Unveiling the cultivable yeast endophyte diversity in muscadine grape berries and their known functional prospects

Muscadine grapes, which are valued for their fresh consumption, boutique wine production with distinctive flavors and aromas, and disease resistance, provide a unique ecological niche for investigating endophytic yeast communities. Despite their potential, the cultivable diversity and functional capabilities of these yeasts remain largely underexplored. In this study, we investigated culturable endophytic yeasts from the unskinned berries of diverse muscadine and bunch grape cultivars. Through molecular identification and morphological analysis, we identified 48 distinct yeast isolates spanning 18 species and 12 genera, with a predominance of non-Saccharomyces yeasts. Hanseniaspora guilliermondii emerged as the most abundant species, followed by members of the Pichia genus. Notably, novel species such as Nakaseomyces nivariensis and Wickerhamiella sorbophila were discovered, expanding the known diversity of grape-associated yeasts. Their documented roles in other grape systems highlight species-specific abilities to enhance wine quality, control pathogens, promote plant growth, and offer broader biotechnological applications. These findings not only deepen our understanding of yeast–grape interactions but also highlight diverse microbial reservoirs with promising applications in agriculture, food science, and biotechnology. This work lays the foundation for leveraging endophytic yeasts to increase crop quality, sustainability, and innovation across multiple domains.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-025-04291-y.

Highlights

Diverse non-Saccharomyces yeast genera were isolated from grape cultivars.

H. guilliermondii was the dominant species, followed by Pichia spp.

Isolates exhibited varied colony and cellular morphologies.

Novel grape-associated species, N. nivariensis and W. sorbophila, were identified.

Literature underscores species-specific roles in wine fermentation and biocontrol.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-025-04291-y.

Introduction

Endophytic microorganisms, which inhabit the internal tissues of plants, play pivotal roles in modulating host plant metabolism and providing resilience against various stresses [1]. Among these microorganisms, fungi, particularly yeasts, have garnered significant attention because of their ability to synthesize diverse bioactive compounds. These unicellular fungi thrive in plant tissues through asexual budding and adapt dynamically to the host’s internal environment, which shapes their community composition and functional attributes [2].

Grapevines are among the most economically significant fruit crops worldwide and are utilized in various forms, including fresh and dried fruit, juice, jam, jelly, and, most notably, wine. Yeasts associated with grapevines are well known for their impact on wine fermentation, as they metabolize grape sugars and other berry components to produce ethanol, carbon dioxide, and a spectrum of secondary metabolites that contribute to wine composition, flavor, and quality [3]. Traditionally, Saccharomyces cerevisiae (S. cerevisiae) has been the dominant yeast species employed in winemaking. However, research has highlighted the importance of non-Saccharomyces yeasts, which contribute to wine aroma, flavor complexity, and overall chemical composition [4]. For example, coinoculation of Hanseniaspora uvarum with S. cerevisiae has been shown to enhance tropical fruit flavors by increasing ethyl ester production and reducing acetic acid content in Cabernet Sauvignon and Chardonnay wines [5]. Similarly, cofermentation of S. cerevisiae with Pichia kluyveri has been reported to increase the production of 3-mercaptohexyl acetate (3-MHA), a key varietal aroma compound, in Sauvignon Blanc wine [6].

In addition to their role in fermentation, grapevine-associated yeasts exhibit biocontrol potential by secreting lytic enzymes, volatile organic compounds (VOCs), and antifungal metabolites and competing for nutrients and space [7, 8]. For example, strains from the genera Pichia and Meyerozyma have been reported to inhibit fungal competitors via VOC-mediated antagonism [9]. Additionally, P. kluyveri has been shown to mitigate spoilage microorganisms during winemaking [10]. Yeasts also produce bioactive secondary metabolites, including phenols, flavonoids, stilbenoids, isoprenoids, and sesquiterpenes, with applications spanning pharmaceuticals, cosmetics, and the food industry. Furthermore, certain yeasts exhibit probiotic potential due to their resistance to antibacterial agents, with S. cerevisiae var. boulardii being the most recognized probiotic yeast in human health [11]. Additionally, given the acidic nature of grapes, grape-associated yeasts may possess traits that allow them to survive in the gastrointestinal tract, making them promising candidates for probiotic applications.

