NATIVE YEAST FROM DISTINCT ORGANS OF GRAPEVINES ESTABLISHED IN QUERETARO, MEXICO, AND THEIR POTENTIAL OENOLOGICAL UTILIZATION

The aim of this study was to isolate, identify and determine the oenological potential of yeasts present in Vitis vinifera organs of grapevines established in Queretaro State, Mexico. The yeast distribution was influenced by the organ and the sampling season, and the yeast populations ranged from 0.8 and 5.5 Log CFU/g. A total of 93 yeasts were isolated, identified by RFLP and confirmed by sequencing of the ITS region, prevailing Aureobasidium cf. melanogenum and Basidiomycota yeast. The identified species with previously reported oenological potential were: Pichia cf. kluyveri and Clavispora cf.opuntiae. Remarkably, P. cf. kluyveri 3.1HM showed killer phenotype and was the most tolerant to sulfur dioxide, and survived 72 h after its inoculation in ‘Tempranillo’ must. C. cf. opuntiae 5.7HM showed β-glucosidase activity, the highest tolerance to 5 % ethanol and 25 °Brix (sugar levels). On the contrary, Rhodotorula isolates were not tolerant to stress conditions, and R. mucilaginosa 8HM did not grow under must conditions. Mixed fermentation using H. uvarum NB108/S. cerevisiae N05 resulted in the highest volatile acidity (0.45 g/L acetic acid), while no differences for total acidity, alcohol strength, residual sugars and total SO2 were found between the mixed fermentations treatments. This study provides an insight into the yeast diversity present in grapevines established in Queretaro, Mexico, and the oenological potential of. P. cf. kluyveri 3.1HM.


INTRODUCTION
Wine is the beverage resulting from the alcoholic fermentation of grape must and it is carried out by a sequential action of yeasts (OIV, 2019a). During the first steps of fermentation the so-called non-Saccharomyces (n-S) dominate (Jolly et al., 2014). Due to their low tolerance to ethanol, diversity of this group gradually decreases as the fermentation advances and Saccharomyces species become dominant and complete the process (Varela and Borneman, 2017).
During their residency on must fermentation, native yeast especially n-S, produce certain secondary metabolites, such as glycerol, esters, higher alcohols, medium chain fatty acids, among others, which positively affect the wine quality, conferring higher complexity to wine (Varela, 2016). Moreover, the inoculation of selected native strains to perform must fermentation is blooming, since the strains may be better adapted to the environmental conditions and could also potentiate the tipicity of wines (Callejón et al., 2010;Capozzi et al., 2015;Padilla et al., 2016a;Miranda et al., 2017).
The isolation and selection of oenological yeasts is generally performed from mature grapes and must in fermentation (Belda et al., 2016;Liu et al., 2016;Padilla et al., 2016b). Besides, in vineyards, yeasts could be found on stems, leaves, and on the surface of berries, mainly in stomata, lenticels and sites where microfissures are present (Bernardi, 2013).This yeast diversity was rarely considered to be studied (Garijo et al., 2011), on the belief that strains with oenological potential could be mainly found in wineries.
The recent blooming of the wine industry in Queretaro, Mexico, demands to explore quite new yeast diversity difficult to have in other winemaking areas, where the age of the vineyards and their proximity to wineries has led to a more selected microbiota. Research concerning the selection of native oenological Saccharomyces  and n-S (Ortiz-  yeasts has been performed in this region. Nevertheless, there is a lack of information about the diversity and distribution of native yeasts in the vineyard and their possible use in winemaking in Mexico, which could lead to the possibility of finding interesting and original strains with enological benefits. Therefore, the aim of this study was to isolate, identify and determine the oenological potential of yeasts present in different organs of grapevines in the region.

