Diversity and extracellular enzyme profiles of yeasts on organic and fungicide treated strawberries

  • Tülay Turgut Genç Çanakkale Onsekiz Mart University, Faculty of Arts and Science, Department of Biology, Çanakkale, Turkey
  • Melih Günay Çanakkale Onsekiz Mart University, Graduate School of Natural and Applied Sciences, Çanakkale, Turkey
Keywords: Strawberry, Fungicide treatment, D1/D2 rDNA, RFLP, Extracellular enzyme, Yeast


Since yeasts can survive under variable environmental conditions using different food sources they have a wide distribution in nature. Fruits are suitable living spaces for yeasts and other microorganisms due to their high and different sugar contents. Strawberry fruit as well as other fruits are very sensitive to pathogenic fungi. Due to their residues on fruits, limitations on the use of fungicides have led to increased use of microorganisms with antagonistic effects as biological control agents. The biological agents to be used are selected mainly from the microorganisms found in the natural microbiota of the fruit. Therefore, in this study yeast biota on strawberry fruit collected from fungicide treated (Klorzon and Topas) and organic fields was determined using molecular identification methods. In addition, extracellular enzyme profiles of the identified yeast species were determined by the APIZYM-based system. There was no difference in the diversity of yeast species on strawberries collected from fungicide treated and organic fields, but the yeast density on organic strawberries was greater than fungicide treated fruits. The identified yeast species on fruits were determined as Metschnikowia pulcherrima (61.7%), Hanseniaspora uvarum (34.0%) and Wickerhamomyces pijperi (4.3%). W. pijperi yeast species was reported on strawberry fruit in our study first time. It was determined that H. uvarum and W. pijperi yeast species showed no α-glucosidase enzyme activity. All yeast strains showed industrially important β-glucosidase enzyme activity.

DOI: http://dx.doi.org/10.5281/zenodo.5539705


Download data is not yet available.


