Antifungal activities of vineyard-habitat wild yeast for grape gray-mold disease and its initial promotion of alcohol fermentation in spontaneous winemaking

doi:10.21203/rs.3.rs-2098543/v1

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Antifungal activities of vineyard-habitat wild yeast for grape gray-mold disease and its initial promotion of alcohol fermentation in spontaneous winemaking

https://doi.org/10.21203/rs.3.rs-2098543/v1

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Microorganisms, including native yeasts, are abundant in vineyard fields. Herein, we studied the possibility of using vineyard-derived wild yeast as a microbial pesticide against Botrytis cinerea, a pathogen that causes grape gray mold disease, to boost the initial alcohol production of spontaneously fermented wine. We identified the Saccharomyces cerevisiae strain KONDO170908, which showed the most effective antifungal activity in an ex vivo yeast dripping experiment on grape berries. This strain was utilized in an in vivo spray test on grape bunches in vineyard fields and was proven to significantly suppress gray mold disease on the grape berries in test plot #16 when the yeast was sprayed during both the flowering and ripening periods (morbidity 11.2% against 15.3% of the control plot, χ² test, P < 0.0001). However, in test plot #17, spraying the yeast during only the ripening period had no effect (morbidity 16.3%). The grapes from each test plot were also submitted for spontaneous wine fermentation. Alcoholic fermentation of the grapes from test plot #16 provided the most active bubbling of CO₂ gas and the highest ethanol production and colony counts over seven days of fermentation. Unique changes in the different strains of S. cerevisiae among the plots were observed throughout the early fermentation stage. Thus, yeast spraying during the flowering period might trigger modification of the entire microbiota and could ultimately contribute to promoting alcohol production in the spontaneously fermented wine.

Antifungal activity

Biocontrol

Botrytis cinerea

Saccharomyces cerevisiae

Spontaneously fermented wine

Gray mold disease is caused by inoculation with the airborne pathogen Botrytis cinerea, affecting over 200 crops worldwide (Williamson et al. 2007). B. cinerea inoculation of wine grapes causes severe damage to the harvest, followed by serious losses in the quantity and quality of wine (Keller et al. 2003; Nigro et al. 2006; Marisol et al. 2012; Cabañas et al. 2020). To date, many fungicides, such as benomyl (Pearson et al. 1980; Yarden et al. 1993), have been developed as chemical pesticides to control B. cinerea. However, B. cinerea is also known as a pathogen that has a particularly short life cycle, produces large amounts of spores and is prone to gene mutation and the emergence of fungicide-resistant strains (Leroux et al. 2002). In recent years, due to the growing interest in organic cultivation, alternative biological control methods using native microorganisms as natural antagonists to pathogens and pests have attracted attention (Freimoser et al. 2019; Pretscher et al. 2018; Cordro-Bueso et al. 2017).

Currently, microbial pesticides using Bacillus bacteria (Ji et al. 2019), lactic acid bacteria (Lamont et al. 2017), Xanthomonas bacteria (Nagai et al. 2017) and wine yeasts (Marisol et al. 2012; Nally et al. 2012; Cordero-Bueso et al. 2017; Pretscher et al. 2018; Chen et al. 2018; De Simone et al. 2020) are being investigated as measures to control gray mold disease. The mechanisms by which microbial pesticides inhibit the growth of pathogens include competition (competition for location and nutrients), antagonistic action, parasitism, lysis and resistance induction (Cordero-Bueso et al. 2017; Freimoser et al. 2019). Microorganisms such as bacteria and yeasts colonizing vegetables and fruits have long been used as natural preservatives in the production of familiar fermented foods such as bread, pickles and wine. These microorganisms have been proven to be safe for humans and have the advantage of being accepted by consumers as safe (Lamont et al. 2017).

