Abstract
Grapevine cultivars are distributed worldwide, nevertheless the fermentation of its grape berries renders distinct wine products that are highly associated to the local fungal community. Despite the symbiotic association between wine and the fungal metabolism, impacting both the terroir and mycotoxin production, few studies have explored the vineyard ecosystem fungal community using both molecular marker sequencing and mycotoxin production assessment. In this study, we investigated the fungal community of three grapevine cultivars (Vitis vinifera L.) in two tropical vineyards. Illumina MiSeq sequencing was performed on two biocompartments: grape berries (GB) and grapevine soil (GS); yielding a total of 578,495 fungal internal transcribed spacer 1 reads, which were used for taxonomic classification. GB and GS fungal communities were mainly constituted by Ascomycota phylum. GS harbors a significant richer and more diverse fungal community than GB. Among GB samples, Syrah grape berries exclusively shared fungal community included wine-associated yeasts (e.g. Saccharomycopsis vini) that may play key roles in wine terroir. Mycotoxin production assessment revealed the high potential of Aspergillus section Flavi and Penicillium section Citrina isolates to produce aflatoxin B1-B2 and citrinin, respectively. This is the first study to employ next-generation sequencing to investigate vineyard associated fungal community in Brazil. Our findings provide valuable insights on the available tools for fungal ecology assessment applied to food products emphasizing the coexistence between classical and molecular tools.
Similar content being viewed by others
Availability of data and materials
Raw data available at NCBI SRA. Bioproject PRJNA686129.
References
Albergaria H, Arneborg N (2016) Dominance of Saccharomyces cerevisiae in alcoholic fermentation processes: role of physiological fitness and microbial interactions. Appl Microbiol Biotechnol 100:2035–2046. https://doi.org/10.1007/s00253-015-7255-0
Anagnostopoulos DA, Kamilari E, Tsaltas D (2019) Contribution of the microbiome as a tool for estimating wine’s fermentation output and authentication. In: Morata A (ed) Advances in grape and wine biotechnology. IntechOpen, London
Asemaninejad A, Weerasuriya N, Gloor GB, Lindo Z, Thorn RG (2016) New primers for discovering fungal diversity using nuclear large ribosomal DNA. PLoS ONE 11:e0159043. https://doi.org/10.1371/journal.pone.0159043
Banos S, Lentendu G, Kopf A, Wubet T, Glockner FO, Reich M (2018) A comprehensive fungi-specific 18S rRNA gene sequence primer toolkit suited for diverse research issues and sequencing platforms. BMC Microbiol 18:190. https://doi.org/10.1186/s12866-018-1331-4
Belda I, Ruiz J, Esteban-Fernandez A, Navascues E, Marquina D, Santos A, Moreno-Arribas MV (2017a) Microbial contribution to wine aroma and Its intended use for wine quality improvement. Molecules 22(2):189. https://doi.org/10.3390/molecules22020189
Belda I, Zarraonaindia I, Perisin M, Palacios A, Acedo A (2017b) From vineyard soil to wine fermentation: microbiome approximations to explain the “terroir” concept. Front Microbiol 8:821. https://doi.org/10.3389/fmicb.2017.00821
Bokulich NA, Thorngate JH, Richardson PM, Mills DA (2014) Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate. Proc Natl Acad Sci USA 111:E139-148. https://doi.org/10.1073/pnas.1317377110
Bokulich NA, Collins TS, Masarweh C, Allen G, Heymann H, Ebeler SE, Mills DA (2016) Associations among wine grape microbiome, metabolome, and fermentation behavior suggest microbial contribution to regional wine characteristics. Mbio. https://doi.org/10.1128/mBio.00631-16
