Main Article Content



Commercial grape production in Washington State, USA relies on the use of fungicides to combat grape pathogens. However, increasing incidence of fungicide resistance is of concern to the wine industry and members of the public. Here, we extend previous studies on the biocontrol potential of native Washington yeasts from Central Washington to yeasts and Botrytis cinerea from the Puget Sound, where Botrytis bunch rot is endemic. Field isolates of native yeasts and B. cinerea were collected from diseased grapes from two vineyards in 2017 and 2018. Multi-locus sequence analysis showed that the Botrytis isolates were related to each other and to B. cinerea accessions in GenBank, but one isolate was more closely related to B. eucalypti. For dual plate inhibition assays, five B. cinerea isolates were selected from different grape cultivars. Ten native yeasts, isolated from the same cultivars, were selected for their ability to inhibit B. cinerea in preliminary screens. The yeasts were identified as Candida californica, Hanseniaspora uvarum (5 isolates), Pichia kluyveri (2 isolates) and Metschnikowia pulcherrima based on sequences of the D1-D3 regions of the nuclear large ribosomal RNA gene. These nine yeasts inhibited up to three of the five pathogen isolates, and inhibition was not identical for the five H. uvarum strains. Furthermore, the Botrytis isolates showed differential sensitivity to the panel of yeasts. The last yeast inhibited all pathogen isolates and resembled Aureobasidium pullulans in morphology and inhibition pattern. As in the previous study, which focused on Mt. pulcherrima and Mt. chrysoperlae, in vitro inhibition was found to be dependent on the genotypes of both the native yeast strain and Botrytis isolate. The present findings indicated that native yeasts can exert inhibition on bunch rot pathogens co-occurring at the same locality, and the development of biological control needs to account for genotypic differences in yeasts and pathogens.

Botrytis cinerea, native yeasts, Candida californica, Hanseniaspora uvarum, Pichia kluyveri, MLSA

Article Details

How to Cite
NEVADA, S. S., LUPIEN, S. L., WATSON, B., & OKUBARA, P. A. (2021). GROWTH INHIBITION OF Botrytis cinerea BY NATIVE VINEYARD YEASTS FROM PUGET SOUND, WASHINGTON STATE, USA. Journal of Biology and Nature, 13(1), 42-53. Retrieved from
Short Research Articles


González-Domínguez E, Caffi T, Ciliberti N, Rossi V. A mechanistic model of Botrytis cinerea on grapevines that includes weather, vine growth stage, and the main infection pathways. PLoS ONE. 2015;10: e0140444.
DOI: 10.1371/journal.pone.0140444

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;3:111-119.
DOI: 10.1093/fqsafe/fyy016

De Simone N, Pace B, Grieco F, Chimienti M, Tyibilika V, Santoro V, et al. Botrytis cinerea and table grapes: A review of the main physical, chemical, and bio-based control treatments in post-harvest. Foods. 2020; 9:1138.
DOI: 10.3390/foods9091138

Moyer MM, Grove GG. Botrytis bunch rot in commercial Washington grape production: Biology and disease management. WSU Extension Publication #FS046e. Washington State University, Pullman, WA; 2011.

Dugan FM, Lupien SL, Grove GG. Incidence, aggressiveness and in planta interactions of Botrytis cinerea and other filamentous fungi quiescent in grape berries and dormant buds in Central Washington State. J. Phytopathol. 2002;150:375–381.

Ky I, Lorrain B, Jourdes M, Pasquier G, Fermaud M, Gény L, et al. Assessment of grey mould (Botrytis cinerea) impact on phenolic and sensory quality of Bordeaux grapes, musts and wines for two consecutive vintages. Aust. J. Grape Wine Res. 2012;18:215-226.
DOI: 10.1111/j.1755-0238.2012.00191.x

Sanzani SM, Schena L, De Cicco V, Ippolito A. Early detection of Botrytis cinerea latent infections as a tool to improve postharvest quality of table grapes. Postharvest Biol. Tech. 2012;68:64-71.
DOI: 10.1016/j.postharvbio.2012.02.003

Kosel J, Raspor P, Cadež N. Maximum residue limit of fungicides inhibits the viability and growth of desirable non-Saccharomyces wine yeasts. Aust. J. Grape Wine Res. 2019,2018; 25:43–52.
DOI: 10.1111/ajgw.12364

Fernández-Ortuño D, Grabke A, Li X, Schnabel G. Independent emergence of resistance to seven chemical classes of fungicides in Botrytis cinerea. Phytopathology. 2015;105:424-432.