Sampling and yeast strains
Samples were collected from six-year-old grapevines (Vitis vinifera L. cv. 'Tempranillo' grafted on SO4 rootstock), established in Los Cues, Queretaro, Mexico. The parts of the vines sampled according to their vegetative cycle (Table I) were randomly collected along three lines of the vineyard (triplicate), put into plastic bags (10 x 20 cm), transported inside a cooler and immediately processed in the laboratory. The commercial strain S. cerevisiae K1-V1116 (Lalvin, Ontario, Canada) and the native selected strain S. cerevisiae N05  were used. Besides, the sensitive to killer phenotype reference strain AH22 (ATCC38626) as well as two n-S Hanseniaspora uvarum (NB27 and NB108) controls  were also used.

Isolation of yeasts
For each triplicate of sample collected, a total of 50 berries of each physiological state, 3 g of inflorescences or buds and 5 g of leaves were placed separately in plastic bags containing 50 mL of peptone diluent (0.1 % peptone of casein). Berries were carefully and manually homogenized for 3 min. The other samples were homogenized separately in a Stomacher at medium velocity during 1 min. Suspensions were placed in 50 mL conical tubes and centrifuged at 1790 g for10 min in order to obtain the pellet containing the microorganisms.
Plates were incubated at 28 °C for 48 h, counts were made, and contrasting colony morphologies were isolated in NYDA.

Identification of yeasts species
For the DNA extraction, a loop of each isolate colony collected from the agar was resuspended in 300 μL of TNES (Tris-HCl 200 mM pH 8.5, NaCl 250 mM, EDTA 25 mM, SDS 0.5%) and, from this step, the methodology described by Crespo-Sempere et al. (2013) was followed.
For PCR, 2 µL of DNA from each isolate were added into 48 µL of reaction mix containing: 10 µM ITS1 (5' TCCGTAGGTGAACCTGCGG 3'), 10 µM ITS4 (5' TCCTCCGCTTATTGATATGC3'), 2mM of dNTPs, buffer 10X, taq polymerase and PCR-grade water. The PCR reaction was carried out in a Veriti TM 96-Well Thermal Cycler (Thermo Fisher Scientific, CA, USA) following the next conditions: 95 °C for 2 min; 35 cycles at 94 °C for 15 s, 55 °C for 30 s; 72 °C for 30 s; 72 °C for 5 min. The PCR products were separated by electrophoresis in 1.3 % agarose gel using 100 V for 30 min. Then, PCR products were separated in three tubes and restriction enzymes Hha, HaeIII and HinfI were independently added; each reaction was incubated at 37 °C for 3 h. The obtained fragments after the enzymatic digestion were separated in a 3 % agarose gel. The size of the restriction fragments was determined comparing them with GeneRuler 100 bp DNA ladder (Thermo Scientific, USA) using Gel analyzer plug-in on the Image J software. Then, all the isolates with the same banding pattern were grouped. The distinct banding patterns were compared with the data base from the platform yeast-id.org in order to presumptively identify isolates at species level (Esteve-Zarzoso et al., 1999).
To corroborate the identification by RFLP, representative isolates of each group (one every five with common pattern) were selected or sequencing. Briefly, DNA was extracted and PCR amplicon of ITS region was obtained as previously mentioned, then separated from remaining primers on a 1.5 % agarose gel electrophoresis; the band was excised from the gel and purified using Illustra GFX PCR DNA and Gel Band purification kit (GE Healthcare, Mexico). Purified amplicons on TE buffer were sent to Macrogen Korea (ABI 3730XL 96-capillary DNA analyzer, Applied Biosystems), and then sequence was compared against nucleotide collection of NCBI BLASTN excluding uncultured and model microorganisms sequences, limiting the search on Type material. The sequences were also compared against Mycobank.org databases. In the cases that the RFLP identification of any strain was different from that obtained by sequencing, it was sent for verification by sequencing.

In vitro selection assays
The isolates previously reported as potentially useful on co-inoculation (Ortiz-  were selected for the following phenotypic evaluation.

β-glucosidase activity
The isolates were streaked by triplicate on esculin glycerol agar plates and incubated at 25 °C for 72 h. Those with β-glucosidase activity hydrolyzed the substrate and a dark brown precipitate was observed around the colony (Pérez et al., 2011).

