87:551–558CrossRefPubMedGoogle Scholar
  35. Klich MA, Lax AR, Bland JM (1991) Inhibition of some mycotoxigenic fungi by iturin a, a peptidolipid produced by Bacillus subtilis. Mycopathologia 116:77–80CrossRefPubMedGoogle Scholar
  36. Kraus J, Lopper JE (1990) Biocontrol of Pythium damping-off of cucumber by Pseudomonas fluorescens pf-5: mechanistic studies. The Second International Workshop on Plant Growth-Promoting Rhizobacteria, Interlaken, pp 172–175Google Scholar
  37. Leelasuphakul W, Hemmaneea P, Chuenchitt S (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 48:113–121CrossRefGoogle Scholar
  38. Liu HB, Tian SP, Qin GZ, Xu Y (2002) Effect of Cryptococcus laurentii on biological control of postharvest diseases in grapes. Agric Sci China 35:831–835Google Scholar
  39. Madden AA, Boyden SD, Soriano JAN, Corey TB, Leff JW, Fierer N, Starks PT (2017) The emerging contribution of social wasps to grape rot disease ecology. Peer Journal 5:e3223CrossRefGoogle Scholar
  40. Manici L, Lazzeri L, Palmieri S (1997) In vitro fungitoxic activity of some glucosinolates and their enzyme-derived products toward plant pathogenic fungi. J Agric Food Chem 45:2768–2773CrossRefGoogle Scholar
  41. Mari M, Guizzardi M, Pratella GC (1996) Biological control of gray mold in pears by antagonistic bacteria. Biol Control 7:30–37CrossRefGoogle Scholar
  42. Masih E, Paul B (2002) Secretion of β-1,3-glucanases by the yeast Pichia membranifaciens and its possible role in the biocontrol of Botrytis cinerea causing grey mold disease of the grapevine. Curr Microbiol 44:391–395CrossRefPubMedGoogle Scholar
  43. Nally MC, Pescea VM, Maturanoa YP, Toroa ME, Combinab M, Castellanos De Figueroa LI, Vazquez F (2013) Biocontrol of fungi isolated from sour rot infected table grapes by Saccharomyces and other yeast species. Postharvest Biol Technol 86:456–462CrossRefGoogle Scholar
  44. Nunes CA (2012) Biological control of postharvest diseases of fruit. Eur J Plant Pathol 133:181–196CrossRefGoogle Scholar
  45. O'Neill TM, Elad Y, Shtienberg D, Cohen A (1996) Control of grapevine grey mold with Trichoderma harzianum T39. Biocontrol Sci Tech 6:139–146CrossRefGoogle Scholar
  46. Paster N, Droby S, Chalutz E, Menasherov M, Nitzan R, Wilson CL (1993) Evaluation of the potential of the yeast Pichia guilliermondii as a biocontrol agent against Aspergillus flavus and fungi of stored soya beans. Mycol Res 97:1201–1206CrossRefGoogle Scholar
  47. Peng G, Sutton JC (1991) Evaluation of microorganisms for biocontrol of Botrytis cinerea in strawberry. Can J Plant Pathol 13:247–257CrossRefGoogle Scholar
  48. Pimenta RS, Morais PB, Rosa CA, Correa A (2009) Utilization of yeast in biological control programs. In: Satyanarayana T, Kun G (eds) Yeast biotechnology: diversity and applications. Springer, Berlin, pp 199–214CrossRefGoogle Scholar
  49. Poppe L, Vanhoutte S, Hofte M (2003) Mode of action of Pantoea agglomerans CPA-2, an antagonist of postharvest pathogens on fruits. Eur J Plant Pathol 109:963–973CrossRefGoogle Scholar
  50. Prusky D (2011) Reduction of the incidence of postharvest quality losses, and future prospects. Food Security 3:463–474CrossRefGoogle Scholar
  51. Rabosto X, Carrau M, Paz A, Boido E, Dellacassa E, Carraul FM (2006) Grapes and vineyard soils as sources of microorganisms for biological control of Botrytis cinerea. Am J Enol Vitic 57:332–338Google Scholar
  52. Raspor P, Miklic-Milek D, Avbelj M, Cadez N (2010) Biocontrol of grey mold disease on grape caused by Botrytis cinerea with autochthonous wine yeasts. Food Technol Biotechnol 48:336–343Google Scholar
  53. Romanazzi G, Lichter A, Gabler FM, Smilanick JL (2012) Recent advances on the use of natural and safe alternatives to conventional methods to control postharvest gray mold of table grapes. Postharvest Biol Technol 63:141–147CrossRefGoogle Scholar
  54. Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratoy press. Cold Spring Harbor, New YorkGoogle Scholar
  55. Santos A, Marquina D (2004) Killer toxin of Pichia membranifaciens and its possible use as a biopreservative agent to control grey mold disease of grapevine. Microbiology 150:2527–2534CrossRefPubMedGoogle Scholar