Saito S, Michailides TJ, Xiao CL. Fungicide-resistant phenotypes in Botrytis cinerea populations and their impact on control of gray mold on stored table grapes in California. Eur. J. Plant Pathol. 2019;154:203–213.

Calvo-Garrido C, Roudet J, Aveline N, Davidou L, Dupin S, Fermaud M. Microbial antagonism toward Botrytis bunch rot of grapes in multiple field tests using one Bacillus ginsengihumi strain and formulated biological control products. Front. Plant Sci. 2019;10:105.
DOI: 10.3389/fpls.2019.00105

Garrido CC, Usall J, Torres R, Teixido N. Effective control of Botrytis bunch rot in commercial vineyards by large-scale application of Candida sake CPA-1 Bio Control. 2017;62:161–173.
DOI: 10.1007/s10526-017-9789-9

Ferreira-Saab M, Formey D, Torres M, Aragón W, Padilla EA, Tromas A, et al. Compounds released by the biocontrol yeast Hanseniaspora opuntiae protect plants against Corynespora cassiicola and Botrytis cinerea. Front. Microbiol. 2018;9:1596.
DOI: 10.3389/fmicb.2018.01596

Freimoser FM, Rueda‑Mejia MP, Tilocca B, Migheli Q. Biocontrol yeasts: mechanisms and applications. World J. Microbiol. Biotech. 2019;35:154.

Parafati L, Vitale A, Restuccia C, Cirvilleri G. Biocontrol ability and action mechanism of food-isolated yeast strains against Botrytis cinerea causing post-harvest bunch rot of table grape. Food Microbiol. 2015;47:85-92.
DOI: 10.1016/

Eder MLR, Conti F, Rosa AL. Differences between indigenous yeast populations in spontaneously fermenting musts from V. vinifera L. and V. labrusca L. grapes harvested in the same geographic location. Front Microbiol. 2018;9:1320.

Chanchaichaovivat A, Ruenwongsa P, Panijpan B. Screening and identification of yeast strains from fruits and vegetables: Potential for biological control of postharvest chilli anthracnose (Colletotrichum capsici). Biol. Cont. 2007;42:326-335.

Wang X, Glawe DA, Kramer E, Weller D, Okubara PA. Biological control of Botrytis cinerea: interactions with native vineyard yeasts from Washington State. Phytopathology. 2018;108:691-701.

Spadaro D, Droby S. Development of biocontrol products for postharvest diseases of fruit: The importance of elucidating the mechanisms of action of yeast antagonists. Trends Food Sci. Technol. 2016;47:39-49.

Bourret TB, Grove GG, Vandemark GJ, Henick-Kling T, Glawe DA. Diversity and molecular determination of wild yeasts in a central Washington State vineyard. N. Am. Fungi. 2013;8:1.

Hiruma K, Saijo Y. Methods for long-term stable storage of Colletotrichum species, in: Duque, P. (Ed.), Environmental Responses, in: Plants. Methods in Molecular Biology, Humana Press, New York. 2016;1398:309-312.

White TJ, Bruns T, Lee SJWT, Taylor JW. 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. Academic Press, Massachusetts. 1990;315–322.

Okubara PA, Schroeder KL, Paulitz TC. Identification and quantification of Rhizoctonia solani and R. oryzae using real-time polymerase chain reaction. Phytopathology. 2008;98:837-847.
DOI: 10.1094/PHYTO-98-7-0837

Tedersoo L, Anslan S, Bahram M, Põlme S, Riit T, Liiv I, et al. Shotgun metagenomes and multiple primer pair-barcode combinations of amplicons reveal biases in metabarcoding analyses of fungi. MycoKeys. 2015;10:1–43.
DOI: 10.3897/mycokeys.10.4852

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res. 1997;25:3389-3402.