Many yeast species, such as Saccharomyces spp., Schizosaccharomyces spp., Torulaspora spp., Candida spp. and Zygosaccharomyces spp. are present on the surface of grapes (De Simone et al. 2020). These yeast species have their own roles in grape quality control and winemaking. Research on the usefulness of the biological control of vineyard wild yeast is increasing year by year (Chen et al. 2018; Pretscher et al. 2018; Freimoser et al. 2019; Cabañas et al. 2020). Among various yeast species, S. cerevisiae, which has excellent alcohol-producing ability, has been studied in various ways as a functional and model organism. S. cerevisiae is also the most studied yeast as a microbial pesticide, as described above, and many strains are reported to suppress the growth of B. cinerea (Freimoser et al. 2019). Its antagonistic activity, first discovered in S. cerevisiae, could inhibit the growth of susceptible strains of the same species by secreting a proteinaceous "killer toxin" (Rogers et al. 1978). This discovery promoted research on microbial pesticide application of yeasts in practical use. The activity is contributed by multiple factors associated with yeast growth: competition between nutrient sources and space, production of volatile secondary metabolites (ethanol, ethyl acetate, carbon dioxide, etc.) and lytic enzymes (Cordero-Bueso et al. 2017). Nevertheless, many studies of the antifungal activity of yeasts against B. cinerea have been limited to in vitro or ex vivo tests. Only a few studies have been applied to vineyard fields in which the application of Candida sake CPA-1 reduced the botrytis bunch rot incidence and severity by 64–67% and 89–90%, respectively (Cañamás et al. 2011; Calvo-Garrido et al. 2013). They also reported that no adverse effects on wine quality were observed when the grapes were subsequently fermented with a commercial yeast (Calvo-Garrido et al. 2013).

Generally, in spontaneously fermented wine, the abundance ratio of the wild yeasts differs during the progress of fermentation, and in the very early stage, the growth of non-Saccharomyces yeasts such as Kloeckera apiculata and Hanseniaspora vineae often appears, and after 2 to 3 days, S. cerevisiae grows and becomes dominant (Ciani et al. 2010). These substitutions of the yeast diversity are well known to contribute to the deep and complex flavors of spontaneously fermented wine. In contrast, the delay of alcohol fermentation boost, without the use of starter yeast, easily cause spoilage, such as sour rot, by acetic acid bacteria (Hall et al. 2018). Enrichment of the diversity and amount of the yeasts colonizing on wine grapes at harvest time will accelerate the initial alcohol fermentation and contribute to safer making of natural wine.

In this study, S. cerevisiae strains were selected by their in vitro inhibitory activity against colony growth of B. cinerea (Hudagula et al. 2021) and were subjected to an ex vivo inoculate test on grape berries. A strain that showed noticeable suppression ability in the ex vivo test was selected for the field test of vineyard grapes. Thus, the S. cerevisiae strain adopted as a microbial pesticide was evaluated against botrytis rot. Finally, we noted that the yeast sprayed on vineyard grapes enriched the microbiota on harvested grapes and/or promoted initial alcohol production in spontaneously fermented wine.

Yeast strains

Five strains of Saccharomyces cerevisiae with different antifungal activities in vitro were selected from 55 yeast strains isolated from vineyards and spontaneously fermented wine precipitates (Hudagula et al. 2021). Including a commercially available strain (Pasteur Blank, Marcq-en-Barœul, France), a total of 6 strains were subjected to the following ex vivo test. The strains were cultured at 25 ℃ for 3 to 7 days in YPD broth (1% yeast extracts, 2% peptone, 2% glucose, 0.01% chloramphenicol and 0.2% sodium propionate) before being used in antifungal tests. The number of yeast cells was counted under a microscopic field using a cell counter (Biomedical Science, Tokyo, Japan) and diluted with the medium to obtain a yeast suspension of 1×10⁸ cells/mL for the ex vivo test.

B. cinerea spore suspension

The B. cinerea NBRC5964 strain (NITE, Tokyo, Japan) was cultured in potato dextrose agar (PDA) medium (Eiken Chemical, Tokyo, Japan) supplemented with 0.01% chloramphenicol. Mycelium discs (6 mm\(\varnothing\)) punched out together with the agar were placed in the center of fresh PDA medium and cultured at 25 ℃ for 2 weeks until spore formation was observed. Ten milliliters of sterile water was added to the spores on the agar medium, scraped off with a spreader, and collected in a 50 mL tube. This spore harvesting was performed twice in total. The combined spore suspensions were sonicated for 10 minutes and filtered through a heat sterilized glass funnel stuffed with glass wool. The obtained filtrate was centrifuged at 7400 ×g for 10 min at 25 ℃, and the supernatant was removed by decantation. Twenty milliliters of sterilized water was added to the sediment, and after dispersion, spores were collected by centrifugation. This spore washing by centrifugation and decantation was performed a total of three times. Finally, the spores were dispersed in sterile water to prepare a suspension of 1×10⁶ cells/mL, stored at 4°C, and used within 3 weeks.

Antifungal Test On Grape Berries (Ex Vivo)

Pretreatment of grapes

Grape bunches (Niagara) purchased at retail stores in Ebetsu City on September 24 and October 20, 2020 were used. The berries removed from the bunches were washed six times with tap water, sterilized by immersing them in 70% ethanol for 1 minute, and air-dried in a safety cabinet.

