Diversity of Yeast and Drosophila Associated with Grape Sour Rot in China

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At present, there is insufficient research on its species distribution and diversity in China, resulting in constrained prevention and control efficacy. In this study, the species diversity of yeast and Drosophila was determined by sequencing the 26S rDNA D1/D2 gene and Drosophila COI region. A total of 9 yeast genera were identified, of which Hanseniaspera spp. (45.76%) had the highest abundance, followed by Starmerella spp. (23.62%) and Saccharomyces spp. (17.34%) 14 yeast species were identified at the species level. Starmerella bacillaris (23.62%), Saccharomyces cerevisiae (17.34%), Hanseniaspora opuntiae (17.34%) and Hanseniaspera uvarum (15.13%) were the dominant yeasts. 6 species of Drosophila have been identified, where the Drosophila melanogaster (62.50%) was the dominant species. This study is the first to analyze the diversity of yeasts and Drosophila in different grape producing regions in China. The research results can provide a scientific basis for clarifying the key driving factors of sour rot and formulating precise prevention and control strategies. Grape Sour rot Yeast Drosophila Molecular identification Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Grape sour rot is a complex disease caused by multiple factors [ 1 – 3 ], which is widespread in the main producing regions of China. It mainly occurs from fruit color turning to maturity, which not only causes fruit decay and juice loss [ 4 ], but also reduces fruit sugar-acid ratio and flavor quality [ 5 , 6 ]. Numerous studies have demonstrated that sour rot in grapes results primarily from the synergistic effects of yeast and Drosophila . The two have formed a special ecological interaction relationship, which collectively promotes the spread of the disease. The yeasts related to grape sour rot predominantly involved Hanseniaspora spp., Saccharomyces spp., Pichia spp., Issatchenki a spp., Candida spp., etc [ 7 – 9 ]. There are significant differences in the pathogenicity of different yeasts. H. uvarum and H. vineae have been identified as the dominant yeasts in numerous grape producing regions [ 10 ]. They have strong acid-producing ability and pectinase properties, and can decompose sugars in grape juice and produce metabolites such as ethanol and acetic acid [ 5 ], resulting in fruit rancidity. S. cerevisiae , Pichia kudriavzevii and others were also confirmed to be involved in enzymatic hydrolysis and alcohol fermentation [ 11 , 12 ]. In addition, the infection of yeast also creates favorable conditions for the infection of other pathogens and aggravates the occurrence of diseases. Drosophila is the key disseminator in the ecological chain. Among them, D. melanogaster and Drosophila suzukii of Drosophila spp. are the most widely distributed and the most harmful [ 13 , 14 ]. Other common species include Drosophila immigrans , Drosophila simulan , Drosophila mojavensis , Drosophila arizonensis , Drosophila ritae , Drosophila starmeri , etc [ 15 – 17 ]. Drosophila damages grapes primarily by disseminating yeasts carried on their body surfaces and within their digestive tracts through contact and feeding [ 18 , 19 ], or the larvae release enzymes to promote the consumption of pulp [ 20 ]. The geographical span of grape producing regions in China is large, and the climatic conditions and cultivation practices are diverse, which affect the dynamic variations in grape microbial communities [ 10 , 21 ]. In addition, the differences in physicochemical properties of different grape varieties also affect the microbial communities [ 22 ]. Currently, research on the species, geographical distribution and Drosophila vector of pathogenic yeast remains limited, which restricts accurate prevention and control. In view of these problems, this study employed 26S rDNA D1/D2 region sequence analysis and COI barcode technology to systematically investigate the diversity of yeasts and Drosophila in sour rot samples from major grape producing regions in China. The findings aim to enhance the effective prevention and control of grape sour rot, holding significant implications for promoting the sustainable development of China's grape industry. 2 Materials and Methods 2.1 Source of sour rot grapes The yeasts and Drosophila isolated in this experiment were sampled from four major grape producing regions in China, involving 12 provinces in Central China and East China (CCEC), North China and Bohai Bay (NCBB), Southwest China (SC), and Northwest China and Loess Plateau (NCLP). A total of 11 grape varieties were involved. The sources are detailed in supplementary materials (Supplementary Tables 1 and 2). 2.2 Isolation and Purification of Yeast The sour rot fruit 5–6 grains were put into the YEPD (containing (g/L): peptone 20, yeast extract fermentation 10, glucose 20) (Beijing Kulaibo Technology Co., Ltd., Beijing, China) YEPD liquid medium, and the shaker was shaken for 0.5 h. The culture medium was diluted with 0.90% sterile sodium chloride solution to the concentrations of 10⁻¹, 10⁻², 10⁻³, respectively, and inoculated into YEPD (containing (g/L): peptone 20, yeast extract fermentation 10, glucose 20, agar 20, penicillin 0.1) solid medium. Three replicates per concentration. Finally, the YEPD solid medium in petri dishes, which were then placed in a constant temperature incubator (Shanghai Fuma Experiment Equipment Co., Ltd., Shanghai, China) at 28 ℃ for 3–5 days. After 3–4 times of repeated purification, a pure culture of the yeast strain was obtained. 2.3 Preservation of Yeast Purified single colony was oscillated culture in YEPD liquid medium for 3 days, and the yeast solution was mixed with 30% sterile glycerol at a ratio of 1:1, then it was stored at − 70°C. All experiments were carried out under the horizontal-laminar airflow clean bench. 2.4 Molecular Identification of Yeast Strains Fresh yeast was collected and suspended in sterile water in a 1.5 mL centrifuge tube. Total genomic DNA was extracted directly from pure hyphae or conidia using the rapid extraction kit of fungal genomic DNA (Omega Bio-Tek, New Orleans, LA, USA) according the manufacturer’s protocol. The internal transcribed spacer region (D1/D2) of the ribosomal DNA (rDNA) was amplified with NL1/NL4 primers (NL1: 5′-GCATATCAATAAGCGGAGGAAAAG-3′; NL4: 5′-GGTCCGTGTTTCAAGACGG-3′) (Sangon biotech Co., Ltd., Shanghai, China) [ 23 ]. Amplification reactions were performed in a 50.0 µL reaction volume containing 25.0 µL 1.1×T3 Super PCR Mix (Beijing Qingke Biotechnology Co., Ltd., Beijing, China), 1.0 µL genomic DNA, 1.5 µL of primer, and 21.0 µL of ddH 2 O according to the polymerase chain reaction (PCR) program described in Morgan et al [ 24 ]. The cycling conditions were as follows: initial denaturation at 95 ℃ for 3 min, followed by 35 cycles of denaturation at 95 ℃ for 20 s, annealing at 55 ℃ for 30 s, and extension72 ℃ for 1 min, and a final extension at 72 ℃ for 7 min. The PCR products were detected by 1% agarose gel electrophoresis and sequenced (Sangon biotech Co., Ltd., Shanghai, China). 