{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:53:04Z","timestamp":1787028784035,"version":"build-2736575974"},"reference-count":54,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T00:00:00Z","timestamp":1782777600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Shanghai Agricultural Science and Technology Innovation Program","award":["K2023008"],"award-info":[{"award-number":["K2023008"]}]},{"name":"Natural Science Foundation of Ningbo City, China","award":["2023J003"],"award-info":[{"award-number":["2023J003"]}]},{"name":"Yunnan Tobacco Company program","award":["2024530000241011"],"award-info":[{"award-number":["2024530000241011"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Life"],"abstract":"<jats:p>Alternaria brassicicola is a necrotrophic fungal pathogen causing black spot disease on cruciferous crops worldwide. In this study, we comprehensively characterized the pathogenic isolate Ab0920a from diseased broccoli in Shanghai using morphological, phylogenetic, host range, fungicide sensitivity, genomic, and functional analyses. Pathogenicity tests on 27 cruciferous varieties revealed a broad host range with varying resistance levels. Fungicide sensitivity assays showed that fluxapyroxad (EC50 = 0.0695 \u00b5g\/mL), prochloraz (0.0711 \u00b5g\/mL), and difenoconazole (0.0863 \u00b5g\/mL) were highly effective, whereas fluazinam was least effective. Genome-wide annotation identified 941 secreted proteins (8.95% of the proteome) and 237 candidate effectors, including 31 small cysteine-rich secreted proteins and 68 homologs of known virulence factors. Conserved effector-associated motifs (e.g., RXLR, [Y\/F\/W]xC) were detected, and carbohydrate-active enzyme CAZyme annotation revealed diverse families potentially involved in plant cell wall degradation. Functional validation using the pSUC2 yeast system confirmed that N-terminal signal peptides of tested effectors are competent for secretion. A PVX-based transient expression assay in Nicotiana benthamiana identified two effectors that suppress Bax-induced programmed cell death, suggesting their potential roles in modulating host immunity. Overall, this study provides comprehensive insights into the pathogen Ab0920a, offering resources for disease management and functional studies on necrotrophic fungal pathogenesis.<\/jats:p>","DOI":"10.3390\/life16071099","type":"journal-article","created":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T16:56:55Z","timestamp":1782838615000},"page":"1099","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Genomic and Functional Characterization of an Alternaria brassicicola Isolate Causing Black Spot Disease on Broccoli Leaves"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-2645-0283","authenticated-orcid":false,"given":"Chunyan","family":"Qi","sequence":"first","affiliation":[{"name":"State Key Laboratory for Quality and Safety of Agro-Products, Institute of Plant Virology, Ningbo University, Ningbo 315211, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5962-4956","authenticated-orcid":false,"given":"Rong","family":"Zeng","sequence":"additional","affiliation":[{"name":"Eco-Environment Protection Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Protection Horticultural Technology, Shanghai 201403, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guangqing","family":"Li","sequence":"additional","affiliation":[{"name":"Horticultural Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Protection Horticultural Technology, Shanghai 201403, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3936-4376","authenticated-orcid":false,"given":"Liqing","family":"Zhang","sequence":"additional","affiliation":[{"name":"Eco-Environment Protection Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Protection Horticultural Technology, Shanghai 201403, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jian","family":"Yang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Quality and Safety of Agro-Products, Institute of Plant Virology, Ningbo University, Ningbo 315211, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peng","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Quality and Safety of Agro-Products, Institute of Plant Virology, Ningbo University, Ningbo 315211, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhujie","family":"Xie","sequence":"additional","affiliation":[{"name":"Horticultural Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Protection Horticultural Technology, Shanghai 201403, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1046\/j.1364-3703.2003.00173.x","article-title":"Alternaria Spp.: From General Saprophyte to Specific Parasite","volume":"4","author":"Thomma","year":"2003","journal-title":"Mol. Plant Pathol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"530","DOI":"10.3852\/12-249","article-title":"The Sections of Alternaria: Formalizing Species-Group Concepts","volume":"105","author":"Lawrence","year":"2013","journal-title":"Mycologia"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"171","DOI":"10.3114\/sim0015","article-title":"Alternaria Redefined","volume":"1","author":"Woudenberg","year":"2013","journal-title":"Stud. Mycol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"799","DOI":"10.1007\/s11557-011-0793-7","article-title":"Nimbya and Embellisia Revisited, with Nov. Comb for Alternaria celosiae and A. perpunctulata","volume":"11","author":"Lawrence","year":"2012","journal-title":"Mycol. Prog."