Yeast d

Grape berries (n = 3) sterilized by ethanol treatment were placed individually in each well of a sterilized 12-well plate (TPP, Trasadingen, Switzerland), and a spot was pricked on the surface using an injection needle (22G ×11/2 ", 0.70 ×38 mm, Terumo, Tokyo, Japan). Ten microliters of the yeast suspension (1×10⁸ cells/mL) was dropped on the scratched position of the grapes and allowed to air-dry for 2 hours in a safety cabinet. As a positive control, 1% benzalkonium chloride solution (Fujifilm Wako) was used instead of the yeast suspension. Subsequently, ten microliters of the spore suspension of B. cinerea (1×10⁶ spores/mL) or sterile saline (negative control) was added dropwise to the same position and cultured at 25 ℃ for 3 weeks.

Judgment criteria

The criteria for antifungal activity were that B. cinerea mycelium growth in grape berries was not observed in all in three berries for 21 days and was set as (+++), in two berries (++), in one berry (+) and none (-) to be recognized.

Antifungal Test On Grape Bunches In Vineyard Field (In Vivo)

Yeast preparation

The Saccharomyces cerevisiae strain (Kondo170908), the isolated yeast that showed the most noticeable antifungal activity in the in vitro (Hudagula et al. 2021) and the ex vivo test in this study, was used. Three milliliters of the yeast primary culture was inoculated into 2 L of YPD broth without chloramphenicol and sodium propionate and the cells were cultured at room temperature for five days and mixed three to five times a day.

Yeast spray

The cultured yeast (5.0 × 10⁷ cells/mL) was sprayed onto grape bunches of ten Pinot Noir seedlings (KONDO Vineyard, Iwamizawa, Japan) using a hand sprayer (approximately 5 mL/bunch), and these grapes were set as test plots. The yeast culture was stored at room temperature throughout the spraying period. Untreated grapes of ten seedlings in the adjacent row were used as a control plot. The yeast suspension was sprayed 4 times during the flowering period (June to July 2021) and 3 times during the ripening period (September 2021) (Fig. 1). Test plot #16 was sprayed with yeast during both periods, test plot #17 was sprayed only during the ripening period, and control plot #15 was not sprayed with yeast.

Gray-mold inoculation

The grape bunches were harvested on October 13, 2021. The grape berries affected by gray-mold disease were collected with tweezers, and the weights of the affected and healthy berries were measured separately. The prevalence of gray-mold inoculation was evaluated in each group. The frequency of morbidity was compared by χ2 tests using GraphPad PRISM8 (GraphPad Software Inc., La Jolla, US).

Making Natural Wine

Fermentation

The harvested and selected healthy berries from each plot (approximately 10 kg) were allowed to make spontaneously fermented wine. The progress of alcohol fermentation was monitored by the observation of CO₂ gas bubbling on the surface of the making wine. Each 40 mL portion of the wine was sampled every week until 4 weeks and stored at -30 ℃ until the measurements of ethanol, fungal colony isolation and the ARISA evaluation of fungal DNA diversity.

Ethanol measurement: The concentration of ethanol in the wine sample was measured by an enzymatic colorimetric assay. One hundred microliters of the thawed wine sample was diluted with 900 µL of distilled water in a 2 mL screw cap tube (BM equipment, Tokyo, Japan) and boiled for 10 minutes. After cooling to room temperature, 0.02 g of activated charcoal powder (Fujifilm Wako, Osaka, Japan) was added and it was vortexed vigorously for 15 seconds. One hundred microliters of the supernatant obtained after centrifugation (20,630 ×g, 5 min, 25 ℃) was transferred to a new 1.5 mL tube. This pretreated wine solution was diluted with distilled water to make up an appropriate concentration range against an ethanol calibration curve. The enzyme mixture was prepared as follows: 12 U/mL alcohol dehydrogenase (ADH), 100 U/mL diaphorase (both Fujifilm Wako) and distilled water were mixed at a ratio of 0.03:0.3:0.17. The substrate solution was prepared by mixing 6 mmol/L NAD⁺, 0.01 mol/L Nitro-TB (both Fujifilm Wako) and 0.2 mol/L Tris buffer (pH 8.9) – 0.1% Triton X-100 (Alfa Aesar, Heysham, England) at a ratio of 1:0.5:3. The enzymatic reactive mixture was prepared by mixing the enzyme and substrate solutions at a ratio of 0.5:4.5 immediately before use. To a 96-well microplate, 50 µL of 0-0.4% ethanol solution as a calibration standard or pretreated/diluted wine solution was dispensed followed by the addition of 50 µL of the enzymatic reactive mixture. The microplate was covered, mixed and allowed to incubate for 30 minutes at 37 ℃, and then the absorbance was measured at 550 nm using a microplate reader Emax (Molecular Devices, San Jose, USA). The ethanol concentration of each sample solution was calculated from the ethanol calibration curve using net absorbance minus the corresponding blank absorbance, where ADH was replaced in the enzyme solution with PBS to avoid interference with the sample matrix.