2.5 Identification of Drosophila species The eggs, larvae, pupae and adults were collected. After cleaning the surface impurities with sterile water, the adult species were identified according to morphology. Total genomic DNA was extracted directly from pure hyphae using the TIANamp Genomic DNA Kit (Tiangen Biotech Co., Ltd., Beijng, China) following the manufacturer’s instructions. After the DNA concentration was qualified by ultraviolet spectrophotometer, it was used as PCR template. Sequences of the primers were as follows: ZBJ-ArtF1c: 5′-AGATATTGGAACWTTATATTTTATTTTTGG-3′/ZBJ-ArtR2c: 5′-TACTAATCAATTTCCAAATCCTCC-3′ (Sangon biotech Co., Ltd., Shanghai, China). Amplification reaction system and cycling conditions were consistent with yeast. 2.6 Statistical Analysis The sequencing sequence was aligned by NCBI BLAST ( https://www.ncbi.nlm.nih.gov/ ) and downloaded the standard strain sequence, Excel summary data, Graphpad 8.0 mapping (Graphpad software, Boston, MA, USA), and MEGA7.0 (LynnonBiosoft, San Ramon, CA, USA) to construct a phylogenetic tree to identify yeast.. 3 Results 3.1 Analysis of genetic diversity of yeast The 26S rDNA D1/D2 sequences of 271 samples from four producing regions in China were aligned by Bioedit, and the haplotypes of all samples were extracted and analyzed by DnaSP 5.0 after cut and alignment. The results showed that 271 samples were distributed across 47 haplotypes. The nucleotide diversity (Pi) was 0.17003, the average nucleotide difference ( k ) was 73.45270, and the haplotype diversity (Hd) was 0.8804. Among them, Hap_35 was the dominant haplotype (59 sequences). Hap_5, Hap_1 and Hap_13 had 44, 40 and 40 sequences, respectively. 30 haplotypes, including Hap_2 and so on were only one sequence. The phylogenetic tree was constructed by MEGA 7.0 software with the Neighbor-Joining method, and it’s reliability was evaluated through Bootstrap analysis with 1,000 replicates. The phylogenetic tree based on the 26S rDNA D1/D2 gene was presented in Fig. 1 . Hanseniaspera spp. has 15 haplotypes, Pichia spp. across 11 haplotypes, Starmerella spp. across 6 haplotypes, Saccharomyces spp. across 6 haplotypes, Zygosaccharomyces spp. across 4 haplotypes, Rhodotorula spp. across 2 haplotypes. Jamesozyma spp., Torulaspora spp., and Schizosaccharomyces spp. were distributed in one haplotype, respectively. 3.2 Isolation and identification of yeast At the genus level, a total of 9 yeast genera were identified across all samples (Fig. 2 A). Among them, Hanseniaspera spp. (45.76%) exhibited the highest relative abundance, followed by Starmerella spp. (23.62%) and Saccharomyces spp. (17.34%). 6 yeast genera including Pichia spp. and so on were less than 10%. At the species level, a total of 14 yeast genera were identified (Fig. 2 B). S . bacillaris (23.62%), S. cerevisiae (17.34%), H. opuntiae (17.34%) and H. uvarum (15.13%) were the dominant yeast species associated with the occurrence of sour rot in China's major grape production regions. 10 yeast species including H. vineae and so on were less than 10%. The difference of yeast abundance in different producing regions were further studied (Fig. 3 A). A total of 11, 10, 2, 3 yeasts were identified in CECC, NCBB, SC and NCLP. Among them, the relative abundance of H . opuntiae (11.44%) in CECC was significantly higher than that in SC (1.85%) ( P < 0.05), but not significantly different from that in NCBB (4.06%). The relative abundance of S. cerevisiae (9.96%) in NCBB was significantly higher than that in CECC (2.21%), but not significantly different from that in SC (5.17%) ( P < 0.05). Although the relative abundance of S. bacillaris in CECC and NCBB was higher, there was no significant difference between them. In addition, there was no significant difference in the relative abundance of other yeasts among different producing regions. In order to clarify the correlation between grape varieties and yeasts, the results of yeasts identified from 11 different grape varieties were analyzed (Fig. 3 B). The relative abundance of S . cerevisiae (8.86%) and H . opuntiae (7.75%) in ‘Summer Black’ was significantly higher than that in other varieties. S . bacillaris (11.81%) was the dominant yeast in ‘Kyoho’, and its relative abundance was significantly higher than that of other varieties. The dominant yeast was S. cerevisiae (6.64%), and Rhodotorula mucilaginosa (2.21%) was unique to this species. 3.3 Isolation and identification of Drosophila Sequencing of the Drosophila COI region revealed 6 species identified across 96 Drosophila samples collected from four grape producing regions (Fig. 4 A). Among them, D . melanogaster (62.50%) was the dominant species in the four grape producing regions, followed by D. simulans (28.13%), and a total of 4 species, including Drosophila bipectinata and so on accounted for less than 5%. There were 1 species of common Drosophila in the four producing regions (Fig. 4 B). D. simulans was not only the common Drosophila in the four producing regions, but also the dominant Drosophila in each producing region, accounting for 63.41%, 61.29%, 66.67% and 61.11% (Fig. 4 C–F), respectively. Drosophila sechellia was exclusive to CECC (Fig. 4 C) and D. suzukii was exclusive to NCBB (Fig. 4 D). 4 Discussion In this study, Hanseniaspora spp. was identified as the dominant yeast genus across samples from four grape producing regions (Fig. 2 A), which is consistent with previous studies [ 25 , 26 ]. Hanseniaspora spp. is frequently reported as the predominant yeast genus in mature and intact grape berries. Species such as H. opuntiae , H. uvarum play a crucial role in the initial stage of grape fermentation [ 27 , 28 ], producing enzymes and aroma compounds that enhance wine flavor [ 29 ]. Starmerella spp. and Saccharomyces spp. which are second only to Hanseniaspora spp. also play the same role. Notably, S. bacillaris exhibits a fructophilic character, while S. cerevisiae displays a glucophilic character, which enables them to coexist for a long time in the fermentation process [ 30 ]. In addition, Rossouw et al. [ 31 ] showed that P. kudriavzwvii also has fermentation ability, but its grow and persist are far less than H. opuntiae and H. uvarum . Grape-related microorganisms are related to the interaction of vineyard geography, climate, soil, and grape cultivation systems in vineyard ecosystems [ 32 – 34 ], Yeasts in different producing regions are more adapted to the local environment and form corresponding dominant flora, and finally form the corresponding dominant yeasts [ 35 ]. Drumonde-Neves et al. [ 33 ] observed significant