Plants with medicinal/nutraceutical value have been a key focus in the search for novel endophytes with multiple applications. In this context, muscadine grape (Vitis rotundifolia), recognized for its high levels of bioactive compounds, natural disease resistance, and unique flavor and aroma in muscadine wine [12, 13], is an ideal candidate for exploring endophytic yeast species. Despite the prevalence of endophytic yeasts within muscadine grapes and their close association with vines, their diversity and functional potential remain underexplored.

This study aims to characterize the diversity of cultivable endophytic yeasts across different muscadine grape cultivars. To provide a comparative perspective, few bunch grape cultivars (Euvitis spp. hybrids) grown in the same vineyard and under similar environmental conditions were also examined to determine whether they harbor similar yeast communities. To accomplish this goal, we enriched and isolated yeast endophytes from the pulp of ripe grape berries, identified them via molecular techniques, characterized their diversity and morphological traits, and assessed their known functional applications in grapevines. By elucidating these microbial communities, this research seeks to identify endophytic yeast species with potential applications in fermentation, biocontrol, and plant and human health, contributing to the advancement of sustainable viticulture and biotechnological innovation.

Materials and methods

Grape berry sample collection and processing

Healthy ripe grape berries from fifteen distinct muscadine grape cultivars and five bunch grape cultivars were harvested and processed as previously described [14]. Owing to their early ripening, bunch grapes were collected in August 2021, whereas muscadine grape cultivars were harvested in September 2021. To maintain consistency in terms of environmental conditions, all the grape samples were collected from the same vineyard.

Yeast enumeration, isolation and purification

Berry surface sterilization and yeast enumeration were carried out as previously described, with minor modifications [14]. Yeast strains were enumerated via YPD media [15] instead of LB media. Colonies with distinct morphologies were selected and streaked onto fresh YPD plates to obtain isolated colonies. These were examined microscopically to confirm purity and single-species identity, while morphologically similar colonies from the same grape sample were excluded. Pure strain stocks were prepared following established protocols [16].

Molecular identification of isolates

Individual colonies from each isolate were cultured in YPD broth for two days, and genomic DNA was extracted using the ZR Fungal/Bacterial DNA Kit (Zymo Research, Irvine, CA, USA). For 18 S rDNA amplification, 20–50 ng of DNA was used with primers 18 S-NS1F (5′–GTAGTCATATGCTTGTCTC–3′) and 18 S-FR1R (5′–AICCATTCAATCGGTAIT–3′). PCR conditions included an initial denaturation at 98 °C for 2 min, followed by 30 cycles of 98 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. Amplification products were verified by agarose gel electrophoresis and purified using Exo-CIP (New England BioLabs). Sanger sequencing was performed using the 18 S-NS1F primer. Resulting sequences were viewed and manually edited in FinchTV v1.4.0 and analyzed using BLASTn against the NCBI database. Species-level identification was based on top hits with 100% query coverage and percent identities above 99%, except for PV568514 (97.89%), PV568524 (98.10%), PV568529 (98.88%), and PV568530 (93.94%).

Microscopic imaging

Yeast cell morphology and structural characteristics were examined via freshly growing cultures on YPD plates. Light microscopy images were captured as described previously [17].

Data analysis

To assess diversity at the species, genus, and family levels, the percentage of each taxon per cultivar was calculated by dividing the number of isolates belonging to a given taxon by the total number of isolates obtained from that cultivar, and multiplying by 100. The resulting values were visualized using bar charts. For relative abundance of yeast species across grape cultivars, the total number of isolates for each species was summed across all cultivars, divided by the total number of all isolates from all cultivars, and multiplied by 100. The results were visualized using pie charts. Similar calculations were performed to estimate relative abundance at the genus and family levels.