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. https://doi.org/10.1093/bioinformatics/btu170
Bougreau M, Ascencio K, Bugarel M, Nightingale K, Loneragan G (2019) Yeast species isolated from Texas High Plains vineyards and dynamics during spontaneous fermentations of Tempranillo grapes. PLoS ONE 14:e0216246. https://doi.org/10.1371/journal.pone.0216246
Chou MY, Vanden Heuvel J, Bell TH, Panke-Buisse K, Kao-Kniffin J (2018) Vineyard under-vine floor management alters soil microbial composition, while the fruit microbiome shows no corresponding shifts. Sci Rep 8:11039. https://doi.org/10.1038/s41598-018-29346-1
Coggins S, Malone BP, Stockmann U, Possell M, McBratney AB (2019) Towards meaningful geographical indications: validating terroirs on a 200 km2 scale in Australia’s lower Hunter Valley. Geoderma Reg 16:e00209. https://doi.org/10.1016/j.geodrs.2019.e00209
Coller E, Cestaro A, Zanzotti R, Bertoldi D, Pindo M, Larger S, Albanese D, Mescalchin E, Donati C (2019) Microbiome of vineyard soils is shaped by geography and management. Microbiome 7:140. https://doi.org/10.1186/s40168-019-0758-7
Creer S, Deiner K, Frey S, Porazinska D, Taberlet P, Thomas WK, Potter C, Bik HM (2016) The ecologist’s field guide to sequence-based identification of biodiversity. Methods Ecol Evol 7:1008–1018. https://doi.org/10.1111/2041-210X.12574
De Filippis F, Laiola M, Blaiotta G, Ercolini D (2017) Different amplicon targets for sequencing-based studies of fungal diversity. Appl Environ Microbiol. https://doi.org/10.1128/AEM.00905-17
de Vries J, Archibald JM (2018) Plant evolution: landmarks on the path to terrestrial life. New Phytol 217:1428–1434. https://doi.org/10.1111/nph.14975
Delgado-Baquerizo M (2019) Obscure soil microbes and where to find them. ISME J 13:2120–2124. https://doi.org/10.1038/s41396-019-0405-0
Dimakopoulou M, Sotirios ET, Polymnia PA, Pietri A, Battilani P, Avramidis N, Markakis EA, Tjamos EC (2008) Phyllosphere grapevine yeast Aureobasidium pullulans reduces Aspergillus carbonarius (sour rot) incidence in wine-producing vineyards in Greece. Biol Control 46:158–165. https://doi.org/10.1016/j.biocontrol.2008.04.015
Drumonde-Neves J, Franco-Duarte R, Lima T, Schuller D, Pais C (2016) Yeast biodiversity in vineyard environments is increased by human intervention. PLoS ONE 11:e0160579. https://doi.org/10.1371/journal.pone.0160579
Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26:2460–2461. https://doi.org/10.1093/bioinformatics/btq461
Einloft TC, Hoeltz M, Teixeira TR, Oldoni VP, Manfroi V, Noll IB (2017) Survey of mycobiota, black Aspergillus and ochratoxin A occurrence on Brazilian wine grapes. Ann Microbiol 67:59–64. https://doi.org/10.1007/s13213-016-1236-0
El Khoury A, Rizk T, Lteif R, Azouri H, Delia ML, Lebrihi A (2008) Fungal contamination and Aflatoxin B1 and Ochratoxin A in Lebanese wine-grapes and musts. Food Chem Toxicol 46:2244–2250. https://doi.org/10.1016/j.fct.2008.02.026
Embrapa Solos (2006) Solos do Nordeste brasileiro. Available at: http://solos.uep.cnps.embrapa.br/. Accessed 20 Jan 2021
Englezos V, Giacosa S, Rantsiou K, Rolle L, Cocolin L (2017) Starmerella bacillaris in winemaking: opportunities and risks. Curr Opin Food Sci 17:30–35. https://doi.org/10.1016/j.cofs.2017.08.007
Ferreira AM, Mendes-Faia A (2020) The role of yeasts and lactic acid bacteria on the metabolism of organic acids during winemaking. Foods 9(9):1231. https://doi.org/10.3390/foods9091231
Filtenborg O, Frisvad JC (1980) A simple screening-method for toxigenic moulds in pure cultures. LWT Food Sci Technol 13:128–130