differences in yeast communities in vineyards on five islands in the Azores. Barata et al. [ 9 ] showed that in the warm and humid grape producing regions, the main yeasts were H. uvarum and S. cerevisiae , while in the dry and hot grape producing regions, the main yeasts were Metschnikowia pulcherrima and so on [ 36 ]. Australia 's Shiraz grapes are prone to fruit cracking at high temperatures, resulting in the rapid propagation of dominant yeasts such as H. uvarum and S. cerevisiae [ 37 ]. Yeast biodiversity was primarily influenced by the grapevine cultivar [ 22 ], with sugar and peel thickness being key determining factors. In the Rhine region of Germany, ‘Riesling’ grapes characterized by high sugar and low acidity, predominantly harbor M. pulcherrima and S. bacillis due to, contributing to a high incidence of sour rot [ 38 ]. In contrast, ‘Cabernet Sauvignon’ grapes have thick, tightly structured skins. Yeasts struggle to proliferate in large quantities, resulting in a low incidence of sour rot. The dominant yeast is S. cerevisiae . The dominant yeasts of ‘Pinot Noir’ were H. uvarum and Candida krusei , and the incidence of sour rot was higher [ 39 ]. This study shows that ‘Summer Black’ skin thin sugar is high, more susceptible than ruby; although the skin of ‘Kyoho’ is thick, the risk of sour rot is increased due to the high humidity (compared with NCLP) in CECC and NCBB regions (Fig. 3 B). D. melanogaster was recognized as a key inducing factor and vector of sour rot disease [ 40 ], prompting extensive research into the damage it causes. In South Korea, D. suzukii was the principal Drosophila pest on some berries [ 41 ]. Man et al. [ 42 ] reported that the effects of cherry varieties on the population dynamics of Drosophila in cherry orchards in northern China. Four species were captured: D. melanogaste r, D. suzukii , Drosophila hydei , and D. immigrans , with D. melanogaste r being the dominant species. The study revealed significant differences in the number of Drosophila species among cherry cultivars. Weißinger et al. [ 43 ] found that five grape varieties with different surface physicochemical properties had different preference for D. suzukii . The above reports are basically consistent with our survey. Regional and between differences in Drosophila species presence and importance may be closely related to differences in climate, local environmental factors, and different cultivars. In nature, insect host selection is mainly mediated by volatiles [ 44 ]. The metabolism of yeast can produce volatile substances such as ethanol, ethyl acetate and 2-phenylethanol, which can attract D. melanogaster [ 45 ]. Kleman et al. [ 46 ] found that H. uvarum had the strongest attraction to D. melanoogaster and D. suzukii , which may be the key factor for Hanseniaspera spp. to become the dominant genus. Becher et al. [ 47 ] also proved that S. cerevisiae has an attractive effect on D. melanogaster , attraction and oviposition were significantly lower if non-fermented grape juice was used, and yeast-free grapes did not support larval development either. Further identified a synthetic mimic of yeast odor, comprising ethanol, acetic acid, acetoin, 2-phenyl- ethanol and 3-methyl-1-butano, which was as attractive for the Drosophila as fermenting grape juice. Therefore, in subsequent studies, exploring the effects of volatiles released by yeast-infected grapes on the host selection behavior of Drosophila will be valuable for elucidating the interactions among insects, microorganisms and grapes. Such research can also contribute to a comprehensive analysis of pathogenic mechanism, and provide a theoretical basis for green pollution-free prevention of grape sour rot. 5 Conclusion This study identified yeasts and Drosophil a species associated with sour rot-affected grapes of China by culture-dependent methods combined with 26S rDNA D1/D2 gene and Drosophil a COI region sequencing analysis, providing comprehensive information on targets for the control of the disease. The yeasts and Drosophil a species associated with sour rot-affected grapes in China 's four major grape producing regions were similar to those found in vineyards around the world. Hanseniaspera spp. and Starmerella spp. were dominant yeasts. S. bacillaris , S. cerevisiae , H. opuntiae and H. uvarum were dominant yeasts. D. melanogaste r was the dominant Drosophil a. This study revealed the effects of different producing regions and different grape varieties on yeast and Drosophil a, providing a scientific basis for the development of targeted strategies for the prevention and control of grape sour rot. Declarations Funding (Acknowledgements) This work was supported by the National Natural Science Foundation of China (32272550), Agriculture Research System of China (CARS-29-bc-5) and Special Regional Collaborative Innovation Project of Xinjiang Uygur Autonomous Region (Science and Technology Aid Xinjiang Program) (2022E0234). Author Contributions Y.Q. L. conceived and designed the experiments. J. H. analyzed the data and wrote the paper. Q.D. F. and Y.F. H. performed the experiments. X.Q. H. and F.F. K. provided constructive suggestions on the experimental design and data analysis. Y.Q. L. revised the paper. All authors read and approved the final manuscript. Data Availability No datasets were generated or analysed during the current study. Competing Interests The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and in-dicate if you modified the licensed material. You do not have permission under this licen-ce to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. 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J Econ Entomol 115(4):999–1007. https://doi.org/10.1093/jee/toac029 Becher PG, Flick G, Rozpędowska E et al (2012) Yeast, not fruit volatiles mediate Drosophila melanogaster attraction, oviposition and development. Funct Ecol 26:822–828. https://doi.org/10.1111/j.1365-2435.2012.02006.x Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 12 Nov, 2025 Read the published version in Microbial Ecology → Version 1 posted Editorial decision: Revision requested 30 Jun, 2025 Reviews received at journal 30 Jun, 2025 Reviews received at journal 26 Jun, 2025 Reviewers agreed at journal 02 Jun, 2025 Reviews received at journal 01 Jun, 2025 Reviewers agreed at journal 31 May, 2025 Reviewers agreed at journal 30 May, 2025 Reviewers agreed at journal 29 May, 2025 Reviewers invited by journal 29 May, 2025 Editor assigned by journal 28 May, 2025 Submission checks completed at journal 28 May, 2025 First submitted to journal 24 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6738604","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":464884884,"identity":"525814e1-c87d-447f-8d1f-4e144340ffae","order_by":0,"name":"Jie Han","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Han","suffix":""},{"id":464884885,"identity":"17ab9d0f-e308-4e4c-848f-f822fe591e0d","order_by":1,"name":"Qiandong