{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T09:27:31Z","timestamp":1786958851331,"version":"build-2736575974"},"reference-count":36,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2026,8,16]],"date-time":"2026-08-16T00:00:00Z","timestamp":1786838400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Municipal Public Welfare Applied Research Project","award":["2022GZ21"],"award-info":[{"award-number":["2022GZ21"]}]},{"DOI":"10.13039\/501100004731","name":"Natural Science Foundation of Zhejiang Province","doi-asserted-by":"publisher","award":["LQ21C160005"],"award-info":[{"award-number":["LQ21C160005"]}],"id":[{"id":"10.13039\/501100004731","id-type":"DOI","asserted-by":"publisher"}]},{"award":["LQ21C160005"],"award-info":[{"award-number":["LQ21C160005"]}],"id":[{"id":"https:\/\/ror.org\/04aqat463","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>Pine wilt disease is a devastating forest disease caused by Bursaphelenchus xylophilus infection. The associated bacterium Pseudomonas fluorescens and its flagellin protein have been implicated in this process, but the role of the flagellin-encoding gene fliC in pathogenesis remains unclear. Current gene knockout strategies in P. fluorescens are limited by low transformation efficiency and cumbersome procedures, which constrain functional studies of this gene. To address this methodological gap, we established a fliC knockout method based on the pT18sacB-sacB counterselection system. Short homologous arms were constructed by splice overlap extension PCR, and the knockout mutant \u0394fliC was obtained through S17-1 \u03bbpir-mediated conjugation and sucrose counterselection. The mutant was verified by junction PCR, internal PCR, and sequencing. Using Pinus massoniana callus as the experimental material, we preliminarily compared the wild-type strain and the \u0394fliC mutant in individual pathogenicity and in promoting complex infection by B. xylophilus. The \u0394fliC mutant showed delayed symptom onset and lower disease severity than the wild type, and its ability to promote complex infection also appeared reduced. These results provide preliminary evidence that fliC contributes to symptom development in this callus assay. The established fliC knockout technique provides a methodological basis for further investigating the role of associated bacteria in pine wilt disease.<\/jats:p>","DOI":"10.3390\/biology15161403","type":"journal-article","created":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T08:05:41Z","timestamp":1786953941000},"page":"1403","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Markerless fliC Knockout in Pseudomonas fluorescens and Preliminary Evaluation on Pinus massoniana Callus"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-4143-0488","authenticated-orcid":false,"given":"Haoran","family":"Liu","sequence":"first","affiliation":[{"name":"College of Life Sciences, Huzhou University, Huzhou 313000, China"},{"name":"School of Life and Health Sciences, Huzhou College, Huzhou 313000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sushuang","family":"Liu","sequence":"additional","affiliation":[{"name":"School of Life and Health Sciences, Huzhou College, Huzhou 313000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jia","family":"Wu","sequence":"additional","affiliation":[{"name":"Anji County Forestry Bureau, Huzhou 313300, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinye","family":"Wu","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Huzhou University, Huzhou 313000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jikai","family":"Ding","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Huzhou University, Huzhou 313000, China"},{"name":"College of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-1004-5291","authenticated-orcid":false,"given":"Liwen","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Huzhou University, Huzhou 313000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yaoyu","family":"Feng","sequence":"additional","affiliation":[{"name":"College of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Linyuan","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Huzhou University, Huzhou 313000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Li","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Huzhou University, Huzhou 313000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e18706","DOI":"10.7717\/peerj.18706","article-title":"Comparative analysis of volume growth processes of Masson pine and Chinese fir forests in different regions of southern China","volume":"13","author":"Chen","year":"2025","journal-title":"PeerJ"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"45103","DOI":"10.1038\/s41598-025-32106-7","article-title":"Long-term effects of resin tapping on growth and modelling of mature Masson pine (Pinus massoniana)","volume":"15","author":"Tang","year":"2025","journal-title":"Sci. Rep."