Killer activity
The killer sensitive strain AH22 (ATCC38626) was pour plated in modified NYDA (methylene blue 0.02%, NaCl 1 % and phosphate-citrate buffer 0.5 M, pH 4.6-4.8) at a final concentration of 5 Log CFU/mL. A volume of 50 μL of overnight culture of isolates to be evaluated were drop inoculated by triplicate and incubated at 25 ºC for 72 h (Lopes and Sangorrin, 2010). Isolates were considered killer when an inhibition halo around the colony limited by a blue border was observed.

Sugar, sulfur dioxide and ethanol tolerance
The growth' kinetics of yeasts were measured using a Varioskan (Thermo Fisher Scientific, USA) turbidimetric analyzer. A solution of NYDB (broth with the same composition of NYDA, without agar), at pH 3.5, was prepared and adjusted with: a) sucrose at 25 °Brix, b) 6 % ethanol, or c) 30 mg/L of sulfur dioxide (SO 2 ) using a solution of potassium metabisulfite. A total of 200 µL of each medium was added to 96-well plates, and the different yeast isolates were separately inoculated by triplicate at a concentration of 1x10 5 CFU/mL and incubated at 25 °C. Low agitation was carried out previously to measure the absorbance at 600 nm every 2 h and the slope of the exponential phase was estimated (Miranda et al., 2017).
Crushed grapes cv. 'Tempranillo' were pasteurized at 60 °C during 20 min (Ribéreau-Gayon et al., 2006) and 700 mL were distributed in 1 L flasks. The must presented the following characteristics: 24.2 °Brix, a total titratable acidity of 7.2 g/L of tartaric acid, and pH of 3.82. Musts were first inoculated separately with the n-S isolates corresponding to species reported in fermented beverages, or used in mixed fermentations at a concentration of 1x10 5 CFU/mL and, after 24 h, with S. cerevisiae N05 at 1x10 6 CFU/mL. Fermentation was performed at 25°C, until must density decreased to 1000. Yeast cell viability was measured every 24 h during fermentation, for Saccharomyces in NYDA, and for n-S in lysine medium . At the end of fermentation, pH, alcohol strength, total titritable acidity, volatile acidity, residual sugars and total SO 2 (OIV, 2019b) were measured. Three replicates for each treatment were evaluated.

Statistical analysis
A unifactorial completely randomized design was used for yeast isolates on their tolerance to sugar, ethanol and SO 2 , as well as for the winemaking assays. In order to establish differences among isolates and evaluate their winemaking performance, ANOVA and Tukey test (p≤0.05) were performed with the obtained data, using the Statistical Program JMP version 11.

Quantification of yeast in the different grape organs
Quantification of yeast in fruit set were less than 1 Log CFU/g, while fruits in veraison reached 3 Log CFU/g and ripe fruits 3.7 Log CFU/g averages (Table  II). Concerning yeast populations in leaves according to their position along the stem, higher viable concentration on basal leaves with respect to medium and apical leaves were obtained in the distinct sampling dates and an increase of yeasts in the leaves through the time was also observed, reaching values higher than 5 Log CFU/g in October.
The values obtained in this study for immature fruits are comparable to those reported by Fleet (2003), ranging from 1 to 3 Log CFU/g, and for healthy ripe fruits, according to Barata et al. (2012) and Mendes et al. (2017). The differences on yeast viable concentration in the leaves according to their location in the stem could be due to that basal leaves are the oldest, having a longer time of exposure to the vineyard's environment than the others (Pinto et al., 2014). The increase of yeast viable concentration over the time on medium and basal leaves coincides with the use of chemical fungicides in this vineyard, since the application of these products is paused two weeks before ripening, allowing a major accumulation of yeasts. However, many other factors, as topographic and climatic conditions, cultivar and viticultural practices performed in the vineyard affect yeast populations (Cordero-Bueso et al., 2011;Bokulich et al., 2013).

