from a Vineyard in Attica, Greece ACCEPTED Running title: YEAST POPULATIONS IN HEALTHY OR BOTRYTISED GRAPES

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1 AEM Accepts, published online ahead of print on February 00 Appl. Environ. Microbiol. doi:./aem.01-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. On Yeast Populations Residing Healthy or Botrytis-Infected Grapes from a Vineyard in Attica, Greece Running title: YEAST POPULATIONS IN HEALTHY OR BOTRYTISED GRAPES Aspasia A. Nisiotou, and George-John E. Nychas* Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Technology, Agricultural University of Athens, Iera Odos,, Athens, Greece *Corresponding author. Mailing address: Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Technology, Agricultural University of Athens, Iera Odos,, Athens, Greece. Phone/Fax: gjn@aua.gr. Key words; Food microbiology, PCR- RFLP, grapes, yeasts, ecology, wine, botrytis 1

2 ABSTRACT Yeast flora associated with healthy and Botrytis-infected grapes was assessed. Molecular identification methods assigned isolates into genera and species. For the first time H. opuntiae was encountered as inhabitant of grape ecosystem. By using DraI, an informative RFLP pattern was generated to distinguish H. opuntiae from its closely related H. guilliermondii. Botrytis infection encouraged an increased population and diversity of yeasts enriched with fermentative or spoilage species

3 Grape berries are common niches for yeasts, especially the interface between soluble nutrients and the septic world. From a biotechnological point of view, they constitute the primordial source of microorganisms for the alcoholic fermentation to occur, providing must with both beneficial and potentially spoilage species. Nevertheless, yeast flora of grapes is surprisingly poorly documented (1, 1). As shown so far, the physiognomy of grape microflora may change in response to factors such as climate variability, grape variety and geographical region (, 1, 1). Biological invasions are of critical concern for widespread community changes. Botrytis is among the most important pathogens that cause grape damage (grey rot) or drying (noble rot), yet its role in yeast ecology has not been studied hitherto. We assessed and compared yeasts present on Botrytis-affected and healthy grapes. For species identification different molecular methods were applied and their robustness is discussed. Grape samples were collected at harvesting (00 vintage) from the experimental vineyard of the Agricultural University of Athens ( ο ' Ν, ο ' Ε, 0m above sea level). Grapevines were treated with ground sulphur rock during spring and no other chemicals were applied thereafter. Mavroliatis and Sefka, two red Vitis vinifera varieties, were included in the analysis. Vines of each variety were cultivated in parallel single rows, 0 m long and 0 m apart. Healthy or rotten bunches for each variety were randomly and aseptically collected from throughout the cultivation rows. The plate-trapped antigen ELISA was applied to confirm Botrytis infection of rotten grapes using the monoclonal antibody BC-1.CA (1) as described (). One hundred grams of randomly collected berries from each sample were aseptically crushed in a Stomacher (Lab Blender 00) and the ph of juice was recorded. D-glucose/D-fructose and ethanol contents were determined by respective enzymatic kits (Boehringer

4 Mannheim/R-Biopharm, Germany). Successive decimal dilutions ( ) in Ringer s solution were prepared and 0 µl were spread on different culture media. For the enumeration and isolation of total yeasts, non-saccharomyces yeasts, Saccharomyces and Dekkera/Brettanomyces spp., samples were spread in triplicate on Wallerstein laboratory nutrient agar (WLNA) (Oxoid LTD), lysine medium agar (LA) (Oxoid LTD), ethanol sulfite agar (ESA) () and Dekkera/Brettanomyces differential medium (DBDMA) (0), respectively, supplemented with mg/l biphenyl and 0 mg/l chloramphenicol (Sigma). Twenty to thirty-five isolates were randomly selected from plates with 0- total colonies and stored at -0 o C until further analysis. Genomic DNA was isolated as described (1). The.S-ITS rrna region and D1/D domain of S rrna gene were PCR amplified using the primer-pairs ITS1/ITS () and NL1/NL (1), respectively, as described (1, 1). For.S-ITS RFLP the restriction endonucleases HinfI, HaeIII, HhaI, DraI (Taqara, Japan) or DdeI (New England Biolabs) were used. Fragments were separated by agarose (%) electrophoresis and detected by ethidium bromide. PCR products of the.s-its or D1/D domain of 1- randomly selected isolates per distinct RFLP pattern were gel- purified (QIAquick PCR Purification Kit, Qiagen) and both DNA strands were directly sequenced (Macrogen, Blast searches were performed at the NCBI/GenBank and ClustalX software (http//www-igbmc.ustrasbg.fr/bioinfo) was used to perform multiple sequence alignments. Nucleotide sequences were deposited in the GenBank under the accession numbers DQ- DQ for the.s-its and DQ-DQ for the D1/D domain of isolates MH01, MH0, MB0, MH0, MB0, MH0, MB, SB, MB1, SH1, SB1, respectively.