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Yang, D., Rui, L., Qiu, Y., Wen, T., Ye, J., and Wu, X. (2024). The Bursaphelenchus xylophilus Effector BxNMP1 Targets PtTLP-L2 to Mediate PtGLU Promoting Parasitism and Virulence in Pinus thunbergii. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25137452"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Zhao, J., Huang, J., Yan, J., and Fang, G. (2020). Economic Loss of Pine Wood Nematode Disease in Mainland China from 1998 to 2017. Forests, 11.","DOI":"10.3390\/f11101042"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"734","DOI":"10.1111\/nph.17594","article-title":"WIND Transcription Factors Orchestrate Wound-Induced Callus Formation, Vascular Reconnection and Defense Response in Arabidopsis","volume":"232","author":"Iwase","year":"2021","journal-title":"New Phytol."},{"key":"ref_6","first-page":"437","article-title":"Advances in Research of Pathogenic Mechanism of Pine Wilt Disease","volume":"4","author":"Wang","year":"2010","journal-title":"Afr. J. Microbiol. Res."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Fu, Y., Liu, H., and Wu, X. (2020). Diversity and Function of Endo-Bacteria in Bursaphelenchus xylophilus from Pinus massoniana Lamb. in Different Regions. Forests, 11.","DOI":"10.3390\/f11050487"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"An, Y., Li, Y., Li, D., Zhang, W., Feng, Y., Liu, Z., Wang, X., Wen, X., and Zhang, X. (2022). The Changes of Microbial Communities and Key Metabolites after Early Bursaphelenchus xylophilus Invasion of Pinus massoniana. Plants, 11.","DOI":"10.3390\/plants11212849"},{"key":"ref_9","unstructured":"Feng, Y. (2025). Effect of Flagellin from Pseudomonas fluorescens on the Expression of Virulence-Associated Genes in Bursaphelenchus xylophilus. [Master\u2019s Thesis, Zhejiang A&F University]."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1111\/j.1439-0329.2005.00417.x","article-title":"Mutualistic Symbiosis between Bursaphelenchus xylophilus and Bacteria of the Genus Pseudomonas","volume":"35","author":"Zhao","year":"2005","journal-title":"For. Pathol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1111\/1758-2229.12202","article-title":"Bacterial Role in Pine Wilt Disease Development-Review and Future Perspectives","volume":"7","author":"Nascimento","year":"2015","journal-title":"Environ. Microbiol. Rep."},{"key":"ref_12","first-page":"151","article-title":"Purification of Flagellin of Pseudomonas fluorescens GcM5-1A Carried by the Pine Wood Nematode, Bursaphelenchus xylophilus, and Its in Vitro Toxicity to a Suspension of Cells of Pinus thunbergii","volume":"16","author":"Guo","year":"2008","journal-title":"Russ. J. Nematol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"729","DOI":"10.1007\/s10658-013-0202-y","article-title":"Effects of Pseudomonas fluorescens Flagellin on Physiological and Biochemical Characteristics in the Suspension Cells of Pinus thunbergii","volume":"136","author":"Xu","year":"2013","journal-title":"Eur. J. Plant Pathol."},{"key":"ref_14","first-page":"243","article-title":"Two Cyclic Dipeptides from Pseudomonas fluorescens GcM5-1A Carried by the Pine Wood Nematode and Their Toxicities to Japanese Black Pine Suspension Cells and Seedlings in Vitro","volume":"39","author":"Guo","year":"2007","journal-title":"J. Nematol."},{"key":"ref_15","unstructured":"Li, S. (2010). Mechanism of Action of Flagellin from Pseudomonas fluorescens GcM5-1A on Pinus thunbergii and Application of Protease Inhibitors in Controlling Monochamus alternatus. [Master\u2019s Thesis, Qingdao University]."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"125404","DOI":"10.1016\/j.ijbiomac.2023.125404","article-title":"The Role of Flagellin F in Vibrio parahaemolyticus-Induced Intestinal Immunity and Functional Domain Identification","volume":"244","author":"Li","year":"2023","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"129395","DOI":"10.1016\/j.ijbiomac.2024.129395","article-title":"Construction of the Flagellin F Mutant of Vibrio parahaemolyticus and Its Toxic Effects on Silver Pomfret (Pampus argenteus) Cells","volume":"259","author":"Li","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"512","DOI":"10.1021\/acsinfecdis.5b00097","article-title":"Permeability Barrier of Gram-Negative Cell Envelopes and Approaches to Bypass It","volume":"1","author":"Zgurskaya","year":"2015","journal-title":"ACS Infect. Dis."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e01205-23","DOI":"10.1128\/mbio.01205-23","article-title":"The Gram-Negative Permeability Barrier: Tipping the Balance of the in and the out","volume":"14","author":"Maher","year":"2023","journal-title":"mBio"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Huang, W., and Wilks, A. (2017). A Rapid Seamless Method for Gene Knockout in Pseudomonas aeruginosa. BMC Microbiol., 17.","DOI":"10.1186\/s12866-017-1112-5"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3485","DOI":"10.1093\/nar\/gkad170","article-title":"Cas9 Off-Target Binding to the Promoter of Bacterial Genes Leads to Silencing and Toxicity","volume":"51","author":"Rostain","year":"2023","journal-title":"Nucleic Acids Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"842","DOI":"10.1021\/acssynbio.4c00757","article-title":"CnRed: Efficient, Marker-Free Genome Engineering of Cupriavidus necator H16 by Adapted Lambda Red Recombineering","volume":"14","author":"Arhar","year":"2025","journal-title":"ACS Synth. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Mosberg, J., Gregg, C., Lajoie, M., Wang, H., and Church, G. (2012). Improving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleases. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0044638"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e605","DOI":"10.1002\/cpz1.605","article-title":"Recombineering in Non-Model Bacteria","volume":"2","author":"Corts","year":"2022","journal-title":"Curr. Protoc."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1820","DOI":"10.1038\/nprot.2015.115","article-title":"Precision-Engineering the Pseudomonas aeruginosa Genome with Two-Step Allelic Exchange","volume":"10","author":"Hmelo","year":"2015","journal-title":"Nat. Protoc."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"111080","DOI":"10.1016\/j.isci.2024.111080","article-title":"An Improved Temperature-Sensitive Shuttle Vector System for Scarless Gene Deletion in Human-Gut-Associated Bifidobacterium Species","volume":"27","author":"Kozakai","year":"2024","journal-title":"iScience"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Cai, L., Li, W., and Zhong, M. (2025). Combining PCR to Detect Junction Fragments and Deleted Exons in the Prenatal Diagnosis of BMD Can Effectively Identify Maternal Cell Contamination. Front. Med., 12.","DOI":"10.3389\/fmed.2025.1599498"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"115540","DOI":"10.1016\/j.diagmicrobio.2021.115540","article-title":"A Deletion in the N Gene May Cause Diagnostic Escape in SARS-CoV-2 Samples","volume":"102","author":"Zannoli","year":"2021","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"5835","DOI":"10.1128\/JCM.41.12.5835.2003","article-title":"Making Internal Amplification Control Mandatory for Diagnostic PCR","volume":"41","author":"Hoorfar","year":"2003","journal-title":"J. Clin. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"15096","DOI":"10.1038\/srep15096","article-title":"The no-SCAR (Scarless Cas9 Assisted Recombineering) System for Genome Editing in Escherichia coli","volume":"5","author":"Reisch","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Yan, J., Lin, N., Wang, X., Chen, X., Wang, H., Lin, Q., Zhou, X., Zhang, L., and Liao, L. (2022). Markerless Gene Deletion in Ralstonia solanacearum Based on Its Natural Transformation Competence. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.977580"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Gorshkov, V., Parfirova, O., Petrova, O., Gogoleva, N., Kovtunov, E., Vorob\u2019ev, V., and Gogolev, Y. (2021). The Knockout of Enterobactin-Related Gene in Pectobacterium atrosepticum Results in Reduced Stress Resistance and Virulence towards the Primed Plants. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22179594"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"765","DOI":"10.47191\/ijmscrs\/v3-i4-35","article-title":"Importance of Virulence Factors in Bacterial Pathogenicity: A Review","volume":"3","author":"Abdulateef","year":"2023","journal-title":"Int. J. Med. Sci. Clin. Res. Stud."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Nedeljkovic, M., Sastre, D., and Sundberg, E. (2021). Bacterial Flagellar Filament: A Supramolecular Multifunctional Nanostructure. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22147521"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Yamaguchi, T., Toma, S., Terahara, N., Miyata, T., Ashihara, M., Minamino, T., Namba, K., and Kato, T. (2020). Structural and Functional Comparison of Salmonella Flagellar Filaments Composed of FljB and FliC. Biomolecules, 10.","DOI":"10.3390\/biom10020246"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"101123","DOI":"10.1016\/j.tfp.2025.101123","article-title":"Insights into Pine Wilt Disease: A Review on Biology and Pathogenicity of Bursaphelenchus xylophilus and B. mucronatus","volume":"24","author":"Silva","year":"2025","journal-title":"Trees For. People"}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/16\/1403\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T08:52:52Z","timestamp":1786956772000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/16\/1403"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,16]]},"references-count":36,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["biology15161403"],"URL":"https:\/\/doi.org\/10.3390\/biology15161403","relation":{},"ISSN":["2079-7737"],"issn-type":[{"value":"2079-7737","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,16]]}}}