Results

Grape cultivar selection, and berry characteristics

The collected muscadine grape cultivars included Black Fry, Carlos, Cowart, Doreen, Hunt, Jumbo, Late Fry, Noble, Pineapple, Scuppernong, Southland, Summit, Tara, Welder and Southern Home. Notably, Southern Home is a hybrid resulting from a cross between muscadine and bunch grape varieties [18]. Upon review of their uses, these cultivars were found to serve a wide range of purposes, including fresh market consumption, juice, jelly, jam, and wine production. In particular, Noble, Carlos, and Welder have been predominantly utilized for winemaking, as well as in juice and jelly production (Andersen et al., 2020). The bunch grape cultivars (Euvitis spp. hybrids) included in this study included Black Spanish, Blanc Du Bois, Blue Lake, MidSouth, and Stover. The grape cultivars presented variations in berry coloration and size (Fig. 1). pH analysis of the ripe berries revealed a range from 2.7 to 3.6, with cv. Noble and Carlos presenting the lowest pH values (Table 1). The total soluble solids, measured in Brix, varied from 13.6 to 18.2 (Table 1). Among the tested cultivars, Dorren and Blue Lake had the lowest Brix value, while the cv. Black Spanish recorded the highest Brix value (Table 1).

Fig. 1.

Photographs showing berries collected from different grape cultivars. A Black Fry, (B) Carlos, (C) Cowart, (D) Doreen, (E) Hunt, (F) Jumbo, (G) Late Fry, (H) Noble, (I) Pineapple, (J) Scuppernong, (K) Southland, (L) Summit, M) Tara, N) Welder and O) Southern Home, P) Black Spanish, Q) Blanc Du Bois, R) Blue Lake, S) MidSouth, and T) Stover

Table 1.

The table summarizes the grape cultivars used in this study, including their grape type, berry color, pH, and Brix levels

Grape type	Grape cultivar	Berry color	Berry pH	Berry brix
Muscadine grapes (V. rotundifolia Michx.)	Black Fry	Black	3.35 ± 0.13	16.3 ± 0.26
	Carlos	Bronze	2.89 ± 0.02	15.56 ± 0.15
	Cowart	Black	3.62 ± 0.17	15.55 ± 0.18
	Doreen	Bronze	3.26 ± 0.07	13.5 ± 0.26
	Hunt	Black	3.32 ± 0.02	15.43 ± 0.25
	Jumbo	Black	3.41 ± 0.07	17.33 ± 0.32
	Late Fry	Bronze	3.21 ± 0.08	16.4 ± 0.43
	Noble	Black	2.75 ± 0.05	15.5 ± 0.26
	Pineapple	Bronze	3.25 ± 0.05	15.33 ± 0.15
	Scuppernong	Bronze	3.49 ± 0.17	15.23 ± 0.11
	Southland	Black	3.65 ± 0.14	15.5 ± 0.1
	Summit	Bronze	3.44 ± 0.21	17.1 ± 0.34
	Tara	Bronze	3.5 ± 0.1	15.33 ± 0.37
	Welder	Bronze	3.38 ± 0.12	17.56 ± 0.41
Interspecific Hybrid (V. rotundifolia × V. vinifera)	Southern Home	Black	3.64 ± 0.12	16.46 ± 0.35
Bunch grapes (Euvitis spp. hybrids)	Black Spanish	Black	3.48 ± 0.02	18.26 ± 0.30
	Blanc Du Bois	Bronze	3.64 ± 0.05	15.3 ± 0.51
	Blue Lake	Black	3.41 ± 0.08	13.66 ± 0.61
	MidSouth	Black	3.48 ± 0.02	16 ± 0.2
	Stover	Bronze	3.66 ± 0.04	17.16 ± 0.15

Isolation and molecular identification revealed the presence of diverse yeast endophytic species across grape cultivars

A total of twenty grape cultivars were selected for the study. The enrichment of endophytic yeast populations in liquid broth followed by successive plating on agar media revealed a diverse range of yeast colonies, distinguishable by their morphological appearances. Further molecular identification of these morphologically distinct and purified isolates via 18 S rRNA gene sequencing resulted in the identification of 48 culturable endophytic yeast isolates under the tested laboratory conditions (Fig. 2A). The composition of the yeast isolates varied to some extent among the different grape cultivars (Fig. 2A, B). Among the 48 isolates, 18 distinct yeast species were identified and deposited in the NCBI database, with the corresponding accession numbers listed in Table 2. The taxonomic classification of these species is detailed in Table S1.