Identification of yeast species
From 93 isolates obtained and their grouping according to their restriction similarity, 14 different banding patterns were found, which corresponded to 10 species identified by PCR-RFLP, while four banding patterns did not match those on the yeastid.org database (Table III).  (Table III).
Then there was a subtle discrepancy between the two methods used, which is ascribed to the fact that RFLP is a more general methodology (polymorphism patterns) identifying the yeasts only presumptively. Nucleotide sequencing should be considered as a more reliable methodology, since it is more precise at the moment of searching the homology of the sequence in the databases (Li et al., 2010). On the other hand, the absence of the Saccharomyces genus can be attributed to the sampling carried out in a young vineyard, located far from other vines and in absence of a winery, where S. cerevisiae prevails (Pérez-Martín et al., 2014;Mendes et al., 2017). Another reason that may explain why no Saccharomyces isolates were detected is that only healthy grapes were sampled. Barata et al. (2012) recovered S. cerevisiae yeasts in post-mature decayed or damaged grapes.
The percentages of relative abundance of the different yeast species found in each organ are shown in Figure  1. Aureobasidium cf. melanogenum predominated throughout the sampling, especially in inflorescences and in the different stages of fruit development. Pichia cf. kluyveri was found in basal leaves and Clavispora cf. opuntiae in medium leaves. Finally, Rhodotorula mucilaginosa and R. nothofagi were only found in leaves.
These results coincide with those of Grube et al. (2011) who isolated Aureobasidium pullulans from stems, leaves and grape berries in 26 %, 41 % and 18 % respectively, while Garijo et al. (2011) found it predominately in the air of the vineyard during two consecutive years. In this study, Naganishia albida and Papiliotrema flavescens, former Cryptococcus albidus and C. flavescens, respectively, were located in berries, coinciding with Li et al. (2010) who 36 detected the last one in the same organ. The species prevalent in the vineyard are highly oxidative yeasts which do not survive in the anaerobic conditions of must (Varela and Borneman, 2016), however they have been reported as biocontrol agents against diverse fungal diseases (Varela and Borneman, 2016;Wang et al., 2018a). Abundância relativa e distribuição de isolados de leveduras nativas encontrados nos diferentes orgãos da videira.
Another species found was Pichia cf. kluyveri, whose use in winemaking has been previously reported by Ma et al. (2017), who evaluated the synthesis of volatile compounds during the co-inoculation of a P. cf. kluyveri with S. cerevisiae, obtaining an increased production of ethyl esters, fatty acids, among others. Concerning R. mucilaginosa, Hu et al. (2016) reported that this species possesses β-glucosidase activity that could promote the release of glycosylated terpens associated with floral aroma. On the other hand, regarding Clavispora genus, C. lusitaniae has been isolated from Mexican fermented beverages as 'mezcal' and 'sotol' (De La Garza-Toledo et al., 2008;Escalante-Minakata et al., 2008). In the present study, the identification of this isolate should be taken with caution, considering the low query coverage (91%) and identity (96%). For the assays regarding winemaking potential, only those isolates belonging to such species previously reported to modify wine quality in mixed fermentation were included, namely P. cf. kluyveri, C. cf. opuntiae, R. mucilaginosa and R. nothofagi (Phaff et al., 1986;Wang et al. 2017;Wang et al. 2018b).

Killer activity and β-glucosidase activity
Isolates 8HM, 4.7HM and 2HE belonging to Rhodotorula genus showed both killer activity and βglucosidase activity (Table V), while P. cf. kluyveri 3.1HM showed killer activity, but not β-glucosidase activity. Finally, both H. uvarum controls (NB27 and NB108) showed β-glucosidase activity, but not killer activity, which coincides with previous studies . The β-glucosidase activity could promote the release of glycolsylate terpenes associated with floral aroma, as Hu et al. (2016) found for other strains of R. mucilaginosa. Meanwhile Ma et al. (2017) obtained an increased production of ethyl esters, fatty acids, among others, while evaluating the synthesis of volatile compounds during the co-inoculation of a P. fermentans strain with S. cerevisiae. As mentioned in this and other previous studies, the evaluated features are strain dependent.

