{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T11:09:54Z","timestamp":1790334594002,"version":"4.1.0"},"reference-count":50,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T00:00:00Z","timestamp":1790294400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100008871","name":"Yunnan Province Agricultural Joint Special Project","doi-asserted-by":"publisher","award":["No. 202501BD070001-116"],"award-info":[{"award-number":["No. 202501BD070001-116"]}],"id":[{"id":"10.13039\/501100008871","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Yunnan Academician Expert Workstation Project","award":["202405AF140032"],"award-info":[{"award-number":["202405AF140032"]}]},{"name":"Provincial Tropical Crop Science and Technology Innovation Special Fund","award":["RF2026"],"award-info":[{"award-number":["RF2026"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Int. J. Mol. Sci.","IJMS"],"abstract":"<jats:p>Gray mold, caused by the necrotrophic fungus Botrytis cinerea, is a devastating disease responsible for substantial global crop losses. The escalating threat of fungicide resistance and environmental concerns necessitates the development of eco-compatible control alternatives. In this study, we investigated the antifungal efficacy of methyl 2-methylbutyrate (M2M), a microbial volatile organic compound (mVOC), against B. cinerea, and elucidated the underlying physiological, transcriptomic, and metabolomic responses. Our results demonstrated that M2M significantly inhibited mycelial growth in a dose-dependent manner, with an EC50 value of 3.49 \u03bcL\/plate. Scanning electron microscopy revealed severe ultrastructural damage in M2M-treated hyphae, characterized by localized swelling, shriveling, and irregular bending. Physiological assays indicated that M2M significantly upregulated the activities of antioxidant enzymes, including catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), while significantly reducing malondialdehyde (MDA) content, suggesting that M2M triggers an oxidative challenge to which B. cinerea responds by activating its antioxidant defense system, thereby suppressing lipid peroxidation and the activities of key metabolic enzymes, such as succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PDH), and lactate dehydrogenase (LDH). These findings suggest that M2M treatment is associated with perturbations in cellular redox homeostasis and central carbon metabolism in B. cinerea. Transcriptomic profiling identified 3664 differentially expressed genes (DEGs), predominantly enriched in carbon metabolism, glyoxylate and dicarboxylate metabolism, redox pathways, and ribosomal biogenesis. Notably, M2M exposure significantly perturbed the transcriptional levels of ROS-scavenging genes (e.g., CAT, POD, GPX, PRX, and GST) and various transcription factors. Untargeted metabolomics revealed 1041 differentially accumulated metabolites (DAMs), with lipids and lipid-like molecules representing the most significantly altered class. Integrated multi-omics analysis revealed that M2M treatment was associated with coordinated changes in lipid metabolism, central carbon metabolism, and redox balance. This study provides mechanistic insights into the antifungal action of M2M and suggests that M2M warrants further investigation as a potential candidate for postharvest gray mold management.<\/jats:p>","DOI":"10.3390\/ijms27198589","type":"journal-article","created":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T10:34:10Z","timestamp":1790332450000},"page":"8589","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Antifungal Activity of Methyl 2-Methylbutyrate Against Botrytis cinerea and the Induced Physiological, Transcriptional, and Metabolic Responses"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5850-8320","authenticated-orcid":false,"given":"Li-Ming","family":"Dai","sequence":"first","affiliation":[{"name":"Tropical Crop Diseases Research Group, Research Center of Plant Protection, Yunnan Key Laboratory of Sustainable Utilization Research on Rubber Tree, Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li-Li","family":"He","sequence":"additional","affiliation":[{"name":"Tropical Crop Diseases Research Group, Research Center of Plant Protection, Yunnan Key Laboratory of Sustainable Utilization Research on Rubber Tree, Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Si-Min","family":"Li","sequence":"additional","affiliation":[{"name":"Tropical Crop Diseases Research Group, Research Center of Plant Protection, Yunnan Key Laboratory of Sustainable Utilization Research on Rubber Tree, Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi-Xian","family":"Liu","sequence":"additional","affiliation":[{"name":"Tropical Crop Diseases Research Group, Research Center of Plant Protection, Yunnan Key Laboratory of Sustainable Utilization Research on Rubber Tree, Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu-Ping","family":"Shi","sequence":"additional","affiliation":[{"name":"Tropical Crop Diseases Research