5 Botrytis infection in rotten samples was verified by ELISA (data not shown). In M, infection led to the situation of noble rot (1), accompanied by sugar content explosion and a slight increase in ph compared to M1 (Table 1). In S, grey rot was apparent and grapes showed diminished and damaged. Sugar content and ph value were lower compared to S1. Ethanol was quite low in all samples ( 0.0 g/l), except in M (1.0 g/l). Similar CFU counts were recorded on WLNA and LA media, whereas no colonies appeared on ESA and DBDMA, suggesting that S. cerevisiae and Dekkera/Brettanomyces spp., respectively, were either absent or at low frequencies. Yeast population exploded by a factor of in noble rotten grapes (M) as compared to M1 a fact that may be ascribed to the release of sugars on the skin (). In grey rotten berries (S) the respective raise was less remarkable (Table 1). A total of 0 isolates were analysed by PCR-RFLP of the.s-its rrna (Fig. 1). By using HinfI, HaeIII, HhaI or DdeI, eight different banding patterns were generated, which according to the dataset of Esteve-Zarzoso et al. () correspond to Hanseniaspora uvarum, H. guilliermondii, Zygosaccharomyces bailii, Issatchenkia terricola, I. occidentalis, Metschikowia pulcherrima, Aureobasidium pullulans and Candida stellata. Sequence analysis confirmed the presence and position of experimental restriction sites. Identification based on sequence relationships to published strains and phylogenetic analysis (data not shown) was in agreement with previous results, with the following two exceptions. Few H. guilliermondii isolates showed higher homology to the recently described H. opuntiae CBS 0 () than to H. guilliermondii CBS ( versus differences out of nt, respectively), and all isolates identified as C. stellata exhibited.% sequence similarity to C. zemplinina (1), while their homology to C. stellata CBS 1 was only %. In view of the above results, the sequences of H. opuntiae and H. guilliermondii were inspected for

6 informative RFLP patterns. In silico analysis and further experimental verification revealed that DraI generates distinct and readily distinguishable banding patterns for their discrimination due to -nt differences in their ITS1 region (Table, Fig. ). After DraI digestions, % of the isolates were identified as H. opuntiae and % as H. guilliermondii. This enzyme was also used for analysis of Candida isolates, as previously suggested (), to identify them as C. zemplinina. Taking together, we suggest the inclusion of DraI in the list of enzymes previously proposed () for rapid discrimination of the novel species H. opuntiae and C. zemplinina from their close relatives H. guilliermondii and C. stellata, respectively. Certain heterogeneity among isolates of M. pulcherrima and H. uvarum was detected, suggesting that different strains of the same species may reside on such a restricted ecosystem. Two M. pulcherrima isolates from M exhibited relatively high sequence divergence ( over 1 nt), being both most closely related to M. pulcherrima AY0 (). In the H. uvarum group, six isolates were divided into two clusters of 1% sequence divergence showing 0% identity or over nt differences to H. uvarum CBS 1 (), respectively. On the contrary, C. zemplinina and I. terricola isolates were clonal. In conclusion, PCR-RFLP combined with sequence analysis of the.s-its assigned isolates to genera and species (Table ). Identification was further corroborated by sequencing the D1/D domain. Genetic heterogeneity was observed among M. pulcherrima ( over nt) and H. uvarum (1 over nt) isolates, which also showed relatively high.s-its divergence. No differences were detected between isolates and published strains, except for MH0 and MH0 having a single-nt difference with sequences of I. terricola U and H. uvarum U (1), respectively.

7 Only two species, H. uvarum and H. opuntiae, were common in all four cases, with the former representing the predominant population (Fig. ). This is the first report to present H. opuntiae as member of grape ecosystem, probably because it has only been described recently (). Other members of the communities occurred in relatively low abundance (<%), with the exception of C. zemplinina in S1 (%) and S (1%). C. zemplinina was also previously related with botrytised musts (1, ) and although encountered in both rotten samples, its high proportion in S1 suggests preference for low glucose/fructose ratio in accordance to its strong fructophillic phenotype (1). H. uvarum, H. guilliermondii, H. opuntiae and I. terricola were encountered both on healthy and infected grapes of Mavroliatis, while M also harboured Z. bailii, M. pulcherrima and C. zemplinina. In Sefka, H. uvarum, H. opuntiae and C. zemplinina were present in both healthy and rotten grapes, while S also harboured I. occidentalis and I. terricola. A. pullulans was the only oxidative yeast-like organism isolated solely from S1. The correlation of A. pullulans with healthy but not with diseased grapes is in accordance to previous findings (, 1). C. zemplinina isolates were highly related to strain EJ1 (1) originating from Californian must, supporting previous suggestion that this species is possibly world spread in geographically distant localities (). Z. bailii was encountered in noble rotten berries. This yeast may reside winery environments, where constitutes real threat for product quality and preservation (, 1). Therefore, its origins must be well established. Concertedly, infected grapes exhibited higher yeast populations and more complex community structure, as a result of the arrival of new fermentative or potentially spoilage species. These observations are in accordance to previous suggestions for damaged grapes (, 1). Interestingly, in a currently undertaken