Fungal colony counts and isolation

The thawed wine sample was diluted 10³-fold or more with sterile PBS, and 0.2 mL each was smeared onto a PDA plate and then cultured at 25 ℃ for 7 days. The colonies that appeared were counted, and the number of active yeast cells was evaluated. Single colonies were inoculated in 3 mL of potato dextrose broth (PDB) medium (Eiken Chemical) containing 0.01% chloramphenicol and cultured at 25 ℃ for 3 to 7 days. Then, the fungal species were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or DNA sequence analysis.

Identification Of Yeast Species By Maldi-tof/ms

Reagent

A matrix HCCA (ɑ-cyano-4-hydroxycinnamic acid) and a calibration standard BTS (bacterial test standard) were prepared according to the manufacturer’s instructions (Bruker, Billerica, USA). A 70% formic acid solution was prepared by adding 21.4 mL of distilled water to 50 mL of formic acid (LC/MS grade, Fujifilm Wako). Acetonitrile (HPLC grade, Nacalai Tesque, Kyoto, Japan) was used as provided.

Protein extraction

Five hundred microliters of the cultured broth was placed in a 1.5 mL tube and centrifuged (9200 ×g, 5 min, 25 ℃), and the supernatant was removed by pipetting. Three hundred microliters of injection water (Otsuka Pharmaceutical, Tokyo, Japan) was added to the cell sediment and dispersed with a vortex, and 900 µL of 99.5% ethanol was added and mixed well. After centrifugation (16000 ×g, 2 min, 25 ℃), the supernatant was completely removed. Twenty microliters of 70% formic acid solution was added to the precipitate and dispersed with a vortex, and then 20 µL of acetonitrile was added and mixed well. After centrifugation (16000 ×g, 2 min, 25°C), 10 µL of the supernatant was placed in a 0.6 mL tube, which was used to prepare a protein extract just before use.

Measurement

One microliter of the protein extract was dropped onto the spot position of the Target Plate (MTP384 target plate polished steel BC, Bruker) and air-dried. One microliter of the HCCA matrix was overlapped on the same spot and air-dried again. In each sample, duplicate spots were prepared, and protein spectra were measured by a MALDI-TOF MS Autoflex with Flex Control software. Yeast species were identified by Biotyper software (above Bruker) using a house-prepared database (Sugawara et al. 2016; Hudagula et al. 2021).

Identification Of Fungal Species By Dna Sequence Analysis

Fungal species not listed in the MALDI-TOF/MS Biotyper database or not providing enough protein signals were submitted to the following DNA sequence analysis with a slight modification of an original protocol (Tu et al. 2019).

DNA extraction

The fungal suspension in broth medium (500 µL) was placed in a 1.5 mL tube and centrifuged (9200 ×g, 5 min, 25 ℃). Then, the supernatant was removed, and cellular pellets were obtained. The cells were suspended in 800 µL of 0.2% sodium dodecyl sulfate (SDS) in PBS solution and transferred to a 2 mL tube with a screw cap (BM equipment) containing different sizes of micro glass beads 300 mg of BZ-01 (0.1 mmφ) and two pieces of BZ-5 (5 mmφ) (both AS ONE, Osaka, Japan). The cells were crushed (2000 r/min − 3 min) using a BC-20 grinding shaker (Central Scientific Trade, Tokyo, Japan) and boiled (100 ℃, 10 min). After centrifugation (20630 ×g, 5 min, 25°C), 400 µL of the supernatant was submitted to a magLEAD 12 gC automatic nucleic acid extractor processed with a magLEAD Consumable Kit and Magtration Reagent MagDEA Dx SV (Precision System Science, Matsudo, Japan). Finally, 50 µL of the purified DNA solution was obtained and stored frozen at -30 ℃ until use.