Fig. 2.

Distribution and diversity of yeast communities across grape cultivars. A Presence‒absence matrix displaying the distribution of 48 yeast species across 20 grape cultivars. The colored dots indicate the presence of specific yeast species in the different grape cultivars. B-D Bar charts illustrating the diversity and composition of yeast communities within each grape cultivar. Each bar is subdivided into segments representing individual taxa at different taxonomic levels: species (B), genus (C), and family (D). E-G Pie charts summarizing the overall relative abundance of yeast communities across all cultivars, represented at the species level (E), genus level (F), and family level (G)

Table 2.

GenBank accession numbers of yeast isolates from this study

Yeast Isolate	Abbreviation	Accession Number
Hanseniaspora guilliermondii AM-72	H. guilliermondii	PV568513
Hanseniaspora uvarum AM-102	H. uvarum	PV568514
Pichia kudriavzevii AM-59	P. kudriavzevii	PV568515
Pichia kluyveri AM-81	P. kluyveri	PV568518
Pichia manshurica AM-132	P. manshurica	PV568517
Pichia terricola AM-79	P. terricola	PV568516
Pichia sporocuriosa AM-86B	P. sporocuriosa	PV568519
Pichia fermentans AM-88	P. fermentans	PV568520
Meyerozyma caribbica AM-54	M. caribbica	PV568521
Nakaseomyces nivariensis AM-62	N. nivariensis	PV568522
Zygoascus hellenicus AM-64	Z. hellenicus	PV568523
Zygosaccharomyces bailii AM-133	Z. bailii	PV568528
Lachancea thermotolerans AM-81D	L.thermotolerans	PV568524
Starmera stellimalicola AM-84	S. stellimalicola	PV568525
Candida parapsilosis AM-85B	C. parapsilosis	PV568526
Wickerhamiella sorbophila AM-111	W. sorbophila	PV568530
Wickerhamomyces anomalus AM-103	W. anomalus	PV568529
Saturnispora diversa AM-87 A	S. diversa	PV568527

Analysis of yeast species diversity revealed that the number of species per cultivar ranged from 1 to 5 (Fig. 2A, B). The Black Spanish cultivar harbored the highest diversity, with five distinct yeast species, followed by the Noble and MidSouth cultivars, each hosting four species. The remaining cultivars included between one and three species each (Fig. 2A and B). Among the identified species, H. guilliermondii emerged as the most widespread, being present in nearly all cultivars except Blanc Du Bois and Blue Lake (Fig. 2A and B). At the genus level, Hanseniaspora was the most common, followed by Pichia (Fig. 2C). Similarly, at the family level, Saccharomycodaceae was the most prevalent, followed by Pichiaceae (Fig. 2D).

A cumulative analysis of all yeast species across cultivars revealed that H. Guilliermondii dominated the yeast community, comprising up to 37% of the total isolates. This was followed by P. kudriavzevii (12%), P. kluyveri (8%), and P. manshurica (8%), while the remaining species contributed between 2 and 4% each (Fig. 2E). At the genus level, Hanseniaspora constituted 41% of the isolates, followed by Pichia (37%), with other genera representing 2% each (Fig. 2F). At the family level, Saccharomycodaceae accounted for 41%, Pichiaceae 39%, and Saccharomycetaceae 12% of the yeast community (Fig. 2G). Minor families, including Trichomonascaceae and Wickerhamomycetaceae, were present at relatively low abundances and were often restricted to specific cultivars, indicating potential niche-specific colonization.