Fredj SMB, Chebil S, Mliki A (2009) Isolation and characterization of ochratoxin A and aflatoxin B1 producing fungi infecting grapevines cultivated in Tunisia. Afr J Microbiol Res 3:523–527
Freire L, Passamani FRF, Thomas AB, Nassur RCMR, Silva LM, Paschoal FN, Pereira GE, Prado G, Batista LR (2017) Influence of physical and chemical characteristics of wine grapes on the incidence of Penicillium and Aspergillus fungi in grapes and ochratoxin A in wines. Int J Food Microbiol 241:181–190. https://doi.org/10.1016/j.ijfoodmicro.2016.10.027
Gadd GM (2006) Fungi in biogeochemical cycles, vol 24. Cambridge University Press, Cambridge
Gamero A, Quintilla R, Groenewald M, Alkema W, Boekhout T, Hazelwood L (2016) High-throughput screening of a large collection of non-conventional yeasts reveals their potential for aroma formation in food fermentation. Food Microbiol 60:147–159. https://doi.org/10.1016/j.fm.2016.07.006
Hall ME, O’Bryon I, Wilcox WF, Osier MV, Cadle-Davidson L (2019) The epiphytic microbiota of sour rot-affected grapes differs minimally from that of healthy grapes, indicating causal organisms are already present on healthy berries. PLoS ONE 14:e0211378. https://doi.org/10.1371/journal.pone.0211378
Hocking AD, Leong SL, Kazi BA, Emmett RW, Scott ES (2007) Fungi and mycotoxins in vineyards and grape products. Int J Food Microbiol 119:84–88. https://doi.org/10.1016/j.ijfoodmicro.2007.07.031
Houbraken J, Frisvad JC, Samson RA (2011) Taxonomy of Penicillium section Citrina. Stud Mycol 70:53–138. https://doi.org/10.3114/sim.2011.70.02
Kachalkin AV, Abdullabekova DA, Magomedova ES, Magomedov GG, Chernov IY (2015) Yeasts of the vineyards in Dagestan and other regions. Mikrobiologiia 84:360–368. https://doi.org/10.1134/S002626171503008X
Knight S, Klaere S, Fedrizzi B, Goddard MR (2015) Regional microbial signatures positively correlate with differential wine phenotypes: evidence for a microbial aspect to terroir. Sci Rep 5:14233. https://doi.org/10.1038/srep14233
Knight SJ, Karon O, Goddard MR (2020) Small scale fungal community differentiation in a vineyard system. Food Microbiol 87:103358. https://doi.org/10.1016/j.fm.2019.103358
Köberl M, Wagner P, Muller H, Matzer R, Unterfrauner H, Cernava T, Berg G (2020) Unraveling the complexity of soil microbiomes in a large-scale study subjected to different agricultural management in Styria. Front Microbiol 11:1052. https://doi.org/10.3389/fmicb.2020.01052
Kok D (2014) A review on grape growing in tropical regions. Turk J Agric Nat Sci 1:1236–1241
Kraková L, Chovanová K, Ženišová K, Turcovská V, Brežná B, Kuchta T, Pangallo D (2012) Yeast diversity investigation of wine-related samples from two different Slovakian wine-producing areas through a multistep procedure. LWT Food Sci Technol 46:406–411. https://doi.org/10.1016/j.lwt.2011.12.010
Loureiro V, Malfeito-Ferreira M (2003) Spoilage yeasts in the wine industry. Int J Food Microbiol 86:23–50. https://doi.org/10.1016/s0168-1605(03)00246-0
Martin FM, Uroz S, Barker DG (2017) Ancestral alliances: plant mutualistic symbioses with fungi and bacteria. Science. https://doi.org/10.1126/science.aad4501
Martinez-Ainsworth NE, Tenaillon MI (2016) Superheroes and masterminds of plant domestication. C R Biol 339:268–273. https://doi.org/10.1016/j.crvi.2016.05.005
McMurdie PJ, Holmes S (2013) phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE 8:e61217. https://doi.org/10.1371/journal.pone.0061217
Medina A, Rodríguez A, Sultan Y, Magan N (2015) Climate change factors and Aspergillus flavus: effects on gene expression, growth and aflatoxin production. World Mycotoxin J 8:171–179. https://doi.org/10.3920/WMJ2014.1726