PCR amplification: The DNA solution was used as a template for PCR amplification of the internal transcribed spacer (ITS) or D1/D2 regions of rDNA using primer pairs Fun-3 (ITS1F: 5'-GTAACAAGGT(T/C)TCCGT/ITS1R: 5'-CGTTCTTCATCGATG) or Fun-5 (NL1: 5'-GCATATCAATAAGCGGAGGAAAAG/NL4: 5'-GGTCCGTGTTTCAAGACGG) (Tu et al. 2019). The PCR mixture contained 14.1 µL of injectable water, 2 µL of 10×Ex Taq buffer, 1.6 µL of 2.5 mmol/L dNTPs, 0.1 µL of Ex Taq (Takara Bio, Kusatsu, Japan), 0.2 µL of the primer mix (each in 50 µmol/L forward and reverse primers) (Fasmac, Atsugi, Japan) and 2 µL of DNA template. The amplification reaction included an initial thermal denaturation at 94°C for 10 min, followed by 30 cycles of heat denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec using a thermal cycler (Thermo Fisher Scientific, Waltham, USA).

Agarose gel electrophoresis

For electrophoresis, 2% agarose gel including Tris acetate EDTA buffer (TAE) and 0.5 µg/mL ethidium bromide (Nippon Gene, Tokyo, Japan) were used with a running buffer of the same components (except for agarose). Ten microliters of the PCR product was electrophoresed at 100 V for 25 minutes, and the amplification band was assessed using a UV gel imaging device FAS-201 (Toyobo, Osaka, Japan).

DNA sequence analysis

PCR products were diluted 10- to 50-fold with injectable water in accordance with the amount of targeted PCR product confirmed by agarose gel electrophoresis. The forward or reverse PCR primers Fun-3 or Fun-5 were diluted to 1.6 pmol/L in injectable water. A total of 14 µL of the mixture, including the diluted PCR products (10 µL) and the primer (4 µL), was submitted for DNA sequencing (Fasmac). The sequences were applied to the Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI), GenBank. The criterion for a species estimation match was a concordance rate of 97% or higher, and the species that provided the maximum score were considered candidate species.

Evaluation Of Fungal Diversity By Arisa

DNA extraction from wine sediments

The thawed wine sample was vortexed sufficiently, and 20 mL was centrifuged (9200 ×g, 10 min, 25 ℃). Then, the supernatant was removed by decantation. The obtained sediments were dispersed in 1 mL of sterile PBS. A 0.2 mL portion of the suspended solution was transferred into a 2 mL screw cap tube containing the glass beads described above, and 800 µL of 2% cetyltrimethylammonium bromide (CTAB)-Tris EDTA NaCl (TEN) buffer solution (Fujifilm Wako) was added. The sediments were crushed, boiled and centrifuged for DNA sequence analysis. Six hundred microliters of the centrifuged supernatant was transferred to a new 2 mL tube, 600 µL of CHCl₃ was added, and the mixture was stirred for 15 seconds. Four hundred microliters of the aqueous supernatant obtained by centrifugation (21000 ×g, 10 min, 25 ℃) was submitted to the magLEAD automatic nucleic acid extractor described above. The extracted DNA solution was further purified using Ethachinmate (Nippon Gene) to remove botanical components that inhibit PCR as follows. To 40 µL of the DNA extract, 60 µL of injection water, 3.3 µL of 3 mol/L sodium acetate buffer and 1 µL of Ethachinmate were added and vortexed. One hundred microliters of 2-propanol was added, vortexed again, and then centrifuged (21000 g, 5 min, 25 ℃). After the supernatant was removed, 200 µL of 70% ethanol was added, vortexed, and centrifuged again (21000 ×g, 5 min, 25 ℃). The supernatant was completely removed, and then 40 µL of Tris-EDTA (TE), pH 8.0 solution was added and the pellet was dissolved by vortexing to finally obtain the purified DNA solution originating from the wine sediments.

PCR amplification for ARISA: The PCR conditions were the same as those described in the sequencing analysis except that the concentration of each primer was 25 µmol/L and the number of PCR cycles was 35. The final DNA solutions were submitted to PCR amplification using a universal HEX fluorescence-labeled primer pair for the fungal ITS regions: HEX-Fun-1 (2234C 5'-GTTTCCGTAGGTGAACCTGC/3126T 5'-ATATGCTTAAGTTCAGCGGGT) and HEX-Fun-2 (1406F 5'-TGYACACACCGCCGT/3126T HEX 5'-ATATGCTTAAGTTCAGCGGGT) (Tu et al. 2019). Ten microliters of the PCR product was submitted to fragment analysis (Fasmac). DNA solutions extracted from fungal colonies to identify the species were also subjected to ARISA, and their specific fragment sizes were determined. The obtained species or strain-specific ARISA fragments were considered identical if the size difference between the corresponding peaks was within ± 0.1%.

