{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T02:17:57Z","timestamp":1787624277350,"version":"build-2736575974"},"reference-count":46,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2025,9,25]],"date-time":"2025-09-25T00:00:00Z","timestamp":1758758400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Agrobacterium tumefaciens (A. tumefaciens), the causal agent of crown gall disease on several plant species, is responsible for substantial yield losses worldwide. The limitations of conventional pesticides in controlling this disease highlight the need for alternative antibacterial solutions. Phage biocontrol can be an option, effectively managing bacterial plant diseases, by reducing pathogen loads while driving evolutionary trade-offs, often enhancing synergy with other antibacterial strategies. In this study, we aimed to explore and develop a sustainable strategy to control A. tumefaciens, by combining Agrobacterium phage PAT1 with the natural antimicrobial peptide \u201cAscaphin 8\u201d and leveraging the fitness trade-offs resulting from phage resistance. In vitro and in planta investigations showed that PAT1 in combination with Ascaphin 8 at the sublethal concentration of 3 \u03bcM could effectively eradicate A. tumefaciens in YPG broth and reduce tumor formation by 46.33% on tomato plants, unlike their individual applications, indicating that the combination was synergistic against A. tumefaciens. This synergy was attributed to the fitness trade-offs in A. tumefaciens induced by phage resistance, which led to increased sensitivity to antimicrobial peptides, slower growth rate, and an 89.96% attenuation of virulence in the PAT1-resistant mutant (AT-M1). Transmission electron microscopy analyses showed that treatment with 1 \u00b5M of Ascaphin 8 induced cytoplasmic condensation in 80% of AT-M1 cells, whereas only 16% of the wild-type CFBP 5770 cells exhibited similar alterations under identical conditions. Furthermore, proteomic analyses performed on AT-M1 and CFBP 5770 revealed that the mutant AT-M1 exhibited a loss of DNA-binding protein HupB and downregulation of SDR family oxidoreductase and superoxide dismutase. These molecular alterations are potentially associated with the reduced virulence and heightened AT-M1 sensitivity. This study investigated the fitness costs associated with phage resistance in A. tumefaciens and laid the first foundation for potential biocontrol of plant bacterial diseases, particularly A. tumefaciens infections, using phage\u2013peptide combination.<\/jats:p>","DOI":"10.3390\/ijms26199355","type":"journal-article","created":{"date-parts":[[2025,9,25]],"date-time":"2025-09-25T08:56:44Z","timestamp":1758790604000},"page":"9355","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Synergistic Biocontrol of Agrobacterium tumefaciens by Phage PAT1 and Ascaphin-8: Enhanced Antimicrobial Activity and Virulence Attenuation via HupB Loss"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2574-4842","authenticated-orcid":false,"given":"Miloud","family":"Sabri","sequence":"first","affiliation":[{"name":"International Centre for Advanced Mediterranean Agronomic Studies (CIHEAM of Bari), Via Ceglie 9, 70010 Valenzano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaoutar","family":"El Handi","sequence":"additional","affiliation":[{"name":"International Centre for Advanced Mediterranean Agronomic Studies (CIHEAM of Bari), Via Ceglie 9, 70010 Valenzano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8832-7072","authenticated-orcid":false,"given":"Cosima Damiana","family":"Calvano","sequence":"additional","affiliation":[{"name":"Interdepartmental SMART Center, Department of Chemistry, University of Bari, Via E. Orabona 4, 70126 Bari, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9286-5543","authenticated-orcid":false,"given":"Mariachiara","family":"Bianco","sequence":"additional","affiliation":[{"name":"Interdepartmental SMART Center, Department of Chemistry, University of Bari, Via E. Orabona 4, 70126 Bari, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1624-2365","authenticated-orcid":false,"given":"Angelo","family":"De Stradis","sequence":"additional","affiliation":[{"name":"National Research Council of Italy (CNR), Institute for Sustainable Plant Protection (IPSP), University of Bari, Via Amendola 165\/A, 70126 Bari, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2211-7907","authenticated-orcid":false,"given":"Toufic","family":"Elbeaino","sequence":"additional","affiliation":[{"name":"International Centre for Advanced Mediterranean Agronomic Studies (CIHEAM of Bari), Via Ceglie 9, 70010 Valenzano, Italy"},{"name":"National Research Council of Italy (CNR), Institute for Sustainable Plant Protection (IPSP), Piazzale Enrico Fermi, 1, 80055 