Mezzasalma V, Sandionigi A, Guzzetti L, Galimberti A, Grando MS, Tardaguila J, Labra M (2018) Geographical and cultivar features differentiate grape microbiota in northern Italy and Spain vineyards. Front Microbiol 9:946. https://doi.org/10.3389/fmicb.2018.00946
Morgan HH, du Toit M, Setati ME (2017) The grapevine and wine microbiome: insights from high-throughput amplicon sequencing. Front Microbiol 8:820. https://doi.org/10.3389/fmicb.2017.00820
Nilsson RH, Larsson KH, Taylor AFS, Bengtsson-Palme J, Jeppesen TS, Schigel D, Kennedy P, Picard K, Glöckner FO, Tedersoo L, Saar I, Kõljalg U, Abarenkov K (2019) The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classifications. Nucleic Acids Res 47:D259–D264. https://doi.org/10.1093/nar/gky1022
Nisiotou A, Mallouchos A, Tassou C, Banilas G (2019) Indigenous yeast interactions in dual-starter fermentations may improve the varietal expression of moschofilero wine. Front Microbiol 10:1712. https://doi.org/10.3389/fmicb.2019.01712
Oksanen J, Blanchet FG, Friendly M, Kindt R, Legendre P, McGlinn D, Minchin PR, O'Hara RB, Simpson GL, Solymos P, Stevens MHH, Szoecs E, Wagner H (2019) Vegan: community ecology package. R package version 2.5.6. http://CRAN.R-project.org/package=vegan
Palmer JM, Jusino MA, Banik MT, Lindner DL (2018) Non-biological synthetic spike-in controls and the AMPtk software pipeline improve mycobiome data. PeerJ 6:e4925. https://doi.org/10.7717/peerj.4925
Paterson RRM, Venancio A, Lima N, Guilloux-Benatier M, Rousseaux S (2018) Predominant mycotoxins, mycotoxigenic fungi and climate change related to wine. Food Res Int 103:478–491. https://doi.org/10.1016/j.foodres.2017.09.080
Perazzolli M, Antonielli L, Storari M, Puopolo G, Pancher M, Giovannini O, Pindo M, Pertot I (2014) Resilience of the natural phyllosphere microbiota of the grapevine to chemical and biological pesticides. Appl Environ Microbiol 80:3585–3596. https://doi.org/10.1128/AEM.00415-14
Perez-Jaramillo JE, Carrión VJ, Bosse M, Ferrão LFV, Hollander M, Garcia AAF, Ramírez CA, Mendes R, Raaijmakers JM (2017) Linking rhizosphere microbiome composition of wild and domesticated Phaseolus vulgaris to genotypic and root phenotypic traits. ISME J 11:2244–2257. https://doi.org/10.1038/ismej.2017.85
Pitt JI, Hocking AD (2009) Fungi and food spoilage. Springer, Boston
Qi TF, Renaud JB, McDowell T, Seifert KA, Yeung KK, Sumarah MW (2016) Diversity of mycotoxin-producing black aspergilli in Canadian vineyards. J Agric Food Chem 64:1583–1589. https://doi.org/10.1021/acs.jafc.5b05584
R Core Team (2017) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/
Rognes T, Flouri T, Nichols B, Quince C, Mahe F (2016) VSEARCH: a versatile open source tool for metagenomics. PeerJ 4:e2584. https://doi.org/10.7717/peerj.2584
Rosa CA, Lachance MA (1998) The yeast genus Starmerella gen. nov. and Starmerella bombicola sp. nov., the teleomorph of Candida bombicola (Spencer, Gorin & Tullock) Meyer & Yarrow. Int J Syst Bacteriol 48(Pt 4):1413–1417. https://doi.org/10.1099/00207713-48-4-1413
Rosa CA, Palacios V, Combina M, Fraga ME, De OR, Magnoli CE, Dalcero AM (2002) Potential ochratoxin A producers from wine grapes in Argentina and Brazil. Food Addit Contam 19:408–414. https://doi.org/10.1080/02652030110092748
Roudil L, Russo P, Berbegal C, Albertin W, Spano G, Capozzi V (2019) Non-Saccharomyces commercial starter cultures: scientific trends, recent patents and innovation in the wine sector. Recent Pat Food Nutr Agric. https://doi.org/10.2174/2212798410666190131103713