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1016\/j.fgb.2004.10.009","article-title":"Alt a 1 Allergen Homologs from Alternaria and Related Taxa: Analysis of Phylogenetic Content and Secondary Structure","volume":"42","author":"Hong","year":"2005","journal-title":"Fungal Genet. Biol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/j.fgb.2003.10.009","article-title":"Identification of Alternaria brassicicola genes expressed in planta during pathogenesis of Arabidopsis thaliana","volume":"41","author":"Cramer","year":"2004","journal-title":"Fungal Genet. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"394","DOI":"10.1016\/j.phytochem.2009.01.005","article-title":"The phytopathogenic fungus Alternaria brassicicola: Phytotoxin production and phytoalexin elicitation","volume":"770","author":"Pedras","year":"2009","journal-title":"Phytochemistry"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2154","DOI":"10.1094\/PDIS-01-24-0056-RE","article-title":"Aggressive Alternaria brassicicola with Reduced Fungicide Sensitivity Can Be Associated with Naturally Infested Broccoli Seeds","volume":"108","author":"Kaur","year":"2024","journal-title":"Plant Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"e70172","DOI":"10.1111\/ppl.70172","article-title":"Leaf epicuticular wax and hormone-mediated resistance to Alternaria brassicicola in broccoli","volume":"177","author":"Gangurde","year":"2025","journal-title":"Physiol. Plant"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1016\/j.phytochem.2006.12.005","article-title":"Camalexin","volume":"68","author":"Glawischnig","year":"2007","journal-title":"Phytochemistry"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e0108325","DOI":"10.1128\/aem.01083-25","article-title":"Cross-resistance in Alternaria brassicicola from naturally infested broccoli seeds against two succinate dehydrogenase inhibitor fungicides","volume":"991","author":"Kaur","year":"2025","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1146\/annurev-cellbio-092910-154055","article-title":"Hormonal Modulation of Plant Immunity","volume":"28","author":"Pieterse","year":"2012","journal-title":"Annu. Rev. Cell Dev. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1146\/annurev.phyto.112408.132637","article-title":"Fungal Effector Proteins","volume":"47","author":"Stergiopoulos","year":"2009","journal-title":"Annu. Rev. Phytopathol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1146\/annurev-arplant-043014-114623","article-title":"Fungal Effectors and Plant Susceptibility","volume":"66","author":"Lanver","year":"2015","journal-title":"Annu. Rev. Plant Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1111\/nph.13794","article-title":"Effectorp: Predicting Fungal Effector Proteins from Secretomes Using Machine Learning","volume":"2","author":"Sperschneider","year":"2016","journal-title":"New Phytol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Sonah, H., Deshmukh, R.K., and B\u00e9langer, R.R. (2016). Computational Prediction of Effector Proteins in Fungi: Opportunities and Challenges. Front. Plant, 7.","DOI":"10.3389\/fpls.2016.00126"},{"key":"ref_17","first-page":"613","article-title":"Phi-Base: A New Database for Pathogen Host Interactions","volume":"48","author":"Urban","year":"2006","journal-title":"Nucleic Acids Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1236","DOI":"10.1093\/bioinformatics\/btu031","article-title":"Interproscan 5: Genome-Scale Protein Function Classification","volume":"30","author":"Jones","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"490","DOI":"10.1093\/nar\/gkt1178","article-title":"The carbohydrate-active enzymes database (CAZy) in 2013","volume":"42","author":"Lombard","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e2400552","DOI":"10.1002\/jobm.202400552","article-title":"A LysM Effector Mediates Adhesion and Plant Immunity Suppression in the Necrotrophic Fungus Botrytis cinerea","volume":"65","author":"Rascle","year":"2025","journal-title":"J. Basic Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1085","DOI":"10.1016\/j.tim.2025.05.001","article-title":"Fungal cell walls: The rising importance of carbohydrate-active enzymes","volume":"33","author":"Yao","year":"2025","journal-title":"Trends Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1368","DOI":"10.1094\/MPMI-19-1368","article-title":"A Functional Screen to Characterize the Secretomes of Eukaryotic Pathogens and Their Hosts in Planta","volume":"19","author":"Lee","year":"2006","journal-title":"Mol. Plant-Microbe Interact."