Fang","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qiandong","middleName":"","lastName":"Fang","suffix":""},{"id":464884886,"identity":"a046b00d-5fc4-47f3-b4e9-7dc40adcf125","order_by":2,"name":"Yifan Hao","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yifan","middleName":"","lastName":"Hao","suffix":""},{"id":464884887,"identity":"b9330deb-1174-4144-a668-ff9b6a095c5f","order_by":3,"name":"Xiaoqing Huang","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqing","middleName":"","lastName":"Huang","suffix":""},{"id":464884888,"identity":"d4085e9d-3347-4559-ad86-1fea9f3e19a4","order_by":4,"name":"Fanfang Kong","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Fanfang","middleName":"","lastName":"Kong","suffix":""},{"id":464884889,"identity":"4f136d9e-9285-43b8-9550-efba9568cbff","order_by":5,"name":"Yongqiang Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYBACPmYogw1EfCBGCxuyFsYZRGlB5jDzEKWFncdM4uOOWtk+9rOHX9vU2MnLNzA/e4DfYTxmkjPPHDdu48lLs845lmy44QCbuQEhLdK8bccS2xhyzIxzG5gZNzDwsEkQp4X/jZmxZUO9/fwG4rTUJLZJ5Bg/Zmw4nNhwgKAWtmLLmW0HjNsk3pgx9hw7nrzhMJsZXi38/Ic33vjYVic7vz/H+MOPmmrb+e3Nz/BqAQIWoILDjA1AGyEqmfErBysBJpM6kBZmotLLKBgFo2AUjDwAAIINP1v7hO4NAAAAAElFTkSuQmCC","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Yongqiang","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-05-24 10:54:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6738604/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6738604/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00248-025-02616-y","type":"published","date":"2025-11-12T15:58:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83750634,"identity":"f716e6e0-2fe5-47a2-8f39-e8f9c9d1a061","added_by":"auto","created_at":"2025-06-02 06:39:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":112840,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic trees of yeast with other related strains based on 26S rDNA D1/D2 gene sequences by the neighbor-joining method.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6738604/v1/4256d2d61710aa6aefd87ff6.png"},{"id":83750628,"identity":"340df967-2751-4c9e-9913-aae2e3f54c20","added_by":"auto","created_at":"2025-06-02 06:39:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":115408,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of yeast genera (A) and species (B).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6738604/v1/84a469c1b3d954080a6f398c.png"},{"id":83750632,"identity":"6cf74641-10fc-495d-b962-3463d92d2df8","added_by":"auto","created_at":"2025-06-02 06:39:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":124657,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification results of yeasts. (A) Heat map of yeast species in different producing regions; (B) heat map of yeast species in different grape varieties.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6738604/v1/f5ec3a4c136837410ecd3c5b.png"},{"id":83750629,"identity":"9a274079-c3e1-4736-b734-05b6e48e5fbd","added_by":"auto","created_at":"2025-06-02 06:39:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":161549,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification results of \u003cem\u003eDrosophila.\u003c/em\u003e(A) Relative abundance of \u003cem\u003eDrosophila\u003c/em\u003especies; (B) Venn graph is based on the genus of different producing regions; (C, D, E, F) the identification results of CECC, NCBB, SC and NCLP in each producing region.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6738604/v1/985ca9128bccaccbe158881c.png"},{"id":96105253,"identity":"c7563a60-a6bc-45ec-8c2b-8e45935eee28","added_by":"auto","created_at":"2025-11-17 16:10:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1117769,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6738604/v1/e9dc6800-c997-48e6-b635-5193db942b8b.pdf"},{"id":83750633,"identity":"fb875d8b-a3d4-45e1-8860-be862c110737","added_by":"auto","created_at":"2025-06-02 06:39:05","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":11242,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6738604/v1/0d0fa75e36e615ace1158960.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Diversity of Yeast and Drosophila Associated with Grape Sour Rot in China","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eGrape sour rot is a complex disease caused by multiple factors [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], which is widespread in the main producing regions of China. It mainly occurs from fruit color turning to maturity, which not only causes fruit decay and juice loss [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], but also reduces fruit sugar-acid ratio and flavor quality [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNumerous studies have demonstrated that sour rot in grapes results primarily from the synergistic effects of yeast and \u003cem\u003eDrosophila\u003c/em\u003e. The two have formed a special ecological interaction relationship, which collectively promotes the spread of the disease. The yeasts related to grape sour rot predominantly involved \u003cem\u003eHanseniaspora\u003c/em\u003e spp., \u003cem\u003eSaccharomyces\u003c/em\u003e spp., \u003cem\u003ePichia\u003c/em\u003e spp., \u003cem\u003eIssatchenki\u003c/em\u003ea spp., \u003cem\u003eCandida\u003c/em\u003e spp., etc [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere are significant differences in the pathogenicity of different yeasts. \u003cem\u003eH. uvarum\u003c/em\u003e and \u003cem\u003eH. vineae\u003c/em\u003e have been identified as the dominant yeasts in numerous grape producing regions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. They have strong acid-producing ability and pectinase properties, and can decompose sugars in grape juice and produce metabolites such as ethanol and acetic acid [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], resulting in fruit rancidity. \u003cem\u003eS. cerevisiae\u003c/em\u003e, \u003cem\u003ePichia kudriavzevii\u003c/em\u003e and others were also confirmed to be involved in enzymatic hydrolysis and alcohol fermentation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In addition, the infection of yeast also creates favorable conditions for the infection of other pathogens and aggravates the occurrence of diseases.