1. Kurup VP. Fungal allergens. Curr Allergy Asthma Rep. 2003; 3: 416-423.
2. Mons E. Occupational asthma in greenhouse workers. Curr Opin Pulm Med. 2004; 10(2): 147-150.
3. Droby S, Chalutz E, Hofstein R, Wilson CL, Wisniewski ME, Fridlender B, et al. Pilot testing of Pichia guilliermondii: a biocontrol agent of postharvest diseases of citrus fruit. Biol Control. 1993; 3: 47-52.
4. Dermesonlouoglou EK, Bimpilas A, Andreou V, Katsaros GJ, Giannakourou MC, Taoukis PS. Process optimization and kinetic modelling of quality of fresh-cut strawberry cubes pretreated by high pressure and osmosis. J Food Proces Preserv. 2016; 41: 13137.
5. Dermesonlouoglou EK, Giannakourou M, Taoukis PS. Kinetic study of the effect of the osmotic dehydration pre-treatment with alternative osmotic solutes to the shelf life of frozen strawberry. Food Bioprod Process. 2016; 99: 212-221.
6. Tournas VH, Katsoudas E. Mould and yeast flora in fresh berries, grapes and citrus fruits. Int J Food Microbiol. 2005; 105: 11-17.
7. Wedge DE, Smith BJ, Quebedeaux JP, Constantin RJ. Fungicide management strategies for control of strawberry fruit rot diseases in Louisiana and Mississippi. Crop Prot. 2007; 26: 1449-1458.
8. Menzel CM, Gomez A, Smith LA. Control of grey mould and stem-end rot in strawberry plants growing in a subtropical environment. Australas Plant Pathol. 2016; 45: 489-498.
9. De la Torre M, Millan M, Perez-Juan P, Morales J, Ortega J. Indigenous yeasts associated with two Vitis vinifera grape varieties cultured in southern Spain. Microbios. 1999; 100: 27-40.
10. Guerra E, Sordi G, Mannazzu I, Clementi F, Fatichenti F. Occurrence of wine yeasts on grapes subjected to different pesticide treatments. Ital J Food Sci. 1999; 3(11): 221-230.
11. Rigotti S, Viret O, Gindrat D. Fungi from symptomless strawberry plants in Switzerland. Phytopathol Mediterr. 2003; 42: 85-88.
12. Debode J, Van Hemelrijck W, Creemers P, Maes M. Effect of fungicides on epiphytic yeasts associated with strawberry. Microbiologyopen. 2013; 2(3): 482-491.
13. Liu X, Kokare C. Microbial enzymes of use in industry. In: Brahmachari G, ed. Biotechnology of microbial enzymes. Academic Press, Cambridge, 2017.
14. Li S, Yang X, Yang S, Zhu M, Wang X. Technology prospecting on enzymes: application, marketing and engineering. Comput Struct Biotechnol J. 2012; 9(2): e201209017.
15. Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evol. 1985; 39: 783-791.
16. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018; 35: 1547-1549.
17. Kristjuhan A, Lõoke M, Kristjuhan K. Extraction of Genomic DNA from Yeasts for PCR-Based Applications. Biotechniques. 2011; 50(5): 325-328.
18. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR Protocols: A Guide to Methods and Applications. San Diego, California, Academic Press, 1990: 315-322.
19. Kurtzman CP, Robnett CJ. Identification and Phylogeny of Ascomycetous Yeasts from Analysis of Nuclear Large Subunit (26S) Ribosomal DNA Partial Sequences. Antonie Leeuwenhoek. 1998; 73: 331-371.
20. Nei M, Kumar S. Molecular Evolution and Phylogenetics. Oxford University Press, New York, 2000.
21. European Commission. European Commission Health & Consumer Protection Directorate general. OECD Issue Paper on Microbial Contaminant Limits for Microbial Pest Control Products. 2012; 65: 43-53.
22. Comitini F, Ciani M. Influence of fungicide treatments on the occurrence of yeast flora associated with wine grapes. Ann. Microbiol. 2008; 58: 489-493.
23. Buck JW., Burpee LL. The effects of fungicides on the phylloplane yeast populations of creeping bentgrass. Can J Microbiol. 2002; 48: 522-529.
24. Cadez N, Zupan J, Raspor P. The effect of fungicides on yeast communities associated with grape berries. FEMS Yeast Res. 2010; 10: 619-630.
25. Fonseca A, Inacio J. Phylloplane yeasts. C. Rosa and G. Peter, eds. Biodiversity and ecophysiology of yeasts. The yeast handbook. Springer, Berlin, 2006: 263-301.
26. Babjeva I, Reshetova I. Yeast Resources in Natural Habitats at Polar Circle Latitude. Food Technol Biotech. 1998; 36(1): 1-5.
27. Baffi MA, Bezerra CS, Arévalo-Villena M, Briones-Pérez AI, Gomes E, Da Silva R. Isolation and molecular identification of wine yeasts from a Brazilian vineyard. Ann Microbiol. 2010; 61: 75-78.
28. Carvalho CM, Meirinho S, Estevinho MLF, Choupina A. Yeast species associated with honey: Different identification methods. Arch Zootec. 2010; 59(225): 103-113.
29. Kurtzman CP, Fell JW. Summary of species characteristics. In: Kurtzman CP & Fell JW (Eds) The Yeasts, A Taxonomic Study, 4th edn. Elsevier Science BV, Amsterdam, 1998: 915-947.