Antifungal activity against B. cinerea in grape berries (ex vivo)

The Kondo170908 strain of wild S. cerevisiae had a strong inhibitory effect (+++) on the mycelial growth of B. cinerea in grape berries (Fig. 2). Two strains, Kondo180304 and Kondo180305, showed moderate inhibitory effects (++). The remaining wild strains Kondo180303 and Kondo180306, as well as the commercially available yeast strain Pasteur Blanc, lacked an inhibitory effect (-).

Antagonistic effect on gray mold disease in vineyard fields (in vivo)

Morbidity of gray mold disease

The harvested grape berries were separated and weighed in each test plot as healthy and expressing gray-mold disease (Table 1). Test plot #16, sprayed with the yeast strain Kondo170908 both at flowering and ripening, showed significant suppression of morbidity to 11.2% against 15.3% of the control plot #15 and 16.3% of test plot #17, which was sprayed with yeast only at ripening (χ² test, p < 0.0001) (Fig. 3). Here, the weights (kg) were multiplied by 1000 based on the preliminary evaluated weights of 1 berry = approximately 1 g to estimate an integral number of grape berries in each group. Unexpectedly, the harvested grape weight of 11.2 kg in test plot #16 was obviously lower than the 15.3 and 16.3 kg in the control plot #15 and test plot #17, respectively. Thus, the yeast spray both in the flowering and ripening periods suppressed 4.1% morbidity of gray mold disease, although it decreased the grape yield by 20% (Table 1, Fig. 3).

Making of spontaneously fermented wine

Alcohol fermentation

On the fourth day of fermentation of grape berries, it was confirmed that in test plots #16 and #17 sprayed with yeast, the generation of carbon dioxide bubbles due to alcoholic fermentation progressed compared to control plot #15 (Fig. 4). In the comparison of ethanol production, on the seventh day of fermentation, the ethanol concentrations were 1.8% in control plot #15 and 0.8% in test plot #17, whereas they were more than twice as high at 4.1% in test plot #16 (Fig. 5). On the 13th day of fermentation, the ethanol concentration of control plot #15 was 6.0%, test plot #17 was 6.8% and test plot #16 was 7.5%. The acceleration of ethanol production, especially in test plot #16 sprayed with yeast at flowering and ripening, was remarkable compared to the other plots during the early stage of the spontaneously making wine. On the 23rd day of fermentation, the ethanol concentrations converged to approximately 12% in all plots.

Fungal colony counts and species identification

Fungal colony counts on the PDA plate of test plot #16 reached a maximum of 7.6×10⁵ cfu/mL, the earliest at the seventh day of fermentation, while the other plots#15 and #17 showed the highest counts after 23 days of fermentation (Fig. 6).

Eight yeast-like colonies in each plate were first submitted to species identification by MALDI-TOF/MS, followed by DNA sequencing analysis for the colonies not identified by MALDI-TOFMS. On the seventh day of fermentation in the wine samples of plots #15 and #16, typical wild yeasts, such as Kloeckera apiculate, Candida pulcherrima and Hanseniaspora uvarum, which are known to work during the beginning stage of wine nakiing (Ciani et al. 2010), were predominantly identified in addition to S. cerevisiae and/or Penicillium sp. (Table 2). After 13 days of fermentation, the yeast species converged to S. cerevisiae in all plots.

Fungal diversity obtained by amplified DNA fragment analysis (ARISA)

The fungal diversity of spontaneously making wine samples from each test plot was compared on Days 7 and 23 of fermentation. The two fragment groups 865/866 and 959/960 bps corresponded to different strains of S. cerevisiae (Hudagula et al. 2021). The fragment 959/960 bp was equivalent to the sprayed yeast strain KONDO170908 (Fig. 7). In test plot #17, unrelated to the fermentation period, a yeast strain providing the same band size as the sprayed strain was abundant. In contrast, in plots #15 and #16, two strains of S. cerevisiae were present, although their existing ratios were different, especially after 7 days of fermentation.

Vineyard fields are abundant habitats for a variety of microorganisms, including native yeasts (Pearson and Taschenberg 1980; De Simone et al. 2020). This study aimed to verify the possibility of using the vineyard-derived wild yeast S. cerevisiae as a microbial pesticide against B. cinerea, the cause of grape gray-mold disease, and we expected to simultaneously find that the yeast contributes to boosting the initial alcohol production of spontaneously fermented wine.

Minimizing the use of chemical pesticides is an urgent issue to reduce the environmental burden accumulated year by year (Freimoser et al. 2019). Spontaneous making of a natural wine without a spike of a starter yeast is critical to control initial alcohol fermentation to prevent the expansion of unfavorable microbes such as acetic acid bacteria (Hall et al. 2018). Here, wild yeast, originating from vineyards, was cultured and sprayed on grapes as a microbial pesticide. At harvest, the sprayed yeast carried with the grapes promises safer wine making by the acceleration of initial alcohol fermentation. The goal was to build and promote the wild yeast circulation system from vineyard to wine making.