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDrosophila\u003c/em\u003e is the key disseminator in the ecological chain. Among them, \u003cem\u003eD. melanogaster\u003c/em\u003e and \u003cem\u003eDrosophila suzukii\u003c/em\u003e of \u003cem\u003eDrosophila\u003c/em\u003e spp. are the most widely distributed and the most harmful [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Other common species include \u003cem\u003eDrosophila immigrans\u003c/em\u003e, \u003cem\u003eDrosophila simulan\u003c/em\u003e, \u003cem\u003eDrosophila mojavensis\u003c/em\u003e, \u003cem\u003eDrosophila arizonensis\u003c/em\u003e, \u003cem\u003eDrosophila ritae\u003c/em\u003e, \u003cem\u003eDrosophila starmeri\u003c/em\u003e, etc [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. \u003cem\u003eDrosophila\u003c/em\u003e damages grapes primarily by disseminating yeasts carried on their body surfaces and within their digestive tracts through contact and feeding [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], or the larvae release enzymes to promote the consumption of pulp [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe geographical span of grape producing regions in China is large, and the climatic conditions and cultivation practices are diverse, which affect the dynamic variations in grape microbial communities [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In addition, the differences in physicochemical properties of different grape varieties also affect the microbial communities [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Currently, research on the species, geographical distribution and \u003cem\u003eDrosophila\u003c/em\u003e vector of pathogenic yeast remains limited, which restricts accurate prevention and control.\u003c/p\u003e \u003cp\u003eIn view of these problems, this study employed 26S rDNA D1/D2 region sequence analysis and COI barcode technology to systematically investigate the diversity of yeasts and \u003cem\u003eDrosophila\u003c/em\u003e in sour rot samples from major grape producing regions in China. The findings aim to enhance the effective prevention and control of grape sour rot, holding significant implications for promoting the sustainable development of China's grape industry.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Source of sour rot grapes\u003c/h2\u003e \u003cp\u003eThe yeasts and \u003cem\u003eDrosophila\u003c/em\u003e isolated in this experiment were sampled from four major grape producing regions in China, involving 12 provinces in Central China and East China (CCEC), North China and Bohai Bay (NCBB), Southwest China (SC), and Northwest China and Loess Plateau (NCLP). A total of 11 grape varieties were involved. The sources are detailed in supplementary materials (Supplementary Tables\u0026nbsp;1 and 2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Isolation and Purification of Yeast\u003c/h2\u003e \u003cp\u003eThe sour rot fruit 5\u0026ndash;6 grains were put into the YEPD (containing (g/L): peptone 20, yeast extract fermentation 10, glucose 20) (Beijing Kulaibo Technology Co., Ltd., Beijing, China) YEPD liquid medium, and the shaker was shaken for 0.5 h. The culture medium was diluted with 0.90% sterile sodium chloride solution to the concentrations of 10⁻\u0026sup1;, 10⁻\u0026sup2;, 10⁻\u0026sup3;, respectively, and inoculated into YEPD (containing (g/L): peptone 20, yeast extract fermentation 10, glucose 20, agar 20, penicillin 0.1) solid medium. Three replicates per concentration. Finally, the YEPD solid medium in petri dishes, which were then placed in a constant temperature incubator (Shanghai Fuma Experiment Equipment Co., Ltd., Shanghai, China) at 28 ℃ for 3\u0026ndash;5 days. After 3\u0026ndash;4 times of repeated purification, a pure culture of the yeast strain was obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preservation of Yeast\u003c/h2\u003e \u003cp\u003ePurified single colony was oscillated culture in YEPD liquid medium for 3 days, and the yeast solution was mixed with 30% sterile glycerol at a ratio of 1:1, then it was stored at \u0026minus;\u0026thinsp;70\u0026deg;C. All experiments were carried out under the horizontal-laminar airflow clean bench.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Molecular Identification of Yeast Strains\u003c/h2\u003e \u003cp\u003eFresh yeast was collected and suspended in sterile water in a 1.5 mL centrifuge tube. Total genomic DNA was extracted directly from pure hyphae or conidia using the rapid extraction kit of fungal genomic DNA (Omega Bio-Tek, New Orleans, LA, USA) according the manufacturer\u0026rsquo;s protocol. The internal transcribed spacer region (D1/D2) of the ribosomal DNA (rDNA) was amplified with NL1/NL4 primers (NL1: 5\u0026prime;-GCATATCAATAAGCGGAGGAAAAG-3\u0026prime;; NL4: 5\u0026prime;-GGTCCGTGTTTCAAGACGG-3\u0026prime;) (Sangon biotech Co., Ltd., Shanghai, China) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmplification reactions were performed in a 50.0 \u0026micro;L reaction volume containing 25.0 \u0026micro;L 1.1\u0026times;T3 Super PCR Mix (Beijing Qingke Biotechnology Co., Ltd., Beijing, China), 1.0 \u0026micro;L genomic DNA, 1.5 \u0026micro;L of primer, and 21.0 \u0026micro;L of ddH\u003csub\u003e2\u003c/sub\u003eO according to the polymerase chain reaction (PCR) program described in Morgan et al [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The cycling conditions were as follows: initial denaturation at 95 ℃ for 3 min, followed by 35 cycles of denaturation at 95 ℃ for 20 s, annealing at 55 ℃ for 30 s, and extension72 ℃ for 1 min, and a final extension at 72 ℃ for 7 min. The PCR products were detected by 1% agarose gel electrophoresis and sequenced (Sangon biotech Co., Ltd., Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Identification of \u003cem\u003eDrosophila\u003c/em\u003e species\u003c/h2\u003e \u003cp\u003eThe eggs, larvae, pupae and adults were collected. After cleaning the surface impurities with sterile water, the adult species were identified according to morphology. Total genomic DNA was extracted directly from pure hyphae using the TIANamp Genomic DNA Kit (Tiangen Biotech Co., Ltd., Beijng, China) following the manufacturer\u0026rsquo;s instructions. After the DNA concentration was qualified by ultraviolet spectrophotometer, it was used as PCR template. Sequences of the primers were as follows: ZBJ-ArtF1c: 5\u0026prime;-AGATATTGGAACWTTATATTTTATTTTTGG-3\u0026prime;/ZBJ-ArtR2c: 5\u0026prime;-TACTAATCAATTTCCAAATCCTCC-3\u0026prime; (Sangon biotech Co., Ltd., Shanghai, China). Amplification reaction system and cycling conditions were consistent with yeast.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e \u003cp\u003eThe sequencing sequence was aligned by NCBI BLAST (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e and downloaded the standard strain sequence, Excel summary data, Graphpad 8.0 mapping (Graphpad software, Boston, MA, USA), and MEGA7.0 (LynnonBiosoft, San Ramon, CA, USA) to construct a phylogenetic tree to identify yeast..\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Analysis of genetic diversity of yeast\u003c/h2\u003e \u003cp\u003eThe 26S rDNA D1/D2 sequences of 271 samples from four producing regions in China were aligned by Bioedit, and the haplotypes of all samples were extracted and analyzed by DnaSP 5.0 after cut and alignment. The results showed that 271 samples were distributed across 47 haplotypes. The nucleotide diversity (Pi) was 0.17003, the average nucleotide difference (\u003cem\u003ek\u003c/em\u003e) was 73.45270, and the haplotype diversity (Hd) was 0.8804. Among them, Hap_35 was the dominant haplotype (59 sequences). Hap_5, Hap_1 and Hap_13 had 44, 40 and 40 sequences, respectively. 