30. Pincus DH, Orenga S, Chatellier S. Yeast identification past, present, and future methods. Med Mycol. 2007; 45: 97-121.
31. Esteve-Zarzoso B, Belloch C, Uruburu F, Querol A. Identification of Yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. Int J Syst Evol Microbiol. 1999; 49: 329-337.
32. Rodrıguez-Vico F, Clemente-Jimenez JM, Mingorance-Cazorla L, Martinez-Rodriguez S, Las Heras-Vazquez FJ. Molecular characterization and oenological properties of wine yeasts isolated during spontaneous fermentation of six varieties of grape must. Food Microbiol. 2003; 21: 149-155.
33. Guillamon JM, Sabate J, Barrio E, Cano J, Querol A. Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region. Arch Microbiol. 1998; 169(5): 387-392.
34. Romano P, Capece A, Siesto G, Romaniello R. Restriction analysis of rDNA regions to differentiate non-Saccharomyces wine species in mixed cultures. Int J Eng Res Technol. 2009; 1(4): 068-071.
35. Gibson BR, Pham T, Wimalasena T, Box WG, Koivuranta K, Storgards E, Smart KA. Evaluation of ITS PCR and RFLP for differentiation and identification of brewing yeast and brewery ‘wild’ yeast contaminants. J Inst Brew. 2011; 117(4): 556–568.
36. Mambuscay LA, Lopez WA, Cuervo RA, Argote FE, Osorio-Cadavid E. Identification of yeast in the native pineapple juice, blackberry and grape [in Spanish]. Biotecnol Sector Agropecuario Agroindustr. 2013; 2: 136-144.
37. De Curtis F, Torriani S, Rossi F, De Cicco V. Selection and use of Metschnikowia pulcherrima as a biological control agent for postharvest rots of peaches and table grapes. Ann Microbiol. 1996; 46: 45-55.
38. Piano S, Neyrotti V, Migheli Q, Gullino ML. Biocontrol capability of Metschnikowia pulcherrima against Botrytis postharvest rot of apple. Postharvest Biol Tec. 1997; 11: 131-140.
39. Spadaro D, Vola R, Piano S, Gullino ML. Mechanisms of action and efficacy of four isolates of the yeast Metschnikowia pulcherrima active against postharvest pathogens on apples. Postharvest Biol Tec. 2002; 24(2): 123-134.
40. Hua L, Yong C, Zhanquan Z, Boqiang L, Guozheng Q, Shiping T. Pathogenic mechanisms and control strategies of Botrytis cinerea causing post-harvest decay in fruits and vegetables. Food Quality Safety. 2018; 2(3): 1-9.
41. Bending GD, Turner MK, Jones JE. Interactions between crop residue and soil organic matter quality and the functional diversity of soil microbial communities. Soil Biol Biochem. 2002; 34: 1073-1082.
42. García-Martos P, Marín P, Hernández-Molina JM, García-Agudo L, Aoufi S, Mira J. Extracellular enzymatic activity in 11 Cryptococcus species. Mycopathologia. 2001; 150(1): 1-4.
43. Baldrian P, Kolařík M, Stursová M, Kopecký J, Valášková V, Větrovský T, Zifčáková L, Snajdr J, Rídl J, Vlček C, Voříšková J. Active and total microbial communities in forest soil are largely different and highly stratified during decomposition. The ISME J. 2012; 6(2): 248-258.
44. Fleet GH, Charoenchai C, Henschke PA, Todd BEN. Screening of non-Saccharomyces wine yeasts for the presence of extracellular hydrolytic enzymes. Aust J Grape Wine Res. 1997; 3(1): 2-8.
45. Dodor DE, Tabatabai MA. Effects of cropping systems and microbial biomass on arylamidase activity in soils. Biol Fert Soils. 2002; 35: 253-261.
46. Nikolaou E, Soufleros EH, Bouloumpasi E, Tzanetakis N. Selection of indigenous Saccharomyces cerevisiae strains according to their oenological characteristics and vinification results. Food Microbiol. 2006; 23(2): 205-211.
47. Nikolaou A, Meriç S, Fatta D. Occurrence patterns of pharmaceuticals in water and wastewater environments. Anal Bioanal Chem. 2007; 387: 1225-1234.
48. Guetsky R, Elad Y, Stienberg D, Dinoor A. Establishment, survival and activity of the biocontrol agents Pichia guilliermondii and Bacillus mycoides applied as a mixture on strawberry plants. Biocontrol Sci Tech. 2002; 12: 705-714.
49. Wszelaki AL, Mitcham EJ. Effect of combinations of hot water dips, biological control and controlled atmospheres for control of gray mold on harvested strawberries. Postharvest Biol Tec. 2003; 27: 255-264.
50. Zhang HY, Wang L, Dong Y, Jiang S, Cao H, Meng RJ. Postharvest biological control of gray mold decay of strawberry with Rhodotorula glutinis. Biol Control. 2007; 40: 287-292.
How to Cite
Turgut Genç, T.; Günay, M. Diversity and Extracellular Enzyme Profiles of Yeasts on Organic and Fungicide Treated Strawberries. European Journal of Biological Research 2021, 11, 480-492.
Research Articles