Group, Research Center of Plant Protection, Yunnan Key Laboratory of Sustainable Utilization Research on Rubber Tree, Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hai-Peng","family":"Su","sequence":"additional","affiliation":[{"name":"Tropical Crop Diseases Research Group, Research Center of Plant Protection, Yunnan Key Laboratory of Sustainable Utilization Research on Rubber Tree, Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhi-Ying","family":"Cai","sequence":"additional","affiliation":[{"name":"Tropical Crop Diseases Research Group, Research Center of Plant Protection, Yunnan Key Laboratory of Sustainable Utilization Research on Rubber Tree, Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Li, R., and Cheng, Y.L. (2023). Recent advances in mechanisms underlying defense responses of horticultural crops to Botrytis cinerea. Horticulturae, 9.","DOI":"10.3390\/horticulturae9111178"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"D\u00edaz, M.A., Pereyra, M.M., Pic\u00f3n-Montenegro, E., Meinhardt, F., and Dib, J.R. (2020). Killer yeasts for the biological control of postharvest fungal crop diseases. Microorganisms, 8.","DOI":"10.3390\/microorganisms8111680"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"107085","DOI":"10.1016\/j.cropro.2024.107085","article-title":"Microbial biological control of Botrytis bunch rot (Botrytis cinerea) of grapevine (Vitis vinifera) crops: A meta-analysis","volume":"190","author":"Poveda","year":"2025","journal-title":"Crop Prot."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Elsherbiny, E.A., Francesco, A.D., and Bennett, J.W. (2024). Editorial: The use of microbial volatile compounds for controlling plant pathogens. Front. Plant Sci., 15.","DOI":"10.3389\/fpls.2024.1502485"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1007\/s10343-023-00951-z","article-title":"Microbial volatile compounds: Prospects for fungal phytopathogens management, mechanisms and challenges","volume":"76","author":"Naik","year":"2024","journal-title":"J. Crop Health"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Razo-Belman, R., and Ozuna, C. (2023). Volatile organic compounds: A review of their current applications as pest biocontrol and disease management. Horticulturae, 9.","DOI":"10.3390\/horticulturae9040441"},{"key":"ref_7","first-page":"103788","article-title":"Volatile organic compounds produced by Aureobasidium pullulans induce electrolyte loss and oxidative stress in Botrytis cinerea and Alternaria alternata","volume":"172","author":"Don","year":"2020","journal-title":"Res. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1016\/j.biocontrol.2014.10.004","article-title":"Production of volatile organic compounds by Aureobasidium pullulans as a potential mechanism of action against postharvest fruit pathogens","volume":"81","author":"Francesco","year":"2015","journal-title":"Biol. Control"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1016\/j.biocontrol.2011.10.014","article-title":"Effects of volatile substances of Streptomyces globisporus JK-1 on control of Botrytis cinerea on tomato fruit","volume":"61","author":"Li","year":"2012","journal-title":"Biol. Control"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e02706-25","DOI":"10.1128\/spectrum.02706-25","article-title":"Multifaceted antifungal mechanisms of volatile organic compounds emitted from Pseudomonas chlororaphis ZL3 against Botrytis cinerea","volume":"14","author":"Wang","year":"2025","journal-title":"Microbiol. Spectr."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Zuo, G.X., Han, Y.X., Dong, Q.Y., Lu, W.T., Gao, C.T., Zhao, N., Liu, S.H., Fan, G.Q., Liu, C.X., and Xiang, W.S. (2025). Antifungal activity and mechanism of volatile organic compounds produced by Bacillus siamensis NEAU-ZGX24 against Botrytis cinerea. Microbiol. Res., 299.","DOI":"10.1016\/j.micres.2025.128265"},{"key":"ref_12","unstructured":"(2024). National Food Safety Standard for Uses of Food Additives (Standard No. GB 2760-2024)."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Lyu, A., Yang, L., Wu, M.D., Zhang, J., and Li, G.Q. (2020). High Efficacy of the volatile organic compounds of Streptomyces yanglinensis 3-10 in suppression of Aspergillus contamination on peanut kernels. Front. Microbiol., 11.","DOI":"10.3389\/fmicb.2020.00142"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/j.ijfoodmicro.2023.110462","article-title":"Mechanisms of methyl 2-methylbutyrate suppression on Aspergillus flavus growth and aflatoxin B1 biosynthesis","volume":"409","author":"Wei","year":"2024","journal-title":"Int. J. Food Microbiol."},{"key":"ref_15","first-page":"12","article-title":"Methyl 2-methylbutyrate produced by Streptomyces saccharicans strain ZBY1-20 inhibits Monilinia fructicola causing peach brown rot","volume":"209","author":"Chen","year":"2010","journal-title":"Biol. Control"},{"key":"ref_16","first-page":"249","article-title":"Inhibition of the spore production of green mould pathogen Penicillium digitatum by microbial volatile organic compounds and its mechanisms","volume":"50","author":"Tao","year":"2023","journal-title":"J. Plant Prot."