{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T05:03:06Z","timestamp":1789534986089,"version":"build-2803163510"},"reference-count":32,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T00:00:00Z","timestamp":1789084800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Hefei Biosynthesis Technology Co., Ltd of China","award":["No. 25H00705"],"award-info":[{"award-number":["No. 25H00705"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Background: Crop fungal diseases cause huge yield losses globally, and the abuse of chemical fungicides has led to severe resistance and environmental risks. Antimicrobial peptides (AMPs) represent ideal green alternatives, but poor salt tolerance and high production costs limit their field application. Results: In this study, we developed a His-tag engineering strategy to enhance salt tolerance of antifungal peptides, and identified a novel peptide Cla-H derived from Styela clava. Cla-H was efficiently expressed via secretory fermentation in Pichia pastoris, and its crude fermentation supernatant could be directly applied to plants. Cla-H exhibited strong and rapid fungicidal activity against Fusarium graminearum and Botrytis cinerea, accompanied by irreversible inhibition of spore germination and observable membrane permeabilization phenotypes. While SYTOX green and PI staining indicated membrane-damaging events, adequate control experiments are still required to firmly establish membrane permeabilization as its primary mode of action. Meanwhile, it showed outstanding thermal and storage stability. Most importantly, His-tag modification significantly improved its salt tolerance, enabling stable activity under high-salt conditions that completely inactivated the parental peptide. In planta assays demonstrated that crude Cla-H fermentation supernatant effectively controlled wheat scab and tomato gray mold. Conclusions: This study provides a promising salt-tolerant antifungal peptide for crop protection and establishes a simple and low-cost technical route for developing next-generation green biofungicides.<\/jats:p>","DOI":"10.3390\/biom16091326","type":"journal-article","created":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T14:39:24Z","timestamp":1789137564000},"page":"1326","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Styela clava-Derived Peptide Cla-H Is a Promising Green Biofungicide for Crop Protection"],"prefix":"10.3390","volume":"16","author":[{"given":"Yuliang","family":"Xu","sequence":"first","affiliation":[{"name":"Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai 200444, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lang","family":"Su","sequence":"additional","affiliation":[{"name":"Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai 200444, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yating","family":"Zhang","sequence":"additional","affiliation":[{"name":"Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai 200444, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Changfu","family":"Li","sequence":"additional","affiliation":[{"name":"Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai 200444, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7597-9821","authenticated-orcid":false,"given":"Yansheng","family":"Zhang","sequence":"additional","affiliation":[{"name":"Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai 200444, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e00449-20","DOI":"10.1128\/mBio.00449-20","article-title":"Threats posed by the fungal kingdom to humans, wildlife, and agriculture","volume":"11","author":"Fisher","year":"2020","journal-title":"mBio"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1038\/nature10947","article-title":"Emerging fungal threats to animal, plant and ecosystem health","volume":"484","author":"Fisher","year":"2012","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"430","DOI":"10.1038\/s41559-018-0793-y","article-title":"The global burden of pathogens and pests on major food crops","volume":"3","author":"Savary","year":"2019","journal-title":"Nat. Ecol. Evol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"739","DOI":"10.1126\/science.aap7999","article-title":"Worldwide emergence of resistance to antifungal drugs challenges human health and food security","volume":"360","author":"Fisher","year":"2018","journal-title":"Science"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1002\/etc.5766","article-title":"Further Limitations of Synthetic Fungicide Use and Expansion of Organic Agriculture in Europe Will Increase the Environmental and Health Risks of Chemical Crop Protection Caused by Copper-Containing Fungicides","volume":"43","author":"Burandt","year":"2024","journal-title":"Environ. Toxicol. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"5318","DOI":"10.1111\/mec.15877","article-title":"Contrasting levels of genetic predictability in the evolution of resistance to major classes of fungicides","volume":"30","author":"Hawkins","year":"2021","journal-title":"Mol. Ecol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Lobo, F., and Boto, A. (2022). Host-Defense Peptides as New Generation Phytosanitaries: Low Toxicity and Low Induction of Antimicrobial Resistance. Agronomy, 12.","DOI":"10.3390\/agronomy12071614"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1002\/ps.1236","article-title":"Cytochrome b gene structure and consequences for resistance to Qo inhibitor fungicides in plant pathogens","volume":"62","author":"Grasso","year":"2006","journal-title":"Pest Manag. Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1016\/j.plantsci.2014.05.012","article-title":"Multitasking antimicrobial peptides in plant development and host defense against biotic\/abiotic stress","volume":"228","author":"Goyal","year":"2014","journal-title":"Plant Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3545","DOI":"10.1007\/s00018-013-1260-1","article-title":"Properties and mechanisms of action of naturally occurring antifungal peptides","volume":"70","author":"Bleackley","year":"2013","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"687","DOI":"10.1038\/s41579-025-01200-y","article-title":"Antimicrobial peptides: Structure, functions and translational applications","volume":"23","author":"Oliveira","year":"2025","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"441","DOI":"10.2217\/fmb-2016-0181","article-title":"Antifungal plant defensins: Increased insight in their mode of action as a basis for their use to combat fungal infections","volume":"12","author":"Cools","year":"2017","journal-title":"Future Microbiol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1016\/j.semcdb.2018.02.004","article-title":"The evolution, function and mechanisms of action for plant defensins","volume":"88","author":"Parisi","year":"2019","journal-title":"Semin. Cell Dev. Biol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Kovaleva, V., Bukhteeva, I., Kit, O.Y., and Nesmelova, I.V. (2020). Plant Defensins from a Structural Perspective. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21155307"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"542","DOI":"10.1111\/mmi.13333","article-title":"Antifungal mechanisms of a plant defensin MtDef4 are not conserved between the ascomycete fungi Neurospora crassa and Fusarium graminearum","volume":"100","author":"Islam","year":"2016","journal-title":"Mol. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Sagaram, U.S., Pandurangi, R., Kaur, J., Smith, T.J., and Shah, D.M. (2011). Structure-activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0018550"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Sagaram, U.S., El-Mounadi, K., Buchko, G.W., Berg, H.R., Kaur, J., Pandurangi, R.S., Smith, T.J., and Shah, D.M. (2013). Structural and functional studies of a phosphatidic acid-binding antifungal plant defensin MtDef4: Identification of an RGFRRR motif governing fungal cell entry. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0082485"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"896","DOI":"10.1111\/mpp.13336","article-title":"Plant defensin MtDef4-derived antifungal peptide with multiple modes of action and potential as a bio-inspired fungicide","volume":"24","author":"Tetorya","year":"2023","journal-title":"Mol. Plant Pathol."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Kerenga, B.K., McKenna, J.A., Harvey, P.J., Quimbar, P., Garcia-Ceron, D., Lay, F.T., Phan, T.K., Veneer, P.K., Vasa, S., and Parisi, K. (2019). Salt-Tolerant Antifungal and Antibacterial Activities of the Corn Defensin ZmD32. Front. Microbiol., 10.","DOI":"10.3389\/fmicb.2019.00795"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2898","DOI":"10.1128\/iai.65.7.2898-2903.1997","article-title":"Effects of pH and salinity on the antimicrobial properties of clavanins","volume":"65","author":"Lee","year":"1997","journal-title":"Infect. Immun."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Luo, Y., and Song, Y. (2021). Mechanism of Antimicrobial Peptides: Antimicrobial, Anti-Inflammatory and Antibiofilm Activities. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms222111401"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1016\/j.bbrc.2023.09.013","article-title":"Effects of structural changes on antibacterial activity and cytotoxicity due to proline substitutions in chimeric peptide HnMc","volume":"679","author":"Park","year":"2023","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1649","DOI":"10.1094\/MPMI-08-19-0224-R","article-title":"Antifungal Potency and Modes of Action of a Novel Olive Tree Defensin Against Closely Related Ascomycete Fungal Pathogens","volume":"32","author":"Li","year":"2019","journal-title":"Mol. Plant-Microbe Interact."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e202108501","DOI":"10.1002\/anie.202108501","article-title":"Histidine-Mediated Ion Specific Effects Enable Salt Tolerance of a Pore-Forming Marine Antimicrobial Peptide","volume":"61","author":"Xian","year":"2022","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Wang, K.J., Cai, J.J., and Li, J.F. (2013). The Effect of Adding Histidine on Biological Activity and Stability of Pc-pis from Pseudosciaena crocea. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0083268"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Mahlapuu, A., H\u00e5kansson, J., Ringstad, L., and Bj\u00f6rn, C. (2024). Enhancing Antimicrobial Peptide Activity through Modifications of Charge, Hydrophobicity, and Structure. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms251910821"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1016\/j.plipres.2011.12.005","article-title":"Role of Lipids in the Interaction of Antimicrobial Peptides with Membranes","volume":"51","author":"Teixeira","year":"2012","journal-title":"Prog. Lipid Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"e0003722","DOI":"10.1128\/mmbr.00037-22","article-title":"Antimicrobial Peptides and Small Molecules Targeting the Cell Membrane of Staphylococcus aureus","volume":"87","author":"Ganesan","year":"2023","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Struyfs, C., Cammue, B.P.A., and Thevissen, K. (2021). Membrane-Interacting Antifungal Peptides. Front. Cell Dev. Biol., 9.","DOI":"10.3389\/fcell.2021.649875"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"7694","DOI":"10.1021\/acs.langmuir.3c00498","article-title":"Interaction of a Histidine-Rich Antimicrobial Saliva Peptide with Model Cell Membranes: The Role of Histidines","volume":"39","author":"Oyen","year":"2023","journal-title":"Langmuir"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"9837","DOI":"10.1021\/jacs.9b00440","article-title":"Structure and Function of Piscidins: Histidine Position, Directionality of Membrane Insertion, and pH-Dependent Permeabilization","volume":"141","author":"Mihailescu","year":"2019","journal-title":"J. Am. Chem. Soc."},{"key":"ref_32","first-page":"32","article-title":"Effect of Poly-Histidine Tag Position toward Inhibition Activity of Secretory Leukocyte Protease Inhibitor as Candidate for Material Wound Healing","volume":"12","author":"Munadziroh","year":"2020","journal-title":"Avicenna J. Med. Biotechnol."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/9\/1326\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T04:20:35Z","timestamp":1789532435000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/9\/1326"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,11]]},"references-count":32,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["biom16091326"],"URL":"https:\/\/doi.org\/10.3390\/biom16091326","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,11]]}}}