Portici, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,9,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1016\/S0168-9452(02)00164-4","article-title":"Silencing Crown Gall Disease in Walnut (Juglans regia L.)","volume":"163","author":"Escobar","year":"2002","journal-title":"Plant Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"703","DOI":"10.1104\/pp.107.111302","article-title":"Salicylic Acid and Systemic Acquired Resistance Play a Role in Attenuating Crown Gall Disease Caused by Agrobacterium tumefaciens","volume":"146","author":"Anand","year":"2008","journal-title":"Plant Physiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"568","DOI":"10.1002\/ps.4331","article-title":"Lipopeptides from a Novel Bacillus Methylotrophicus 39b Strain Suppress Agrobacterium Crown Gall Tumours on Tomato Plants","volume":"73","author":"Ghorbel","year":"2017","journal-title":"Pest Manag. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1111\/nph.17810","article-title":"Agrobacterium tumefaciens Fitness Genes Involved in the Colonization of Plant Tumors and Roots","volume":"233","author":"Torres","year":"2022","journal-title":"New Phytol."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Liu, S., Zheng, Y., Yu, K., Gao, S., Zhao, X., Cao, A., and Han, Q. (2024). Technology for Distribution and Control of Agrobacterium tumefaciens in Cherry Tree Soil. Agriculture, 14.","DOI":"10.3390\/agriculture14111857"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Attai, H., Boon, M., Phillips, K., Noben, J.-P., Lavigne, R., and Brown, P.J.B. (2018). Larger Than Life: Isolation and Genomic Characterization of a Jumbo Phage That Infects the Bacterial Plant Pathogen, Agrobacterium tumefaciens. Front. Microbiol., 9.","DOI":"10.3389\/fmicb.2018.01861"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"4155","DOI":"10.1128\/AEM.02847-10","article-title":"Biocontrol of Ralstonia Solanacearum by Treatment with Lytic Bacteriophages","volume":"77","author":"Fujiwara","year":"2011","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Clavijo-Coppens, F., Ginet, N., Cesbron, S., Briand, M., Jacques, M.-A., and Ansaldi, M. (2021). Novel Virulent Bacteriophages Infecting Mediterranean Isolates of the Plant Pest Xylella fastidiosa and Xanthomonas albilineans. Viruses, 13.","DOI":"10.3390\/v13050725"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Rendueles, C., Duarte, A.C., Escobedo, S., Fern\u00e1ndez, L., Rodr\u00edguez, A., Garc\u00eda, P., and Mart\u00ednez, B. (2022). Combined Use of Bacteriocins and Bacteriophages as Food Biopreservatives. A Review. Int. J. Food Microbiol., 368.","DOI":"10.1016\/j.ijfoodmicro.2022.109611"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"111","DOI":"10.4161\/bact.1.2.14590","article-title":"Pros and Cons of Phage Therapy","volume":"1","author":"Abedon","year":"2011","journal-title":"Bacteriophage"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Fang, Q., Feng, Y., McNally, A., and Zong, Z. (2022). Characterization of Phage Resistance and Phages Capable of Intestinal Decolonization of Carbapenem-Resistant Klebsiella Pneumoniae in Mice. Commun. Biol., 5.","DOI":"10.1038\/s42003-022-03001-y"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Bartnik, P., Lewtak, K., Fio\u0142ka, M., Czaplewska, P., Narajczyk, M., and Czajkowski, R. (2022). Resistance of Dickeya solani Strain IPO 2222 to Lytic Bacteriophage \u03a6D5 Results in Fitness Tradeoffs for the Bacterium during Infection. Sci. Rep., 12.","DOI":"10.1038\/s41598-022-14956-7"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Yang, Y., Shen, W., Zhong, Q., Chen, Q., He, X., Baker, J.L., Xiong, K., Jin, X., Wang, J., and Hu, F. (2020). Development of a Bacteriophage Cocktail to Constrain the Emergence of Phage-Resistant Pseudomonas aeruginosa. Front. Microbiol., 11.","DOI":"10.3389\/fmicb.2020.00327"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Oechslin, F. (2018). Resistance Development to Bacteriophages Occurring during Bacteriophage Therapy. Viruses, 10.","DOI":"10.3390\/v10070351"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Koderi Valappil, S., Shetty, P., Deim, Z., Terhes, G., Urb\u00e1n, E., V\u00e1czi, S., Patai, R., Polg\u00e1r, T., Pertics, B.Z., and Schneider, G. (2021). Survival Comes at a Cost: A Coevolution of Phage and Its Host Leads to Phage Resistance and Antibiotic Sensitivity of Pseudomonas aeruginosa Multidrug Resistant Strains. Front. Microbiol., 12.","DOI":"10.3389\/fmicb.2021.783722"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"5464","DOI":"10.1038\/s41467-023-41156-2","article-title":"Visualizing the Membrane Disruption Action of Antimicrobial Peptides by Cryo-Electron Tomography","volume":"14","author":"Chen","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1016\/j.bcp.2016.09.018","article-title":"Antimicrobial