},{"key":"ref_17","unstructured":"Dai, L.M., Li, L.L., Cai, Z.Y., Liu, Y.X., Shi, Y.P., and Deng, Y.Y. (2024). Application of Methyl 2-Methylbutyrate in Inhibiting Plant Pathogens. (CN 117882723 A)."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"113343","DOI":"10.1016\/j.postharvbio.2024.113343","article-title":"2-Methylbutyric acid functions as a potential antifungal fumigant by inhibiting Botrytis cinerea and inducing resistance to gray mold in cherry tomatoes","volume":"222","author":"Liang","year":"2025","journal-title":"Postharv. Biol. Technol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1007\/s11306-007-0099-6","article-title":"Assessment of PLSDA cross validation","volume":"4","author":"Westerhuis","year":"2008","journal-title":"Metabolomics"},{"key":"ref_20","unstructured":"Eriksson, L., Johansson, E., Kettaneh-Wold, N., Trygg, J., Wikstr\u00f6m, C., and Wold, S. (2006). Multi- and Megavariate Data Analysis: Part I: Basic Principles and Applications, Umetrics Academy. [2nd ed.]."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/j.tplants.2022.08.024","article-title":"Killing softly: A roadmap of Botrytis cinerea pathogenicity","volume":"28","author":"Bi","year":"2023","journal-title":"Trends Plant Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1186\/s42483-024-00262-9","article-title":"Understanding Botrytis cinerea infection and gray mold management: A review paper on deciphering the rose\u2019s thorn","volume":"6","author":"Ullah","year":"2024","journal-title":"Phytopathol. Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"432","DOI":"10.1094\/PHYTO-10-20-0475-IA","article-title":"Botrytis spp.: A contemporary perspective and synthesis of recent scientific developments of a widespread genus that threatens global food security","volume":"111","author":"Richards","year":"2021","journal-title":"Phytopathology"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"628","DOI":"10.1094\/PDIS-06-20-1191-RE","article-title":"Resistance to boscalid in Botrytis cinerea from greenhouse-grown tomato","volume":"105","author":"Liu","year":"2021","journal-title":"Plant Dis."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"101606","DOI":"10.1016\/j.funbio.2025.101606","article-title":"Application of fungal volatile organic compounds for enhancing plant growth and controlling plant pathogens","volume":"129","author":"Elsherbiny","year":"2025","journal-title":"Fungal Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1007\/s11274-023-03598-0","article-title":"Research progress of volatile organic compounds produced by plant endophytic bacteria in control of postharvest diseases of fruits and vegetables","volume":"39","author":"Ling","year":"2023","journal-title":"World J. Microbiol. Biotechnol."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Song, G., Du, S.L., Sun, H.L., Liang, Q.W., Wang, H.H., Yan, M.L., and Zhang, J.H. (2022). Antifungal mechanism of (E)-2-hexenal against Botrytis cinerea growth revealed by transcriptome analysis. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.951751"},{"key":"ref_28","first-page":"761","article-title":"Antifungal mechanism of isooctyl alcohol on Botrytis cinerea causing tomato gray mold","volume":"52","author":"Wang","year":"2025","journal-title":"Acta Hortic. Sin."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Ebadzadsahrai, G., Higgins, K.E.A., Soby, S.D., and Bean, H.D. (2020). Inhibition of fungal growth and induction of a novel volatilome in response to Chromobacterium vaccinii volatile organic compounds. Front. Microbiol., 11.","DOI":"10.3389\/fmicb.2020.01035"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"235","DOI":"10.5423\/PPJ.RW.01.2024.0001","article-title":"Antioxidant systems of plant pathogenic fungi: Functions in oxidative stress response and their regulatory mechanisms","volume":"40","author":"Park","year":"2024","journal-title":"Plant Pathol. J."},{"key":"ref_31","first-page":"453","article-title":"Malondialdehyde (MDA) as a lipid peroxidation marker","volume":"57","author":"Gawe","year":"2004","journal-title":"Wiad. Lek."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"He, H., Cao, Z.F., Wang, T., Tang, C.Y., Li, Y.L., and Li, X.Z. (2024). Metabolomics combined with physiology and transcriptomics reveal the response of Samsoniella hepiali to key metabolic pathways and its degradation mechanism during subculture. Antioxidants, 13.","DOI":"10.3390\/antiox13070780"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"316","DOI":"10.1016\/j.numecd.2005.05.003","article-title":"A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress","volume":"15","author":"Stewart","year":"2005","journal-title":"Nutr. Metab. Cardiovasc. Dis."