Sugar, total sulfur dioxide and ethanol tolerance
The tolerance of isolates to limiting factors relevant for winemaking was, as expected, strain dependant ( Figure 2). Yeasts belonging to Rhodotorula genus were the least tolerant to the three stress conditions tested. The inability of the Rhodotorula genus to tolerate stressful conditions is explained by the fact that must is composed by aerobic yeasts, which could be present and disappear in the first stages of fermentation due to the combined effect of SO 2 , low pH, high ethanol and limited nitrogen concentrations (Jolly et al., 2014). Nevertheless, it is interesting to emphasize their practically null sugar tolerance, despite the fact that, according to these authors, it is a genus normally present in must. On the other hand, C. cf. opuntiae 5.7 HE and both H. uvarum controls (NB27 and NB108) outstood in sugar tolerance, which agrees with their previous characterization carried by Ortiz-Barrera et al. (2015). Moreover, Pichia cf. kluyveri 3.1HM was the most tolerant yeast to 30 mg/L SO 2 . Several authors have observed that Pichia sp. possesses a wide range of SO 2 tolerance (from 50 mg/L to 140 mg/L) (Barata et al., 2008;Wang et al., 2018b). Finally, C. cf. opuntiae 5.7HE was the most tolerant to 5 % ethanol, which is consistent with the fact that Clavispora genus has been found in two 'mezcal' distilleries (Kyrchmayr et al., 2017), and has been described as a vigorous fermenter (Phaff et al., 1986).  Taxa de crescimento em diferentes condições de stress. Dados em triplicado, apresentados como média ± desvio padrão. Colunas (condições de stress) marcadas com a mesma letra não são significativamente diferentes (teste de Tukey,p>0,05).
The isolate showing outstanding tolerance to limit factors, Pichia cf. kluyveri (3.1HM) and C. cf. opuntiae (5.7HE), were selected for the winemaking assays. In addition, the best R. mucilaginosa isolate (8HM) was considered, in the hypothesis that its killer and β-glucosidase activity might produce an important effect on wine. Finally, one n-S yeast previously studied and reported , namely H. uvarum NB108, was used as control.
For the differential quantification of Saccharomyces cerevisiae N05 and n-S yeast during fermentations, lysine medium, selective for n-S, was used.
Nevertheless, Saccharomyces was also able to grow due to their capacity to stock nitrogen in its vacuoles (Watson, 1976). Therefore, differential quantification of yeast in this medium was only possible for R. mucilaginosa (salmon color) and P. cf. kluyveri (rugged), both showing contrasting morphologies with S. cerevisiae. Figure 3 shows the growth kinetics for P. cf. kluyveri 3.1HM and S. cerevisiae N05 (PS), and that for R. mucilaginosa 8HM and S. cerevisiae N05 (RS).The differences between the inoculum and viable counts of yeast in the inoculated must could be explained by the stress caused on microorganisms by the particularly complex environment, unusual for them. P. cf. kluyveri 3.1HM population increased to a final count of approximately 8 Log CFU/mL two days after its inoculation. At the same time (24 hours after its inoculation) S.cerevisae N05 population arrived to 5 Log CFU/mL. Afterwards, S. cerevisiae adapted and actively multiplied, increasing 4 Log CFU/mL in 24 h, and reaching a population close to 9 Log CFU/mL, while P. cf. kluyveri 3.1HM reached the stationary phase ( Figure 3A). On the other treatment, R. mucilaginosa 8HM was not detected after 24 h from its inoculation and S. cerevisiae N05 arrived to more than 6 Log CFU/mL 24 h after its inoculation ( Figure  3B). On mixed fermentations, P. cf. kluyveri 3.1HM rapidly adapted to the medium which coincides with its relative tolerance to SO 2 , ethanol and sugar ( Figure  2). Meanwhile, R. mucilaginosa 8HM evidenced its incapacity to adapt under must conditions (sugar, SO 2 , pH and nitrogen deficiency), previously observed in the tolerance assays ( Figure 2).
Finally, the behavior of S. cerevisiae N05 was similar in both mixed fermentations, with a slightly lower viability 24 hours after having been inoculated in presence of P. cf. kluyveri 3.1HM, in relation to that obtained at the same time in presence of R. mucilaginosa 8HM, probably due to some competition offered by this n-S isolate in the first case, associated to the higher concentration of P. cf. kluyveri (~2 Log CFU/mL) than that of S. cerevisiae at this time. As any other organism, yeast competes for space and resources in a shared environment with other yeasts, which is observed in the mixed fermentation with R. mucilaginosa 8HM, where only S. cerevisiae prevailed, taking advantage of space and/or nutrients available, which contrast with its behavior in the presence P. cf. kluyveri 3.1HM.