8 survey in Peloponnesus vineyards it was found that botrytised grapes possessed a similar fermentative yeast community to the presently described, even though respective healthy berries harboured a completely different structure composed solely of oxidative species (A. A. Nisiotou and G. J. Nychas, unpublished data). Botrytis infection introduces structural changes in grapes that increase sugar accessibility and create new niches. In this context, resource availability may enhance population-level diversification and create opportunities for new species to become established. In addition, microbial communities are dynamic consortia of species populations and therefore possible biological attributes of the system, such as interactive associations between Botrytis or other filamentous fungi often accompanying grey rot and yeasts may not be excluded (). Further research on grape microbial ecology will help to address many still unanswered questions as to the impact of microbial consortia on the dynamic structure of yeast communities in grapes. ACKNOWLEDGEMENT We gratefully acknowledge R. Burns (SAPS, Homerton College, Cambridge, UK) for generous gifts of Botrytis-specific monoclonal and phosphatase conjugated antibodies. We thank P. Hatzopoulos and L. Cocolin for helpful discussions, and E. Tsakalidou and G. Banilas for critical reading of the manuscript. A.A.N. was awarded a scholarship from the State Scholarship Foundation (IKY).

9 REFERENCES Burke, D., D. Dawson, and T. Stearns Methods in yeast genetics, a Cold Spring Harbor laboratory course manual, p. -. Cold Spring Harbor Laboratory Press, New York, N.Y.. Cadez, N., G. A. Poot, P. Raspor, and M. T. Smith. 00. Hanseniaspora meyeri sp. nov., Hanseniaspora clermontiae sp. nov., Hanseniaspora lachancei sp. nov. and Hanseniaspora opuntiae sp. nov., novel apiculate yeast species. Int. J. Syst. Evol. Microbiol. :.. Clemente-Jimenez, J. M., L. Mingorance-Cazorla, S. Martinez-Rodriguez, F. J. Las Heras-Vazquez, and F. Rodriguez-Vico. 00. Molecular characterization and oenological properties of wine yeasts isolated during spontaneous fermentation of six varieties of grape must. Food Microbiol. 1: Combina, M., L. Mercado, P. Borgo, A. Elia, V. Jofré, A. Ganga, C. Martinez, and C. Catanis. 00. Yeasts associated to Malbec grape berries from Mendoza, Argentina. J. Appl. Microbiol. : -1.. Dewey, F. M., S. E. Ebeler, D. O. Adams, A. C. Noble, and U. M. Meyer Quantification of Botrytis in grape juice determined by a monoclonal antibody-based immunoassay. Am. J. Enol. Vitic. 1: -.. Esteve-Zarzoso, B., C. Belloch, F. Uruburu, and A. Querol. 1. Identification of yeasts by RFLP analysis of the.s rrna gene and the two ribosomal internal transcribed spacers. Int. J. Syst. Bacteriol. : -.

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13 FIGURE LEGENDS FIG. 1. Representative restriction patterns of the.s-its region of yeast isolates obtained with HinfI. M: 0 bp molecular marker; 1-: H. uvarum; - and : H. opuntiae; : undigested PCR product from C. zemplinina; -: C. zemplinina; -: I. terricola. FIG.. (A) Informative restriction fragment polymorphism in the.s-its amplicons of H. opuntiae and H. guilliermondii generated by DraI digestion. M, molecular marker; 1, H. opuntiae;, H. guilliermondii. (B) A site of nucleotide sequence alignment of the.s rrna ITS region of H. opuntiae and H. guilliermondii showing the DraI informative recognition site ( -TTTAAA- ) due to -nucleotide differences as marked in white boxes. FIG.. Yeast species populations on healthy (M1 and S1) and Botrytis-infected (M and S) grapes of Mavroliatis and Sefka cultivars, respectively

14 Sample code M1 M S1 S Table 1. Physicochemical characteristics and yeast log CFU/g counts of grape samples. Variety/ Status of berries Mavroliatis / Healthy Mavroliatis / Noble rotten Sefka / Healthy Sefka / Grey rotten D-glucose + D-fructose content (g/l) D-glucose / D-fructose ratio ph Yeast log CFU/g counts α on WLNA LA (0.). (0.) (0.0). (0.) (0.).1 (0.) (0.). (0.) α Yeast counts are shown as the mean of triplicates with standard deviation in parentheses. 1

15 Table. Sizes of the.s-its rdna amplicons and the restriction fragments of the yeast isolates Species PCR Restriction fragments (bp) Product (bp) HhaI HaeIII HinfI DdeI DraI Z. bailii H. uvarum H. guilliermondii H. opuntiae A. pullulans C. stellata C. zemplinina I. occidentalis I. terricola M. pulcherrima

16 Fig. 1 1

17 A B Fig. 1

18 0% % 0% % 0% M 1 M S 1 S H. uvarum H. guilliermondii I. terricola Z. bailii C. zemplinina A. pullulans H. opuntiae M. pulcherrima I. occidentalis Fig. 1