30 haplotypes, including Hap_2 and so on were only one sequence.\u003c/p\u003e \u003cp\u003eThe phylogenetic tree was constructed by MEGA 7.0 software with the Neighbor-Joining method, and it\u0026rsquo;s reliability was evaluated through Bootstrap analysis with 1,000 replicates. The phylogenetic tree based on the 26S rDNA D1/D2 gene was presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cem\u003eHanseniaspera\u003c/em\u003e spp. has 15 haplotypes, \u003cem\u003ePichia\u003c/em\u003e spp. across 11 haplotypes, \u003cem\u003eStarmerella\u003c/em\u003e spp. across 6 haplotypes, \u003cem\u003eSaccharomyces\u003c/em\u003e spp. across 6 haplotypes, \u003cem\u003eZygosaccharomyces\u003c/em\u003e spp. across 4 haplotypes, \u003cem\u003eRhodotorula\u003c/em\u003e spp. across 2 haplotypes. \u003cem\u003eJamesozyma\u003c/em\u003e spp., \u003cem\u003eTorulaspora\u003c/em\u003e spp., and \u003cem\u003eSchizosaccharomyces\u003c/em\u003e spp. were distributed in one haplotype, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Isolation and identification of yeast\u003c/h2\u003e \u003cp\u003eAt the genus level, a total of 9 yeast genera were identified across all samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Among them, \u003cem\u003eHanseniaspera\u003c/em\u003e spp. (45.76%) exhibited the highest relative abundance, followed by \u003cem\u003eStarmerella\u003c/em\u003e spp. (23.62%) and \u003cem\u003eSaccharomyces\u003c/em\u003e spp. (17.34%). 6 yeast genera including \u003cem\u003ePichia\u003c/em\u003e spp. and so on were less than 10%. At the species level, a total of 14 yeast genera were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebacillaris\u003c/em\u003e (23.62%), \u003cem\u003eS. cerevisiae\u003c/em\u003e (17.34%), \u003cem\u003eH. opuntiae\u003c/em\u003e (17.34%) and \u003cem\u003eH. uvarum\u003c/em\u003e (15.13%) were the dominant yeast species associated with the occurrence of sour rot in China's major grape production regions. 10 yeast species including \u003cem\u003eH. vineae\u003c/em\u003e and so on were less than 10%.\u003c/p\u003e \u003cp\u003eThe difference of yeast abundance in different producing regions were further studied (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). A total of 11, 10, 2, 3 yeasts were identified in CECC, NCBB, SC and NCLP. Among them, the relative abundance of \u003cem\u003eH\u003c/em\u003e. \u003cem\u003eopuntiae\u003c/em\u003e (11.44%) in CECC was significantly higher than that in SC (1.85%) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not significantly different from that in NCBB (4.06%). The relative abundance of \u003cem\u003eS. cerevisiae\u003c/em\u003e (9.96%) in NCBB was significantly higher than that in CECC (2.21%), but not significantly different from that in SC (5.17%) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Although the relative abundance of \u003cem\u003eS. bacillaris\u003c/em\u003e in CECC and NCBB was higher, there was no significant difference between them. In addition, there was no significant difference in the relative abundance of other yeasts among different producing regions.\u003c/p\u003e \u003cp\u003eIn order to clarify the correlation between grape varieties and yeasts, the results of yeasts identified from 11 different grape varieties were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The relative abundance of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae\u003c/em\u003e (8.86%) and \u003cem\u003eH\u003c/em\u003e. \u003cem\u003eopuntiae\u003c/em\u003e (7.75%) in \u0026lsquo;Summer Black\u0026rsquo; was significantly higher than that in other varieties. \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebacillaris\u003c/em\u003e (11.81%) was the dominant yeast in \u0026lsquo;Kyoho\u0026rsquo;, and its relative abundance was significantly higher than that of other varieties. The dominant yeast was \u003cem\u003eS. cerevisiae\u003c/em\u003e (6.64%), and \u003cem\u003eRhodotorula mucilaginosa\u003c/em\u003e (2.21%) was unique to this species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Isolation and identification of \u003cem\u003eDrosophila\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eSequencing of the \u003cem\u003eDrosophila\u003c/em\u003e COI region revealed 6 species identified across 96 \u003cem\u003eDrosophila\u003c/em\u003e samples collected from four grape producing regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Among them, \u003cem\u003eD\u003c/em\u003e. \u003cem\u003emelanogaster\u003c/em\u003e (62.50%) was the dominant species in the four grape producing regions, followed by \u003cem\u003eD. simulans\u003c/em\u003e (28.13%), and a total of 4 species, including \u003cem\u003eDrosophila bipectinata\u003c/em\u003e and so on accounted for less than 5%. There were 1 species of common \u003cem\u003eDrosophila\u003c/em\u003e in the four producing regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). \u003cem\u003eD. simulans\u003c/em\u003e was not only the common \u003cem\u003eDrosophila\u003c/em\u003e in the four producing regions, but also the dominant \u003cem\u003eDrosophila\u003c/em\u003e in each producing region, accounting for 63.41%, 61.29%, 66.67% and 61.11% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;F), respectively. \u003cem\u003eDrosophila sechellia\u003c/em\u003e was exclusive to CECC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) and \u003cem\u003eD. suzukii\u003c/em\u003e was exclusive to NCBB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn this study, \u003cem\u003eHanseniaspora\u003c/em\u003e spp. was identified as the dominant yeast genus across samples from four grape producing regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), which is consistent with previous studies [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. \u003cem\u003eHanseniaspora\u003c/em\u003e spp. is frequently reported as the predominant yeast genus in mature and intact grape berries. Species such as \u003cem\u003eH. opuntiae\u003c/em\u003e, \u003cem\u003eH. uvarum\u003c/em\u003e play a crucial role in the initial stage of grape fermentation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], producing enzymes and aroma compounds that enhance wine flavor [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. \u003cem\u003eStarmerella\u003c/em\u003e spp. and \u003cem\u003eSaccharomyces\u003c/em\u003e spp. which are second only to \u003cem\u003eHanseniaspora\u003c/em\u003e spp. also play the same role. Notably, \u003cem\u003eS. bacillaris\u003c/em\u003e exhibits a fructophilic