  56. Santos A, Marquina D, Leal JA, Peinado JM (2000) (1,6)-β-D-glucan as cell wall receptor for Pichia membranifaciens killer toxin. Appl Environ Microbiol 66:1809–1813CrossRefPubMedPubMedCentralGoogle Scholar
  57. Schena L, Nigro F, Pentimone I, Ligorio A, Ippolito A (2003) Control of postharvest rots of sweet cherries and table grapes with endophytic isolates of Aureobasidium pullulans. Postharvest Biol Technol 30:209–220CrossRefGoogle Scholar
  58. Senthil R, Prabakar K, Rajendran L, Karthikeyan G (2011) Efficacy of different biological control agents against major postharvest pathogens of grapes under room temperature storage conditions. Phytopathol Mediterr 50:55–64Google Scholar
  59. Serra R, Bragab A, Venancio A (2005) Mycotoxin-producing and other fungi isolated from grapes for wine production, with particular emphasis on ochratoxin a. Res Microbiol 156:515–521CrossRefPubMedGoogle Scholar
  60. Sherman F, Fink G, Hicks J (1986) Methods in yeast genetics. Cold spring harbour laboratory press. Cold Spring Harbor, New YorkGoogle Scholar
  61. Stapleton JJ, Grant RS (1992) Leaf removal for nonchemical control of the summer bunch rot complex of wine grapes in the San Joaquin Valley. Plant Dis 76:205–208CrossRefGoogle Scholar
  62. Swadling I, Jeffries P (1996) Isolation of microbial antagonists for biocontrol of grey mold disease of strawberries. Biocontrol Sci Tech 6:125–136CrossRefGoogle Scholar
  63. Thomidis T, Pantazis S, Konstantinoudis K (2016) Evaluation of serenade max to control fruit rot of grapes. J Agric Sci 8:212–214Google Scholar
  64. Vargas M, Garrido F, Zapata N, Tapia M (2012) Isolation and selection of epiphytic yeast for biocontrol of Botrytis cinerea Pers. on table grapes. Chilean Journal of Agricultural Research 72:332–337CrossRefGoogle Scholar
  65. Visconti A, Perrone G, Cozzi G, Solfrizzo M (2008) Managing ochratoxin a risk in the grape-wine food chain. Food Additives and Contaminants-Part A 25:193–220CrossRefGoogle Scholar
  66. Wilson CL, Wisniewski ME (1989) Biological control of postharvest diseases of fruit and vegetables: an emerging technology. Annu Rev Phytopathol 27:425–441CrossRefGoogle Scholar
  67. Wilson CL, Wisniewski ME (1994) Biological control of postharvest diseases of fruit and vegetables. In: Theory and practice pp 63–75. CRC Press, Boca RatonGoogle Scholar
  68. Wisniewski M, Wilson CL (1992) Biological control of postharvest diseases of fruits and vegetables: recent advances. Horticulture Science 27:94–98Google Scholar
  69. Wisniewski M., Biles C., Droby S., 1990. The use of the yeast Pichia guilliermondii as a biocontrol agent: characterization of attachment to Botrytis cinerea. Biological Control of Postharvest Diseases of Fruits and Vegetables pp 167–183. Agricultural Research Service document ARS-92. U.S. Department of Agriculture, Washington, D.C,, USAGoogle Scholar
  70. Yashiro E, Spear RN, McManus PS (2011) Culture-dependent and culture-independent assessment of bacteria in the apple phyllosphere. J Appl Microbiol 110:1284–1296CrossRefPubMedGoogle Scholar
  71. Zahavi T, Cohen L, Weiss B, Schena L, Daus A, Kaplunov T, Zutkhi J, Ben-Arie R, Droby S (2000) Biological control of Botrytis, Aspergillus and Rhizopus rots on table and wine grapes in Israel. Postharvest Biol Technol 20:115–124CrossRefGoogle Scholar
  72. Zhimo VY, Bhutia DD, Saha J (2016) Biological control of postharvest fruit diseases using antagonistic yeasts in India. J Plant Pathol 98:275–283Google Scholar
  73. Zolan M, Pukkila P (1986) Inheritance of DNA methylation in Coprinus cinereus. Mol Cell Biol 6:195–200CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Società Italiana di Patologia Vegetale (S.I.Pa.V.) 2018

Authors and Affiliations

  • Kazem Kasfi
    • 1
  • Parissa Taheri
    • 1
  • Behrooz Jafarpour
    • 1
  • Saeed Tarighi
    • 1
  1. 1.Department of Plant Protection, Faculty of AgricultureFerdowsi University of MashhadsMashhadIran

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