H. guilliermondii was identified as a dominant and commonly present species in both muscadine and bunch grape cultivars

To investigate whether muscadine and bunch grape cultivars presented differences in yeast species composition and community structure, we analyzed the distribution and proportion of yeast species unique to each group. The Muscadine cultivars harbored a total of 15 distinct yeast species, with H. guilliermondii being the most dominant (41%), followed by P. kudriavzevii (13%) and P. manshurica (10%) (Fig. 3A). In contrast, bunch grape cultivars contained only 7 yeast species, among which P. kluyveri was the most prevalent (30%), followed by H. guilliermondii (20%), with the remaining species contributing 10% each (Fig. 3B). A comparative analysis revealed that four yeast species—H. guilliermondii, P. kudriavzevii, P. kluyveri, and P. terricola—were shared between both the muscadine and bunch cultivars, whereas the remaining species were group-specific (Fig. 3C).

Fig. 3.

Distribution of yeast species in muscadine vs. bunch grapes. A–B Pie charts representing the relative abundance of yeast species isolated from muscadine (A) and bunch (B) grape cultivars. C) Venn diagram illustrating the distribution of yeast species between the muscadine and bunch cultivars. The overlapping region indicates species common to both groups, whereas nonoverlapping areas represent group-specific species. D Heatmap displaying the presence (dark color) and absence (light color) of individual yeast species across the 20 grape cultivars. Hierarchical clustering along both axes reveals patterns of species co-occurrence and cultivar-specific associations. Each row represents a grape cultivar, and each column corresponds to a specific yeast species

Furthermore, hierarchical clustering of all cultivars on the basis of the presence of yeast species clearly revealed patterns of co-occurrence and cultivar-specific associations (Fig. 3D). H. guilliermondii was broadly distributed across most cultivars, whereas many species presented more limited or cultivar-specific occurrences. The clustering also revealed notable grouping patterns, such as bunch cultivars: MidSouth, Blue Lake, and Black Spanish formed a distinct cluster, with Blanc du Bois closely associated. Stover clustered with the rest of the muscadine cultivars, reflecting a probable correlation between cultivar type and associated yeast communities.

Yeast isolates displayed variations in colony morphology and cellular structure

Morphological and microscopic examination of 18 distinct yeast isolates revealed considerable variation in colony appearance, cell shape, and size, as illustrated in Fig. 4. Closer observation of individual colonies revealed textures ranging from smooth to rough, with coloration varying from white to cream. Colony elevations were observed as either flat or raised. Microscopic examination further highlighted variations in cell morphology, including spherical and ellipsoidal shapes, as well as differences in cell size and the presence of budding structures. The species-specific characteristics shown in Fig. 4 aid in visual differentiation and preliminary identification of the isolated yeast species.

Fig. 4.

Morphological characteristics of endophytic yeast isolates. Each species is shown in three panels: A Growth patterns of individual yeast isolates on YPD agar plates after 2 days of incubation. B Close-up images of individual colonies showing colony surface texture and pigmentation. C Differential interference contrast (DIC) microscopy images of yeast cell morphology. Scale bar = 5 μm

Discussion

Muscadine grapes are increasingly recognized for their unique value as fresh fruit, winemaking, and notable resilience to environmental stressors. While endophytic yeasts have been acknowledged as ecologically significant in grapevine systems, their diversity and functional potential within muscadine cultivars remain understudied. This work evaluated cultivable endophytic yeasts inhabiting the berry pulp of muscadine and bunch grape cultivars. Our findings provide important insights into the taxonomic diversity, cultivar-specific associations, morphological characteristics, and biotechnological potential of endophytic yeasts.

Diversity and distribution of endophytic yeasts across cultivars

Our isolation and molecular characterization of yeast isolates revealed a rich assemblage of twelve genera, predominantly composed of non-Saccharomyces taxa as detailed in Table 2; Fig. 2. Morphological analysis highlighted distinct features among the strains, reflecting the phenotypic variability within the collection. This observed diversity underscores the ecological adaptability of yeasts and emphasizes the value of integrating morphological and molecular approaches for accurate taxonomic identification. Notably, most of these identified species have previously been reported in grape and fermentation-related environments [9, 19–21].