character, while \u003cem\u003eS. cerevisiae\u003c/em\u003e displays a glucophilic character, which enables them to coexist for a long time in the fermentation process [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In addition, Rossouw et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] showed that \u003cem\u003eP. kudriavzwvii\u003c/em\u003e also has fermentation ability, but its grow and persist are far less than \u003cem\u003eH. opuntiae\u003c/em\u003e and \u003cem\u003eH. uvarum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eGrape-related microorganisms are related to the interaction of vineyard geography, climate, soil, and grape cultivation systems in vineyard ecosystems [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], Yeasts in different producing regions are more adapted to the local environment and form corresponding dominant flora, and finally form the corresponding dominant yeasts [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Drumonde-Neves et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] observed significant differences in yeast communities in vineyards on five islands in the Azores. Barata et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] showed that in the warm and humid grape producing regions, the main yeasts were \u003cem\u003eH. uvarum\u003c/em\u003e and \u003cem\u003eS. cerevisiae\u003c/em\u003e, while in the dry and hot grape producing regions, the main yeasts were \u003cem\u003eMetschnikowia pulcherrima\u003c/em\u003e and so on [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Australia 's Shiraz grapes are prone to fruit cracking at high temperatures, resulting in the rapid propagation of dominant yeasts such as \u003cem\u003eH. uvarum\u003c/em\u003e and \u003cem\u003eS. cerevisiae\u003c/em\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eYeast biodiversity was primarily influenced by the grapevine cultivar [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], with sugar and peel thickness being key determining factors. In the Rhine region of Germany, \u0026lsquo;Riesling\u0026rsquo; grapes characterized by high sugar and low acidity, predominantly harbor \u003cem\u003eM. pulcherrima\u003c/em\u003e and \u003cem\u003eS. bacillis\u003c/em\u003e due to, contributing to a high incidence of sour rot [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In contrast, \u0026lsquo;Cabernet Sauvignon\u0026rsquo; grapes have thick, tightly structured skins. Yeasts struggle to proliferate in large quantities, resulting in a low incidence of sour rot. The dominant yeast is \u003cem\u003eS. cerevisiae\u003c/em\u003e. The dominant yeasts of \u0026lsquo;Pinot Noir\u0026rsquo; were \u003cem\u003eH. uvarum\u003c/em\u003e and \u003cem\u003eCandida krusei\u003c/em\u003e, and the incidence of sour rot was higher [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This study shows that \u0026lsquo;Summer Black\u0026rsquo; skin thin sugar is high, more susceptible than ruby; although the skin of \u0026lsquo;Kyoho\u0026rsquo; is thick, the risk of sour rot is increased due to the high humidity (compared with NCLP) in CECC and NCBB regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cem\u003eD. melanogaster\u003c/em\u003e was recognized as a key inducing factor and vector of sour rot disease [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], prompting extensive research into the damage it causes. In South Korea, \u003cem\u003eD. suzukii\u003c/em\u003e was the principal \u003cem\u003eDrosophila\u003c/em\u003e pest on some berries [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Man et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] reported that the effects of cherry varieties on the population dynamics of \u003cem\u003eDrosophila\u003c/em\u003e in cherry orchards in northern China. Four species were captured: \u003cem\u003eD. melanogaste\u003c/em\u003er, \u003cem\u003eD. suzukii\u003c/em\u003e, \u003cem\u003eDrosophila hydei\u003c/em\u003e, and \u003cem\u003eD. immigrans\u003c/em\u003e, with \u003cem\u003eD. melanogaste\u003c/em\u003er being the dominant species. The study revealed significant differences in the number of \u003cem\u003eDrosophila\u003c/em\u003e species among cherry cultivars. Wei\u0026szlig;inger et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] found that five grape varieties with different surface physicochemical properties had different preference for \u003cem\u003eD. suzukii\u003c/em\u003e. The above reports are basically consistent with our survey. Regional and between differences in \u003cem\u003eDrosophila\u003c/em\u003e species presence and importance may be closely related to differences in climate, local environmental factors, and different cultivars.\u003c/p\u003e \u003cp\u003eIn nature, insect host selection is mainly mediated by volatiles [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The metabolism of yeast can produce volatile substances such as ethanol, ethyl acetate and 2-phenylethanol, which can attract \u003cem\u003eD. melanogaster\u003c/em\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Kleman et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] found that \u003cem\u003eH. uvarum\u003c/em\u003e had the strongest attraction to \u003cem\u003eD. melanoogaster\u003c/em\u003e and \u003cem\u003eD. suzukii\u003c/em\u003e, which may be the key factor for \u003cem\u003eHanseniaspera\u003c/em\u003e spp. to become the dominant genus. Becher et al. [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] also proved that \u003cem\u003eS. cerevisiae\u003c/em\u003e has an attractive effect on \u003cem\u003eD. melanogaster\u003c/em\u003e, attraction and oviposition were significantly lower if non-fermented grape juice was used, and yeast-free grapes did not support larval development either. Further identified a synthetic mimic of yeast odor, comprising ethanol, acetic acid, acetoin, 2-phenyl- ethanol and 3-methyl-1-butano, which was as attractive for the \u003cem\u003eDrosophila\u003c/em\u003e as fermenting grape juice.\u003c/p\u003e \u003cp\u003eTherefore, in subsequent studies, exploring the effects of volatiles released by yeast-infected grapes on the host selection behavior of \u003cem\u003eDrosophila\u003c/em\u003e will be valuable for elucidating the interactions among insects, microorganisms and grapes. Such research can also contribute to a comprehensive analysis of pathogenic mechanism, and provide a theoretical basis for green pollution-free prevention of grape sour rot.