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Petre, B., and Mcdowell, S. (2014). How Do Filamentous Pathogens Deliver Effector Proteins into Plant Cells?. PLoS Biol., 12.","DOI":"10.1371\/journal.pbio.1001801"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2094","DOI":"10.1111\/mpp.12682","article-title":"Improved Prediction of Fungal Effector Proteins from Secretomes with Effectorp 2.0","volume":"19","author":"Sperschneider","year":"2018","journal-title":"Mol. Plant Pathol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1101\/sqb.2012.77.015933","article-title":"Effector Biology of Plant-Associated Organisms: Concepts and Perspectives","volume":"77","author":"Win","year":"2012","journal-title":"Cold Spring Harb. Symp. Quant. Biol."},{"key":"ref_26","first-page":"16","article-title":"Molecular Characterization of Alternaria Alternata Field Isolates Highly Resistant to the Carboxamide Fungicide Boscalid","volume":"6","author":"Avenot","year":"2008","journal-title":"Phytopathology"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1002\/jemt.24710","article-title":"Microscopic characterization of the infectious process, ROS production, and fungi cellular death of Alternaria alternata on tangerine resistant to QoIs","volume":"88","author":"Dorigan","year":"2025","journal-title":"Microsc. Res. Tech."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1186\/s42483-021-00092-z","article-title":"Biochemical and molecular characterization of Alternaria alternata isolates highly resistant to procymidone from broccoli and cabbage","volume":"3","author":"Wang","year":"2021","journal-title":"Phytopathol. Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"6968","DOI":"10.1038\/s41467-022-34630-w","article-title":"Muscle5: High-Accuracy Alignment Ensembles Enable Unbiased Assessments of Sequence Homology and Phylogeny","volume":"13","author":"Edgar","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1972","DOI":"10.1093\/bioinformatics\/btp348","article-title":"trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses","volume":"25","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1093\/molbev\/msu300","article-title":"IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies","volume":"32","author":"Nguyen","year":"2015","journal-title":"Mol. Biol. Evol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1641","DOI":"10.1093\/molbev\/msp077","article-title":"Fasttree: Computing Large Minimum Evolution Trees with Profiles Instead of a Distance Matrix","volume":"26","author":"Price","year":"2009","journal-title":"Mol. Biol. Evol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"254","DOI":"10.1111\/j.1744-7917.2012.01519.x","article-title":"Data Processing System (DPS) software with experimental design, statistical analysis and data mining developed for use in entomological research","volume":"20","author":"Tang","year":"2013","journal-title":"Insect Sci."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1080\/12298093.2018.1468054","article-title":"Alternaria brassicifolii sp. nov. Isolated from Brassica rapa subsp. pekinensis in Korea","volume":"21","author":"Deng","year":"2018","journal-title":"Mycobiology"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1023","DOI":"10.1038\/s41587-021-01156-3","article-title":"Signalp 6.0 Predicts All Five Types of Signal Peptides Using Protein Language Models","volume":"40","author":"Teufel","year":"2022","journal-title":"Nat. Biotechnol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"646","DOI":"10.1093\/bioinformatics\/17.7.646","article-title":"Evaluation of Methods for the Prediction of Membrane Spanning Regions","volume":"17","author":"Croning","year":"2001","journal-title":"Bioinformatics"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e201900429","DOI":"10.26508\/lsa.201900429","article-title":"Detecting Sequence Signals in Targeting Peptides Using Deep Learning","volume":"2","author":"Armenteros","year":"2019","journal-title":"Life Sci. Alliance"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"444","DOI":"10.1093\/nar\/gkae1082","article-title":"Interpro: The Protein Sequence Classification Resource in 2025","volume":"53","author":"Blum","year":"2025","journal-title":"Nucleic Acids Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1093\/nar\/gkae997","article-title":"The Pfam Protein Families Database: Embracing AI\/ML","volume":"53","author":"Andreeva","year":"2025","journal-title":"Nucleic Acids Res."