From its clear antifungal activities against B. cinerea in in vitro (Hudagula et al. 2021) and ex vivo (Fig. 2) studies, the KONDO170908 strain was selected as a candidate microbial pesticide for vineyard gray-mold disease. The yeast spray test in the vineyard field proved a statistically significant suppression of gray-mold disease on grape berries in test plot #16, where the yeast was sprayed during both the flowering and ripening periods (Figs. 1, 3). Test plot #17, sprayed with yeast only in the ripening period, did not show any suppression and resulted in nearly the same morbidity as control plot #15. In our preliminary study in the previous season (2020), we also experienced no effect of the yeast spray provided only in the ripening period on the suppression of morbidity (data not shown). B. cinerea inoculation of grape bunches was reported to occur most frequently during the flowering period and is sustained through ripening in berries (Keller et al. 2003), which is in good agreement with the results of our field tests. Thus, the yeast spray at the ripening period is unlikely to be effective for the prevention of gray mold disease on grape bunches in vineyard fields, although the results of the ex vivo test clearly showed suppression of B. cinerea growth (Fig. 2). This discrepancy can be explained by the different mechanisms between ex vivo and field in vivo antifungal actions in addition to nonidentical pretreatment on the surface of the berry skin with (ex vivo) or without (in vivo) forced injuries. The main inhibitory actions of biological control yeasts on pathogenic fungi are recognized as competitions between nutrient sources and space or the production of some toxic substances (Fremoser et al. 2019). Furthermore, it is also well known that many environmental factors, such as weather conditions, will modify the effectiveness obtained under laboratory conditions (Cañamás et al. 2011).

A previously reported approach to control B. cinerea in grape bunches in vineyard fields focused on the survival of the antagonistic yeast Candida sake CPA-1 (Cañamás et al. 2011; Calvo-Garrido et al. 2013). A formula using a fatty acid-based additive from flowering to harvest times was shown to be effective in prolonging the biocontrol ability of C. sake CPA-1 and then reducing the incidence and severity of botrytis bunch rot by 64 and 90%, respectively (Calvo-Garrido et al. 2013). In contrast, our yeast recipe, based on simple culture broth, was selected to avoid using any extra additives. The reported magnitude of effectiveness is much larger than our 4.1% decrease in morbidity (Table 1, Fig. 3). This variance is attributable to the different recipes or formulas used for the biocontrol agents. Furthermore, there were different approaches to judge gray mold disease between their visual inspection of botrytis bunch rot and our quantitative evaluation of counting healthy and affected berries in each grape bunch. The incidence of bunches affected by gray mold diseases in untreated plots was reported to be 80–90% on Macabeu wine grapes (Calvo-Garrido et al. 2013), while our incidence was more than 90% on Pinot noir wine grapes. The control incidences of gray mold diseases, as well as the efficacy of the yeast spray, may also be influenced by the differences in terroir and grape species.

On the other hand, the yield of harvested grapes was 11.6 kg in test plot #16, where the yeast was sprayed during the flowering period, which was 20 to 30% lower than the 14.4 of the control and 16.0 kg in the other test plot (Table 1). The spray of liquid yeast broth may have affected grape fruiting at the critical time of flowering. It is well known that natural rainfall during the flowering period often decreases the fruiting of grapes. To determine the optimal use of the yeast spray, more detailed studies will be necessary both for achieving suppression of gray-mold damage and by maintaining the harvest yield of grapes.