Physical and chemical analyses of wines
Differences among treatments were only found for pH and volatile acidity, being the control S. cerevisiae N05 with H. uvarum NB108 (HS) the highest in both (3.75 and 0.45 g/L acetic acid, respectively) (Table  VI). Differences in pH could be explained by the different concentrations of succinic, lactic, piruvic and malic acids and other acids produced by the distinct n-S yeasts during fermentation (Whiting, 1976;Chidi et al., 2018). Nevertheless, no differences between treatments in TTA were apparent. The higher volatile acidity obtained in the co-inoculation of Hanseniaspora uvarum NB108 agrees with Jolly et al. (2014) who reported that Hanseniaspora species produce high quantities of acetic acid and ethyl acetate, which make them less attractive for winemaking. Ortiz-  performed mix fermentations with S. cerevisiae and different Hanseniaspora strains, finding values of volatile acidity lower than 0.2 g/L acetic acid, which differ from our results, probably due to differences in winemaking conditions.
Concerning the other analyzed variables, residual sugars, less than 2 g/L, and alcohol strength, higher than 13 %, correspond to dry wines and confirm complete fermentations. Moreover, total SO 2 values are under the limits accepted for quality wines (OIV, 2019a). These results show that n-S yeast evaluated in this study do not have any negative impact in the chemical characteristics of wines and could be used in commercial mix fermentations. Nevertheless, the most recognized contribution of n-S lays down on subtle sensory attributes, which seem to provide complexity and tipicity to wine (Jolly et al., 2014). Mean and standard deviation from three replicates are shown. Means followed by the same letter in a column are not significantly different (Tukey test, p>0.05). Mix fermentation treatments of S. cerevisiae N05 with: R. mucilaginosa 8 HM (RS), P. cf. kluyveri 3.1 HM (PS), C. cf. opuntiae 5.7 HM (CS); as mix control with H. uvarum NB108 (HS) and general control: S. cerevisiae alone (N05).

CONCLUSIONS
Yeast populations in grapevines vary highly depending of the organ and the sampling season, and all the isolates correspond to non-Saccharomyces yeasts. The prevalent genera in the vineyard are the highly oxidative genus Aureobasidium and basidiomycota yeasts.
Pichia cf. kluyveri 3.1HM showed killer activity, was the most tolerant to sulfite and survives along the exponential phase of S. cerevisie. Clavispora cf. opuntiae 5.7HM showed the highest tolerance to ethanol and sugar, as well as β-glucosidase activity.
Both strains should be further studied in mixed fermentations for the production of regional wines. On the other hand, the different Rhodotorula spp.isolates evaluated lacked tolerance to the stress conditions evaluated and R. mucilaginosa 8HM was not able to grow in the must environment.
This study provided insights on the diversity of yeast present in distinct parts of grapevines established in Queretaro, Mexico, and revealed the oenological potential of Pichia cf. kluyveri 3.1HM.