Interestingly, two yeast species, N. nivariensis and W. sorbophila, were detected, neither of which, to our knowledge, has been previously reported in grapevine-associated microbiota. Given their prior identification in clinical and dairy environments [22, 23], their presence in grape pulp may suggest either rare, overlooked members of the grape endosphere or environmental introductions. Future studies using metagenomic and longitudinal approaches will be critical to understanding their origin, persistence, and ecological function.

Among all the isolates, H. guilliermondii was the most abundant, followed by Pichia species. These findings align with prior reports highlighting Hanseniaspora dominance in grape juice environments [24] and the prevalence of Pichia species in grapes [7]. Nevertheless, our methodology of targeting only cultivable endophytes from pulp tissue likely underrepresents total yeast diversity, especially skin-associated or nonculturable taxa. Repeated sampling across seasons and environments is crucial for a more comprehensive understanding.

Cultivar-specific species-level analysis revealed distinct yeast community structures between muscadine and bunch grapes. H. guilliermondii predominated in muscadine grapes, whereas P. kluyveri was more prevalent in hybrid bunch grapes. Venn analysis further supported cultivar-specific patterns, showing that four yeast species were shared between both grape types, whereas muscadines and bunches harbored seven and four unique species, respectively. In hierarchical clustering analysis, bunch grape cultivars such as MidSouth, Blue Lake, and Black Spanish formed a discrete cluster, with Blanc Du Bois being closely associated, indicating shared microbial profiles. In contrast, Stover clustered closely with muscadines, suggesting that genotype-driven traits may influence microbial assembly. Although all the cultivars were grown under similar environmental conditions, the variation in Brix and pH values among the bunch grapes was less pronounced than that among the muscadine grapes. However, inherent differences in berry characteristics, such as sucrose concentration, phenolic content, or other yet unidentified traits, may influence patterns of endophytic colonization. For example, muscadine grapes have been reported to accumulate relatively high levels of sucrose in ripe berries [25] and exhibit distinct differences in phenolic content [26].

These findings underscore the significant influence of host genotype in structuring grape-associated microbiomes and highlight the need for broader cultivar representation and integrative studies employing shotgun metagenomics and microbial ecology frameworks to fully unravel host–microbe interactions in grapevine systems.

Functional insights into isolated species

Endophytic yeasts are increasingly recognized not only as passive colonizers but also as active contributors to plant health and biotechnological innovation. We examined the known functions of these identified yeast isolates from other grapevines. Notably, most of the species isolated from this study demonstrated significant roles in fermentation processes and biocontrol activities, as summarized in Tables 3 and 4, respectively.

Table 3.