Samson RA, Houbraken J, Thrane U, Frisvad JC, Andersen B (2010) Food and indoor fungi. CBS-Fungal Biodiversity Centre, Utrecht
Selosse MA, Strullu-Derrien C, Martin FM, Kamoun S, Kenrick P (2015) Plants, fungi and oomycetes: a 400-million year affair that shapes the biosphere. New Phytol 206:501–506. https://doi.org/10.1111/nph.13371
Serra R, Lourenco A, Alipio P, Venancio A (2006) Influence of the region of origin on the mycobiota of grapes with emphasis on Aspergillus and Penicillium species. Mycol Res 110:971–978. https://doi.org/10.1016/j.mycres.2006.05.010
Shelest E, Voigt K (2014) Genomics to study basal lineage fungal biology: phylogenomics suggests a common origin. In: Esser K (ed) Fungal genomics, 2nd edn. Springer, Berlin, pp 31–54
Teixeira A, Scherer-Warren M, Hernandez F, Andrade R, Leivas J (2013) Large-scale water productivity assessments with MODIS images in a changing semi-arid environment: a Brazilian case study. Remote Sens 5:5783–5804. https://doi.org/10.3390/rs5115783
Varga J, Frisvad JC, Kocsube S, Brankovics B, Toth B, Szigeti G, Samson RA (2011) New and revisited species in Aspergillus section Nigri. Stud Mycol 69:1–17. https://doi.org/10.3114/sim.2011.69.01
Vitulo N, Lemos WJF Jr, Calgaro M, Confalone M, Felis GE, Zapparoli G, Nardi T (2018) Bark and grape microbiome of Vitis vinifera: influence of geographic patterns and agronomic management on bacterial diversity. Front Microbiol 9:3203. https://doi.org/10.3389/fmicb.2018.03203
Wang C, Garcia-Fernandez D, Mas A, Esteve-Zarzoso B (2015) Fungal diversity in grape must and wine fermentation assessed by massive sequencing, quantitative PCR and DGGE. Front Microbiol 6:1156. https://doi.org/10.3389/fmicb.2015.01156
Wani ZA, Kumar A, Sultan P, Bindu K, Riyaz-Ul-Hassan S, Ashraf N (2017) Mortierella alpina CS10E4, an oleaginous fungal endophyte of Crocus sativus L. enhances apocarotenoid biosynthesis and stress tolerance in the host plant. Sci Rep 7:8598. https://doi.org/10.1038/s41598-017-08974-z
Wei YJ, Wu Y, Yan YZ, Zou W, Xue J, Ma WR, Wang W, Tian G, Wang LY (2018) High-throughput sequencing of microbial community diversity in soil, grapes, leaves, grape juice and wine of grapevine from China. PLoS ONE 13:e0193097. https://doi.org/10.1371/journal.pone.0193097
Willis AD (2019) Rarefaction, alpha diversity, and statistics. Front Microbiol 10:2407. https://doi.org/10.3389/fmicb.2019.02407
Wilson K (2001) Preparation of genomic DNA from bacteria. Curr Protoc Mol Biol. https://doi.org/10.1002/0471142727.mb0204s56
Xun W, Huang T, Zhao J, Ran W, Wang B, Shen Q, Zhang R (2015) Environmental conditions rather than microbial inoculum composition determine the bacterial composition, microbial biomass and enzymatic activity of reconstructed soil microbial communities. Soil Biol Biochem 90:10–18. https://doi.org/10.1016/j.soilbio.2015.07.018
Zarraonaindia I, Owens SM, Weisenhorn P et al (2015) The soil microbiome influences grapevine-associated microbiota. Mbio. https://doi.org/10.1128/mBio.02527-14
Acknowledgements
We acknowledge the grape producers who allowed the grape and soil collection on their properties, we also thank CAPES and EMBRAPA Semiárido for the financial support.
Funding
Not applicable.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Dutra-Silva, L., Pereira, G.E., Batista, L.R. et al. Fungal diversity and occurrence of mycotoxin producing fungi in tropical vineyards. World J Microbiol Biotechnol 37, 112 (2021). https://doi.org/10.1007/s11274-021-03081-8
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11274-021-03081-8