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1727","DOI":"10.1007\/s11274-011-0983-0","article-title":"Lead and cadmium-induced oxidative stress impacting mycelial growth of Oudemansiella radicata in liquid medium alleviated by microbial siderophores","volume":"28","author":"Cao","year":"2012","journal-title":"World J. Microbiol. Biotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"102544","DOI":"10.1016\/j.pmpp.2024.102544","article-title":"Essential oil compounds and their impact on grape (Vitis vinifera L. cv. Narince) physiology under Botrytis cinerea infection","volume":"136","author":"Kaya","year":"2025","journal-title":"Physiol. Mol. Plant Pathol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"116476","DOI":"10.1016\/j.bmc.2021.116476","article-title":"The research progress in and perspective of potential fungicides: Succinate dehydrogenase inhibitors","volume":"50","author":"Li","year":"2021","journal-title":"Bioorg. Med. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"15506","DOI":"10.1021\/acs.jafc.5c16394","article-title":"Antifungal activity and mechanisms of 2-nonanone, a volatile organic compound produced by Bacillus velezensis TCS001, against Botrytis cinerea","volume":"74","author":"Jin","year":"2026","journal-title":"J. Agric. Food Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1508","DOI":"10.1094\/MPMI-05-19-0124-R","article-title":"NADPH oxidase is crucial for the cellular redox homeostasis in fungal pathogen Botrytis cinerea","volume":"32","author":"Li","year":"2019","journal-title":"Mol. Plant-Microbe Interact."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1760","DOI":"10.1186\/s40643-021-00489-w","article-title":"Increasing NADPH impairs fungal H2O2 resistance by perturbing transcriptional regulation of peroxiredoxin","volume":"9","author":"Li","year":"2022","journal-title":"Bioresour. Bioprocess."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"987","DOI":"10.1094\/MPMI-22-8-0987","article-title":"Does Botrytis cinerea ignore H2O2-induced oxidative stress during infection? Characterization of Botrytis activator protein 1","volume":"22","author":"Temme","year":"2009","journal-title":"Mol. Plant-Microbe Interact."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Lu, P., Wang, K., Wang, J.Q., Xia, C.B., Yang, S., Ma, L., and Shi, H.J. (2023). A novel zinc finger transcription factor, BcMsn2, is involved in growth, development, and virulence in Botrytis cinerea. Front. Microbiol., 14.","DOI":"10.3389\/fmicb.2023.1247072"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Jiao, P., Cuiting, H., and Jae-Hyuk, Y. (2018). Lipid biosynthesis as an antifungal target. J. Fungi, 4.","DOI":"10.3390\/jof4020050"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2561826","DOI":"10.1080\/21505594.2025.2561826","article-title":"Fatty acids in filamentous pathogenic fungi: Multidimensional regulation and pathogenic mechanisms","volume":"16","author":"Lin","year":"2025","journal-title":"Virulence"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"127253","DOI":"10.1016\/j.micres.2022.127253","article-title":"Antifungal mechanisms of volatile organic compounds produced by Pseudomonas fluorescens ZX as biological fumigants against Botrytis cinerea","volume":"267","author":"Yue","year":"2023","journal-title":"Microbiol. Res."},{"key":"ref_45","unstructured":"Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J. (1990). Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. PCR Protocols: A Guide to Methods and Applications, Academic Press."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"e107","DOI":"10.1002\/imt2.107","article-title":"Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using fastp","volume":"2","author":"Chen","year":"2023","journal-title":"Imeta"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1650","DOI":"10.1038\/nprot.2016.095","article-title":"Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown","volume":"11","author":"Pertea","year":"2016","journal-title":"Nat. Protoc."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"923","DOI":"10.1093\/bioinformatics\/btt656","article-title":"FeatureCounts: An efficient general purpose program for assigning sequence reads to genomic features","volume":"30","author":"Liao","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1186\/s13059-014-0550-8","article-title":"Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2","volume":"15","author":"Love","year":"2014","journal-title":"Genome Biol."},{"key":"ref_50","first-page":"100141","article-title":"ClusterProfiler 4.0: A universal enrichment tool for interpreting omics data","volume":"2","author":"Wu","year":"2021","journal-title":"Innovation"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/19\/8589\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T10:36:25Z","timestamp":1790332585000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/19\/8589"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,25]]},"references-count":50,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2026,10]]}},"alternative-id":["ijms27198589"],"URL":"https:\/\/doi.org\/10.3390\/ijms27198589","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,25]]}}}