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThis study identified yeasts and \u003cem\u003eDrosophil\u003c/em\u003ea species associated with sour rot-affected grapes of China by culture-dependent methods combined with 26S rDNA D1/D2 gene and \u003cem\u003eDrosophil\u003c/em\u003ea COI region sequencing analysis, providing comprehensive information on targets for the control of the disease. The yeasts and \u003cem\u003eDrosophil\u003c/em\u003ea species associated with sour rot-affected grapes in China 's four major grape producing regions were similar to those found in vineyards around the world. \u003cem\u003eHanseniaspera\u003c/em\u003e spp. and \u003cem\u003eStarmerella\u003c/em\u003e spp. were dominant yeasts. \u003cem\u003eS. bacillaris\u003c/em\u003e, \u003cem\u003eS. cerevisiae\u003c/em\u003e, \u003cem\u003eH. opuntiae\u003c/em\u003e and \u003cem\u003eH. uvarum\u003c/em\u003e were dominant yeasts. \u003cem\u003eD. melanogaste\u003c/em\u003er was the dominant \u003cem\u003eDrosophil\u003c/em\u003ea. This study revealed the effects of different producing regions and different grape varieties on yeast and \u003cem\u003eDrosophil\u003c/em\u003ea, providing a scientific basis for the development of targeted strategies for the prevention and control of grape sour rot.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding (Acknowledgements)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32272550), Agriculture Research System of China (CARS-29-bc-5) and Special Regional Collaborative Innovation Project of Xinjiang Uygur Autonomous Region (Science and Technology Aid Xinjiang Program) (2022E0234).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.Q. L. conceived and designed the experiments. J. H. analyzed the data and wrote the paper. Q.D. F. and Y.F. H. performed the experiments. X.Q. H. and F.F. K. provided constructive suggestions on the experimental design and data analysis. Y.Q. L. revised the paper. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Access\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and in-dicate if you modified the licensed material. You do not have permission under this licen-ce to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article\u0026rsquo;s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article\u0026rsquo;s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by-nc-nd/4.0/.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSteel CC, Blackman JW, Schmidtke LM (2013) Grapevine bunch rots: impacts on wine composition, quality, and potential procedures for the removal of wine faults. 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Funct Ecol 26:822\u0026ndash;828. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2435.2012.02006.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2435.2012.02006.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"meco","sideBox":"Learn more about [Microbial Ecology](https://www.springer.com/journal/248)","snPcode":"248","submissionUrl":"https://submission.nature.com/new-submission/248/3","title":"Microbial Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Grape, Sour rot, Yeast, Drosophila, Molecular identification","lastPublishedDoi":"10.21203/rs.3.rs-6738604/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6738604/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSour rot is a complex disease in grape production, which is mainly caused by the combined effects of yeast and \u003cem\u003eDrosophila\u003c/em\u003e, resulting in fruit decay, quality decline and serious economic losses. At present, there is insufficient research on its species distribution and diversity in China, resulting in constrained prevention and control efficacy. In this study, the species diversity of yeast and \u003cem\u003eDrosophila\u003c/em\u003e was determined by sequencing the 26S rDNA D1/D2 gene and \u003cem\u003eDrosophila\u003c/em\u003e COI region. A total of 9 yeast genera were identified, of which \u003cem\u003eHanseniaspera\u003c/em\u003e spp. (45.76%) had the highest abundance, followed by \u003cem\u003eStarmerella\u003c/em\u003e spp. (23.62%) and \u003cem\u003eSaccharomyces\u003c/em\u003e spp. (17.34%) 14 yeast species were identified at the species level. \u003cem\u003eStarmerella bacillaris\u003c/em\u003e (23.62%), \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e (17.34%), \u003cem\u003eHanseniaspora opuntiae\u003c/em\u003e (17.34%) and \u003cem\u003eHanseniaspera uvarum\u003c/em\u003e (15.13%) were the dominant yeasts. 6 species of \u003cem\u003eDrosophila\u003c/em\u003e have been identified, where the \u003cem\u003eDrosophila melanogaster\u003c/em\u003e (62.50%) was the dominant species. This study is the first to analyze the diversity of yeasts and \u003cem\u003eDrosophila\u003c/em\u003e in different grape producing regions in China. The research results can provide a scientific basis for clarifying the key driving factors of sour rot and formulating precise prevention and control strategies.\u003c/p\u003e","manuscriptTitle":"Diversity of Yeast and Drosophila Associated with Grape Sour Rot in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-02 06:38:33","doi":"10.21203/rs.3.rs-6738604/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-30T15:57:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-30T09:06:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-26T13:25:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213340055589252048892276224157069864309","date":"2025-06-02T12:07:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-01T21:30:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"88696006249765370629843353245681346583","date":"2025-05-31T14:12:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30595663737331697213622276143277429614","date":"2025-05-30T08:50:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"198954271544554506677088270845896879775","date":"2025-05-29T18:34:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-29T16:48:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-28T12:45:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-28T12:39:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Ecology","date":"2025-05-24T10:51:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"meco","sideBox":"Learn more about [Microbial Ecology](https://www.springer.com/journal/248)","snPcode":"248","submissionUrl":"https://submission.nature.com/new-submission/248/3","title":"Microbial Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f0f9d8bf-96e7-444b-a678-9c2e440aecc3","owner":[],"postedDate":"June 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:06:10+00:00","versionOfRecord":{"articleIdentity":"rs-6738604","link":"https://doi.org/10.1007/s00248-025-02616-y","journal":{"identity":"microbial-ecology","isVorOnly":false,"title":"Microbial Ecology"},"publishedOn":"2025-11-12 15:58:19","publishedOnDateReadable":"November 12th, 2025"},"versionCreatedAt":"2025-06-02 06:38:33","video":"","vorDoi":"10.1007/s00248-025-02616-y","vorDoiUrl":"https://doi.org/10.1007/s00248-025-02616-y","workflowStages":[]},"version":"v1","identity":"rs-6738604","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6738604","identity":"rs-6738604","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}