},{"key":"ref_40","first-page":"441","article-title":"Dbcan: A Web Resource for Automated Carbohydrate-Active Enzyme Annotation","volume":"41","author":"Yin","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1016\/S0378-1119(97)00330-2","article-title":"A Genetic Selection for Isolating cDNA Encoding Secreted Proteins","volume":"198","author":"Jacobs","year":"1997","journal-title":"Gene"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Meng, S., Brown, D.E., Ebbole, D.J., Torto-Alalibo, T., Oh, Y.Y., Deng, J., Mitchell, T.K., and Dean, R.A. (2009). Gene Ontology annotation of the rice blast fungus, Magnaporthe oryzae. BMC Microbiol., 9.","DOI":"10.1186\/1471-2180-9-S1-S8"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1930","DOI":"10.1105\/tpc.107.056093","article-title":"RXLR-Mediated Entry of Phytophthora sojae Effector Avr1b into Soybean Cells Does Not Require Pathogen-Encoded Machinery","volume":"20","author":"Dou","year":"2008","journal-title":"Plant Cell"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1038\/nprot.2007.15","article-title":"High-Efficiency Yeast Transformation Using the Liac\/Ss Carrier DNA\/Peg Method","volume":"2","author":"Gietz","year":"2007","journal-title":"Nat. Protoc."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1141","DOI":"10.1080\/15572536.2004.11833024","article-title":"Molecular characterization of Embellisia and Nimbya species and their relationship to Alternaria, Ulocladium and Stemphylium","volume":"95","author":"Pryor","year":"2003","journal-title":"Mycologia"},{"key":"ref_46","unstructured":"Simmons, E. (2007). Alternaria: An Identification Manual, Fully Illustrated and with Catalogue Raisonn\u00e9 1796\u20132007, Amer Society for Microbiology."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1093\/nar\/gkq1189","article-title":"CDD: A Conserved Domain Database for the Functional Annotation of Proteins","volume":"39","author":"Anderson","year":"2011","journal-title":"Nucleic. Acids Res."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1038\/nature06203","article-title":"A Translocation Signal for Delivery of Oomycete Effector Proteins into Host Plant Cells","volume":"450","author":"Whisson","year":"2007","journal-title":"Nature"},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Godfrey, D., B\u00f6hlenius, H., Pedersen, C., Zhang, Z.G., Emmersen, J., and Thordal-Christensen, H. (2010). Powdery Mildew Fungal Effector Candidates Share N-Terminal Y\/F\/Wxc-Motif. BMC Genom., 11.","DOI":"10.1186\/1471-2164-11-317"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1543","DOI":"10.1126\/science.1194573","article-title":"Genome Expansion and Gene Loss in Powdery Mildew Fungi Reveal Tradeoffs in Extreme Parasitism","volume":"330","author":"Spanu","year":"2010","journal-title":"Science"},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Adhikari, B.N., Hamilton, J.P., Zerillo, M.M., Tisserat, N., L\u00e9vesque, C.A., and Buell, C.R. (2013). Comparative Genomics Reveals Insight into Virulence Strategies of Plant Pathogenic Oomycetes. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0075072"},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Dai, M.D., Su, Z.Z., Zhu, X.M., Li, L., Ye, Z.R., Tan, X.F., Kong, D.D., Liu, X.H., and Lin, F.C. (2024). Genome-Wide Identification and Characterization of Effector Candidates with Conserved Motif in Falciphora Oryzae. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25010650"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"7956","DOI":"10.1073\/pnas.96.14.7956","article-title":"Bax-Induced Cell Death in Tobacco Is Similar to the Hypersensitive Response","volume":"96","author":"Lacomme","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"876","DOI":"10.1002\/ps.566","article-title":"Mechanisms of resistance to fungicides in field strains of Botrytis cinerea","volume":"58","author":"Leroux","year":"2010","journal-title":"Pest Manag. Sci."}],"container-title":["Life"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-1729\/16\/7\/1099\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,3]],"date-time":"2026-07-03T12:17:35Z","timestamp":1783081055000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-1729\/16\/7\/1099"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,30]]},"references-count":54,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["life16071099"],"URL":"https:\/\/doi.org\/10.3390\/life16071099","relation":{},"ISSN":["2075-1729"],"issn-type":[{"value":"2075-1729","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,30]]}}}