Regarding the early stages of making of spontaneously fermented wines, alcoholic fermentation preceded the most in test plot #16, where the yeast was sprayed during both the flowering and ripening periods, since the most active CO₂ gas bubbling (Fig. 4), the highest ethanol production (Fig. 5) and the most abundant colony counts (Fig. 6) were observed until seven days of fermentation. These results may suggest that the sprayed yeast population itself did not survive at the harvest time and did not necessarily directly influence the initial alcohol fermentation since test plot #17, which was sprayed only during the ripening period, did not promote fermentation, resulting in the same tendency as control plot #15. To explain this unexpected discrepancy, the following speculation is proposed. The yeast spray at the flowering period triggered modification of the entire microbiota inside immature berries, not limited to B. cinerea alone. The microbial stimulation in premature fruiting promoted an alternatively dominant yeast population through the growing and ripening periods of the berries over several months and could finally contribute to promoting alcohol fermentation at harvest time. The unique proportion of the two S. cerevisiae strains of fragment sizes 865/866 and 959/960 bps in the spontaneously fermented wines could also support this hypothesis (Fig. 7). Here, the fragment size of the sprayed strain KONDO170908 was 959.79 bp. The peak ratios of these strains on the seventh day of fermentation were contrasted sharply between plot #16 and the other plots. At the early stage of making, alcohol fermentation in test plot #16 was considered mainly derived from strain 865/866 bp and not from the spiked strain 959/960 bp due to the existing ratio of these strains after seven days of fermentation (Fig. 7 − 1). The relative peak height of strain 865/866 bp was deceased and close to the existing ratio of control plot #15 at the terminal stage after 23 days of fermentation (Fig. 7 − 2). These data will explain why the yeast strain sprayed 20 days ahead of the harvest time (Fig. 1) did not contribute to the initial alcohol production of the plot #16 wine, but alternative strains derived during ripening could promote alcohol fermentation.

The yeast diversity, especially at the early stage of making spontaneously fermented wine, is important to dominate the quality of matured wines both by adverse influences, such as unfavorable ester flavors, and by superior deep and composed impressions (Ciani et al. 2009; Cordero-Bueso et al. 2017). The yeast species identified on the PDA colonies show a time-dependent transition of the complexed microbiota of the initial fermentation to a simple composition (Table 2). After seven days of fermentation, K. apiculata was more often identified as well as S. cerevisiae. Due to the lack of species diversity, all colonies were S. cerevisiae. In test plot #17, only one major peak, 959/960 bp, of the S. cerevisiae strain was identified both after seven and 23 days of fermentation (Fig. 7). The fragment 959/960 bp corresponds to the sprayed strain; however, even in control plot #15 without the yeast spray, the same-sized fragment was also detected. From the beginning, the sprayed yeast strain was separated from the same vineyard origin (Hudagula et al. 2021); therefore, this strain is considered a widespread habitat, although its alcohol fermentation ability may be inferior to that of another yeast strain with a fragment of 865/866 bp (Figs. 5, 7).

Contributions to the organic cultivation of grapes, avoiding the damage of gray mold disease and to the achievement of superior making by acceleration of early alcohol fermentation in spontaneously fermented wine, were the goals of this study. A vineyard habitat-yeast strain selected from the laboratory in in vitro and ex vivo inhibition tests for B. cinerea growth was submitted to an in vivo field test in the vineyard. The selected S. cerevisiae strain KONDO170908 was proven to have a suppressive effect on gray mold disease when the yeast was sprayed in the flowering period in addition to the ripening period. However, the yeast spray unexpectedly caused a 20% decrease in the harvest yields of the grapes. Thus, the initial goal was achieved in part, but it is necessary to verify more detailed conditions such as the timing and composition of the yeast spray or exposure amounts to improve the effectiveness and maintain the harvest yields. Furthermore, a basic study will be necessary to clarify the mechanism by which the sprayed yeast strain modifies the original microbiota inside grape berries and ultimately promotes alcohol fermentation in spontaneously fermented wine.

Funding This study was supported in part by Grants-in-Aid for Regional R&D Proposal-Based Program from Northern Advancement Center for Science & Technology of Hokkaido Japan.

Conflicts of interest The authors declare no conflicts of interest.

Availability of data and material Not available.

Code availability Not available.

Authors' contributions Hu Hudagula, Ryosuke Kondo, and Akihiro Yamaguchi conceived the study. Hu Hudagula and Akihiro Yamaguchi drafted the manuscript. Hu Hudagula colledted the data. Naoko Maeno, Noriko Minami, Soichiro Takahashi, Kuniko Yoshida, and Ryosuke Kondo provided technical supports. Katsuki Ohtani and Yasuhiro Funatsu provided a critical revision of the manuscript. All the authors contributed equally to the manuscript and read and approved the final version of the manuscript.

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Table 1-2 is available in the Supplemental Files section.

No competing interests reported.

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Antifungal activities of vineyard-habitat wild yeast for grape gray-mold disease and its initial promotion of alcohol fermentation in spontaneous winemaking

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Abstract

Figures

Introduction

Materials And Methods

Yeast strains

Antifungal Test On Grape Berries (Ex Vivo)

Antifungal Test On Grape Bunches In Vineyard Field (In Vivo)

Making Natural Wine

Identification Of Yeast Species By Maldi-tof/ms

Identification Of Fungal Species By Dna Sequence Analysis

Evaluation Of Fungal Diversity By Arisa

Results

Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

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