Peptides (AMPs): Ancient Compounds That Represent Novel Weapons in the Fight against Bacteria","volume":"133","author":"Ageitos","year":"2017","journal-title":"Biochem. Pharmacol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1453","DOI":"10.1038\/s41467-023-36994-z","article-title":"Discovering Highly Potent Antimicrobial Peptides with Deep Generative Model HydrAMP","volume":"14","author":"Szymczak","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1016\/j.bsheal.2022.02.003","article-title":"Biosynthesis, Bioactivity, Biotoxicity and Applications of Antimicrobial Peptides for Human Health","volume":"4","author":"Wei","year":"2022","journal-title":"Biosaf. Health"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"203","DOI":"10.26444\/aaem\/105390","article-title":"Bacteriophages, Phage Endolysins and Antimicrobial Peptides\u2014The Possibilities for Their Common Use to Combat Infections and in the Design of New Drugs","volume":"26","author":"Mirski","year":"2019","journal-title":"Ann. Agric. Environ. Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"100254","DOI":"10.1016\/j.microb.2025.100254","article-title":"Examining Alternative Approaches to Antibiotic Utilisation: A Critical Evaluation of Phage Therapy and Antimicrobial Peptides Combination as Potential Alternatives","volume":"6","author":"Alisigwe","year":"2025","journal-title":"Microbe"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"323","DOI":"10.36253\/phyto-15623","article-title":"Isolation, Characterization and Genomic Analysis of a Novel Lytic Bacteriophage Infecting Agrobacterium tumefaciens","volume":"63","author":"Sabri","year":"2024","journal-title":"Phytopathol. Mediterr."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1925","DOI":"10.1007\/s10499-020-00567-4","article-title":"Bacteriophages for Aquaculture Disease Control","volume":"28","author":"Ninawe","year":"2020","journal-title":"Aquac. Int."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Tang, M., Huang, Z., Zhang, X., Kong, J., Zhou, B., Han, Y., Zhang, Y., Chen, L., and Zhou, T. (2023). Phage Resistance Formation and Fitness Costs of Hypervirulent Klebsiella pneumoniae Mediated by K2 Capsule-Specific Phage and the Corresponding Mechanisms. Front. Microbiol., 14.","DOI":"10.3389\/fmicb.2023.1156292"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/j.peptides.2007.10.026","article-title":"Activities of the Frog Skin Peptide, Ascaphin-8 and Its Lysine-Substituted Analogs against Clinical Isolates of Extended-Spectrum Beta-Lactamase (ESBL) Producing Bacteria","volume":"29","author":"Eley","year":"2008","journal-title":"Peptides"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"5725","DOI":"10.1038\/s41467-022-33294-w","article-title":"Bacteriophage-Antibiotic Combination Therapy against Extensively Drug-Resistant Pseudomonas aeruginosa Infection to Allow Liver Transplantation in a Toddler","volume":"13","author":"Chatzis","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"79","DOI":"10.3390\/encyclopedia4010007","article-title":"Bacteriocins and Bacteriophages as Dual Biological Players for Food Safety Applications","volume":"4","author":"Barache","year":"2024","journal-title":"Encyclopedia"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Manohar, P., Loh, B., Nachimuthu, R., and Leptihn, S. (2024). Phage-Antibiotic Combinations to Control Pseudomonas aeruginosa\u2013Candida Two-Species Biofilms. Sci. Rep., 14.","DOI":"10.1038\/s41598-024-59444-2"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Gu Liu, C., Green, S.I., Min, L., Clark, J.R., Salazar, K.C., Terwilliger, A.L., Kaplan, H.B., Trautner, B.W., Ramig, R.F., and Maresso, A.W. (2020). Phage-Antibiotic Synergy Is Driven by a Unique Combination of Antibacterial Mechanism of Action and Stoichiometry. mBio, 11.","DOI":"10.1128\/mBio.01462-20"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Osman, A.-H., Kotey, F.C.N., Odoom, A., Darkwah, S., Yeboah, R.K., Dayie, N.T.K.D., and Donkor, E.S. (2023). The Potential of Bacteriophage-Antibiotic Combination Therapy in Treating Infections with Multidrug-Resistant Bacteria. Antibiotics, 12.","DOI":"10.3390\/antibiotics12081329"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"8807","DOI":"10.1128\/JB.00960-07","article-title":"Multiple Superoxide Dismutases in Agrobacterium tumefaciens: Functional Analysis, Gene Regulation, and Influence on Tumorigenesis","volume":"189","author":"Saenkham","year":"2007","journal-title":"J. Bacteriol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1111\/mpp.12777","article-title":"The Histone-like Protein HupB Influences Biofilm Formation and Virulence in Xanthomonas citri Ssp. citri through the Regulation of Flagellar Biosynthesis","volume":"20","author":"Conforte","year":"2019","journal-title":"Mol. Plant Pathol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Singh, N., Sharma, N., Singh, P., Pandey, M., Ilyas, M., Sisodiya, L., Choudhury, T., Gosain, T.P., Singh, R., and Atmakuri, K. (2022). HupB, a Nucleoid-Associated Protein, Is Critical for Survival of Mycobacterium tuberculosis under Host-Mediated Stresses and for Enhanced Tolerance to Key First-Line Antibiotics. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.937970"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3895","DOI":"10.1007\/s00018-008-8588-y","article-title":"Medium- and Short-Chain Dehydrogenase\/Reductase Gene and Protein Families: The SDR Superfamily: Functional and Structural Diversity within a Family of Metabolic and Regulatory Enzymes","volume":"65","author":"Kavanagh","year":"2008","journal-title":"Cell Mol. Life Sci."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"El Handi, K., Sabri, M., Valentini, F., De Stradis, A., Achbani, E.H., Hafidi, M., El Moujabber, M., and Elbeaino, T. (2022). Exploring Active Peptides with Antimicrobial Activity in Planta against Xylella fastidiosa. Biology, 11.","DOI":"10.3390\/biology11111685"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Sabri, M., El Handi, K., Valentini, F., De Stradis, A., Achbani, E.H., Benkirane, R., and Elbeaino, T. (2023). Exploring Antimicrobial Peptides Efficacy against Fire Blight (Erwinia amylovora). Plants, 12.","DOI":"10.3390\/plants12010113"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1016\/j.ijantimicag.2012.02.003","article-title":"Database Screening and in Vivo Efficacy of Antimicrobial Peptides against Meticillin-Resistant Staphylococcus aureus USA300","volume":"39","author":"Menousek","year":"2012","journal-title":"Int. J. Antimicrob. Agents"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1007\/978-1-60327-164-6_7","article-title":"Enumeration of Bacteriophages by Double Agar Overlay Plaque Assay","volume":"501","author":"Kropinski","year":"2009","journal-title":"Methods Mol. Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1271\/bbb.110972","article-title":"Purification and Characterization of Multiple Bacteriocins and an Inducing Peptide Produced by Enterococcus faecium NKR-5-3 from Thai Fermented Fish","volume":"76","author":"Ishibashi","year":"2012","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Bar\u00f3, A., Badosa, E., Montesinos, L., Feliu, L., Planas, M., Montesinos, E., and Bonaterra, A. (2020). Screening and Identification of BP100 Peptide Conjugates Active against Xylella fastidiosa Using a Viability-qPCR Method. BMC Microbiol., 20.","DOI":"10.1186\/s12866-020-01915-3"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1307","DOI":"10.1007\/s42161-020-00593-4","article-title":"Stem and Crown Gall Disease Caused by Agrobacterium tumefaciens on Golden Euonymus in Iran","volume":"102","author":"Ganjeh","year":"2020","journal-title":"J. Plant Pathol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1002\/cyto.a.20069","article-title":"Mechanism and Use of the Commercially Available Viability Stain, BacLight","volume":"61","author":"Stocks","year":"2004","journal-title":"Cytom. A"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"5438","DOI":"10.1021\/acs.analchem.6b00859","article-title":"Quantitative Evaluation of Filter Aided Sample Preparation (FASP) and Multienzyme Digestion FASP Protocols","volume":"88","year":"2016","journal-title":"Anal. Chem."},{"key":"ref_44","first-page":"7.3.1","article-title":"SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)","volume":"6","author":"Gallagher","year":"2012","journal-title":"Curr. Protoc."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"120882","DOI":"10.1016\/j.talanta.2020.120882","article-title":"An Easily Transferable Protocol for In-Situ Quasi-Non-Invasive Analysis of Protein Binders in Works of Art","volume":"215","author":"Calvano","year":"2020","journal-title":"Talanta"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"108443","DOI":"10.1016\/j.foodcont.2021.108443","article-title":"Determination of Hidden Milk Allergens in Meat-Based Foodstuffs by Liquid Chromatography Coupled to Electrospray Ionization and High-Resolution Tandem Mass Spectrometry","volume":"131","author":"Bianco","year":"2022","journal-title":"Food Control"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/19\/9355\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:49:25Z","timestamp":1760035765000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/19\/9355"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,25]]},"references-count":46,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2025,10]]}},"alternative-id":["ijms26199355"],"URL":"https:\/\/doi.org\/10.3390\/ijms26199355","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,9,25]]}}}