Functional insights into yeast species isolates related to wine fermentation

Species	Functions	Reference
H. guilliermondii	Exhibits strong β-glucosidase activity and enhances wine sensory characteristics by increasing volatile terpenes and higher alcohols. Sequential inoculation with S. cerevisiae improves the aroma profile of the final beverage.	[27–29]
H. uvarum	Produces β-D-glucosidase, esterase, and volatile compounds that enhance wine fermentation. In mixed fermentations, it increases ethyl esters, reduces acetic acid levels, and improves overall wine quality.	[5]
P. kudriavzevii	Enhances wine aroma through the production of esters, higher alcohols, and volatile acids. Co-fermentation with S. cerevisiae increases ethyl esters, glycerol, and aroma compounds like phenylethyl and isoamyl alcohol. Improves antioxidant activity, polyphenol content, and tolerates high sugar and low pH, making it suitable for sweet and acidic wines.	[30–32]
P. kluyveri	Enhances aromatic compounds including thiols, terpenes, and fruity esters. Co-fermentation with S. cerevisiae increases levels of 3MHA, 3SHA, 2-phenylethyl acetate, and glycerol, contributing passionfruit and floral notes.	[6, 32, 33]
P. manshurica	Considered a spoilage yeast. Known to increase volatile phenols and off-odors.	[34]
P. terricola	Controls spoilage microorganisms. Enhances volatile content due to β-glucosidase activity. Releases glycosidically bound aroma precursors. Coinoculation with S. cerevisiae improves wine aromatic profiles.	[35–37]
P. sporocuriosa	Identified as part of grape yeast diversity. Functional application not yet well studied.	[38]
P. fermentans	Exhibits oxidative metabolism, producing organic acids, acetaldehyde, ethyl acetate, and isoamyl acetate, contributing to wine aroma. In mixed cultures, increases wine polysaccharide content.	[39]
M. caribbica	Shows weak fermentation capacity and intermediate phenotypes during in vitro glucose fermentation.	[40]
N. nivariensis	Not previously reported in grapes.
Z. hellenicus	Associated with wine spoilage and quality deterioration.	[41]
Z. bailii	Costarter with S. cerevisiae increases ethyl ester production. Shows high fermentative vigor, low acetic acid production, and malic acid degradation. Known spoilage yeast resistant to preservatives; may cause refermentation in sweet wine.	[41–44]
L. thermotolerans	Possesses moderate fermentative power; requires co-fermentation with S. cerevisiae. Reduces acetic acid production under aerobic conditions, improving wine quality.	[45–47]
S. stellimalicola	Also known as Candida stellimalicola. Increase aldehyde and ketone content in sequential fermentation.	[48]
C. parapsilosis	Shows intermediate fermentation ability.	[49, 50]
W. sorbophila	Not yet reported in grape environments.
W. anomalus	Mixed starter fermentations with S. cerevisiae improve aroma through production of acetate esters, and contribute floral and fruity notes. Also aids in haze prevention.	[51]
S. diversa	Exhibits low fermentative capacity and lower alcohol yield but higher volatile acidity compared to S. cerevisiae.	[52]

Table 4.

Functional insight of yeast species in relation to biocontrol potential

Species	Functions	Reference
H. guilliermondii	Colonizes grape wound sites and inhibits Aspergillus spp. in Thompson Seedless cultivar. Exhibits antagonistic activity against Botrytis cinerea, A. carbonarius, and P. expansum.	[7, 53]
H. uvarum	Acts as a biocontrol agent against B. cinerea, P. expansum, and A. carbonarius through lytic enzyme production that hydrolyzes fungal cell walls. Also effective against A. tubingensis and P. commune.	[7, 54, 55]
P. kudriavzevii	Reduces gray mold in grapes by producing VOCs. Exhibits antagonistic activity against A. niger and A. alternata.	[56, 57]
P. kluyveri	Functions as a bioprotective agent in wine fermentation, reducing the need for sulfites. Shows antagonistic activity against B. cinerea and M. laxa, and inhibits hyphal growth of B. cinerea and P. expansum.	[7, 10]
P. manshurica	Functions as a biocontrol agent for postharvest grape diseases.	[58]
P. terricola	Inhibits Penicillium glabrum, a spoilage fungus in grapes and wine.	[59]
P. sporocuriosa	Identified as part of grape yeast diversity. Functional application not yet well studied	[38]
P. fermentans	Potential to reduce hyphal growth of grape pathogens like B. cinerea and P. expansum.	[7]
M. caribbica	Produces VOCs with antifungal effects against A. fumigatus, F. poae, P. chrysogenum, Mucor spp., and B. cinerea.	[9]
N. nivariensis	Not yet reported in grape environments.
Z. hellenicus	No evidence of biocontrol activity reported.
Z. bailii	No evidence of biocontrol activity reported.
L. thermotolerans	Exhibits antagonistic activity against Mucor, Aspergillus, and Botrytis spp. Controls growth of A. carbonarius and A. niger and reduces ochratoxin A accumulation.	[21, 60]
S. stellimalicola	No evidence of biocontrol activity reported.
C. parapsilosis	No evidence of biocontrol activity reported.
W. sorbophila	No evidence of biocontrol activity reported.
W. anomalus	Acts as a biocontrol agent during winemaking, targeting Dekkera/Brettanomyces via toxin secretion. Antagonistic activity against B. cinerea.	[51, 57]
S. diversa	Exhibits antagonistic activity against a broad range of fungal pathogens, significantly reducing B. cinerea growth.	[61]