Sievers F, Higgins DG. Clustal Omega, accurate alignment of very large numbers of sequences. Methods Mol. Biol. 2014;1079:105-116.
DOI: 10.1007/978-1-62703-646-7_6

Staats M, van Baarlen P, van Kan JAL. Molecular phylogeny of the plant pathogenic genus Botrytis and the evolution of host specificity. Mol. Biol. Evol. 2005;22:333-346.
DOI: 10.1093/molbev/msi020

Zhou YJ, Zhang J, Wang XD, Yang L, Jiang DH, Li GQ, et al. Morphological and phylogenetic identification of Botrytis sinoviticola, a novel cryptic species causing gray mold disease of table grapes (Vitis vinifera) in China. Mycologia. 2014;106:43-56.
DOI: 10.3852/13-032

Garfinkel AR, Coats KP, Sherry DL, Chastagner GA. Genetic analysis reveals unprecedented diversity of a globally-important plant pathogenic genus. Sci. Rep. 2019;9:6671.
Available: 1

Felsenstein J. PHYLIP -- Phylogeny Inference Package (Version 3.2). Cladistics. 1989;5:164-166.

Page RDM. TreeView: An application to display phylogenetic trees on personal computers. Bioinformatics. 1996;12:357-358.

Darriba D, Taboada GL, Doallo R, Posada D. ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics. 2011;27: 1164-1165.
DOI: 10.1093/bioinformatics/btr088

Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 2012;61:539-542.
DOI: 10.1093/sysbio/sys029

Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28:1647-1649.
DOI: 10.1093/bioinformatics/bts199

Mavrodi OV, Walter N, Elateek S, Taylor CG, Okubara PA. Suppression of Rhizoctonia and Pythium root rot of wheat by new strains of Pseudomonas. Biol. Cont. 2012;62:93–102.

Gostinčar C, Ohm RA, Kogej T, Sonjak S, Turk M, Zajc J, et al. Genome sequencing of four Aureobasidium pullulans varieties: biotechnological potential, stress tolerance, and description of new species. BMC Genomics. 2014;5:549.

Liu QL, Li GQ, Li JQ, Chen SF. Botrytis eucalypti, a novel species isolated from diseased Eucalyptus seedlings in South China. Mycol. Progress. 2016;15:1057–1079.
DOI: 10.1007/s11557-016-1229-1

Rabosto X, Carrau M, Paz A, Boido E, Dellacassa E, Carrau F. Grapes and vineyard soils as sources of microorganisms for biological control of Botrytis cinerea. Am. J. Enol. Vit. 2006;57:332-338.

Jolly NP, Varela C, Pretorius IS. Not your ordinary yeast: non-Saccharomyces yeasts in wine production uncovered. FEMS Yeast Res. 2014;14:215–237.
DOI: 10.1111/1567-1364.12111

Liu HM, Guo JH, Luo L, Liu P, Wang BQ, Cheng YJ, et al. Improvement of Hanseniaspora uvarum biocontrol activity against gray mold by the addition of ammonium molybdate and the possible mechanisms involved. Crop Prot. 2010;29:277–282.
DOI: 10.1016/j.cropro.2009.10.020

Pretscher J, Fischkal T, Branscheidt S, Jäger L, Kahl S, Schlander M, et al. Yeasts from different habitats and their potential as biocontrol agents. Fermentation. 2018;4:31.
DOI: 10.3390/fermentation4020031

Mewa-Ngongang M, du Plessis HW, Ntwampe SKO, Chidi BS, Hutchinson UF, Mekuto L, et al. The use of Candida pyralidae and Pichia kluyveri to control spoilage microorganisms of raw fruits used for beverage production. Foods. 2019; 8:454.

DOI: 10.3390/foods8100454

Fedele G, Brischetto C, Rossi V. Biocontrol of Botrytis cinerea on grape berries as influenced by temperature and humidity. Front. Plant Sci. 2020;11:1232.
DOI: 10.3389/fpls.2020.01232

Bardin M, Leyronas C, Troulet C, Morris CE. Striking similarities between Botrytis cinerea from non-agricultural and from agricultural habitats. Front. Plant Sci. 2018;9:1820.
DOI: 10.3389/fpls.2018.01820

Bozoudi D, Tsaltas D. The multiple and versatile roles of Aureobasidium pullulans in the vitivinicultural sector. Fermentation. 2018;4:85.
DOI: 10.3390/fermentation4040085

Don SMY, Schmidtke LM, Gambetta JM, Steel CC. Aureobasidium pullulans volatilome identified by a novel, quantitative approach employing SPME-GC-MS, suppressed Botrytis cinerea and Alternaria alternata in vitro. Sci. Rep. 2020;10:4498.