This literature review revealed that species-specific metabolic traits contribute distinct characteristics to wine, driven by unique biochemical activities exhibited during fermentation (Table 3). For example, P. kluyveri enhances wine aroma by producing volatile esters and varietal thiols such as 3-MHA, imparting fruity and floral notes. P. terricola exhibits strong β-glucosidase activity, releasing aromatic compounds from glycosylated precursors. P. fermentans increases the production of higher alcohols, glycerol, and polysaccharides, improving the quality and texture of wine, whereas P. manshurica tends to generate volatile phenols and off-odors, negatively affecting aroma. These findings highlight the need for careful strain selection, optimized inoculation timing, and precise fermentation management to fully leverage these yeasts in mixed fermentations. As such, these yeasts present both opportunity and complexity for winemakers aiming to move beyond traditional S. cerevisiae-dominated fermentations.

Consistent with the species-specific impacts observed in wine fermentation, biocontrol mechanisms also vary among species (Table 4). For example, P. kudriavzevii produces VOCs that suppress gray mold, P. fermentans reduces fungal hyphal growth, and P. manshurica suppresses postharvest grape disease. These mechanisms reflect a variety of antifungal strategies, each potentially suited to different environmental contexts and target pathogens. Therefore, targeted, strain-specific evaluations are essential to identify optimal applications and ensure efficacy under commercial agricultural conditions.

In addition to their roles in enology and biocontrol, many of these yeast species (isolated from grape and other sources) have also demonstrated diverse applications in industrial and agricultural contexts. For example, W. anomalus has been implicated in biofuel production [62], H. uvarum has utility in genetic engineering platforms [63], and P. kudriavzevii is emerging as a novel probiotic candidate [64]. Moreover, P. kudriavzevii and P. terricola have been associated with plant growth promotion and increased crop resilience [65]. Collectively, these findings underscore the versatile functional landscape of endophytic yeasts and their growing relevance in advancing biobased innovations across multiple sectors (Fig. 5).

Fig. 5.

Overview of the functions of endophytic yeast species. The figure illustrates muscadine grape berries in various hues (left panel), harboring diverse yeast species (central panel), and highlights the associated positive roles of these endophytes in wine fermentation, biocontrol, and other applications (right panel)

Conclusions

This study offers the first comprehensive report of cultivable endophytic yeasts in muscadine and hybrid grape cultivars, revealing a diverse yeast community predominantly composed of non-Saccharomyces genera. We identified both common and unique yeast species across genotypes, with host cultivar influencing community structure. The discovery of novel species such as N. nivariensis and W. sorbophila has expanded the known ecological range of grape-associated yeasts. Many of the isolated yeast species are recognized for their significant potential in biotechnological applications, including winemaking, biocontrol, plant growth promotion, probiotic use, and genetic engineering. These findings position muscadine grapes and their yeast communities as critical resources for sustainable agriculture and biotechnology innovation. Future research, incorporating broader sampling, seasonal variation studies, and field trials, will be crucial to understand the potential of these yeasts for crop resilience, disease mitigation, and novel bioprocessing solutions.

Abbreviations

VOC

Volatile Organic Compounds

DIC

Differential Interference Contrast

3-MHA

3-Mercaptohexyl acetate

Authors’ contributions

MA contributed to the concept, design, data curation, funding acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, writing-original draft, writing-review, and editing. MBS contributed to the concept, funding acquisition, resources, and reviewing the draft. Both authors read and approved the final manuscript.

Funding

This work was supported by USDA/NIFA Capacity Building Grants # 2021-38821-34711, 2021-38821-34580, and 2023-38821-39591.

Data availability

The datasets supporting the findings of this study have been deposited in the NCBI under accession numbers; PV568513, PV568515, PV568518, PV568517, PV568516, PV568519, PV568520, PV568521, PV568522, PV568523, PV568528, PV568524, PV568525, PV568526, PV568530, PV568529, and PV568527.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.