Biscogniauxia mediterranea is an opportunistic fungal pathogen associated with charcoal canker disease in both juvenile and mature oak trees. This study investigated the genetic diversity, population structure, and pathogenic variability of B. mediterranea populations in the Zagros forests of Iran using inter-simple sequence repeat (ISSR) markers, with the goal of supporting effective forest disease management strategies. A total of 159 isolates were obtained from symptomatic oak trees in four ecologically distinct provinces: Fars, Ilam, Kermanshah, and Kohgiluyeh & Boyer-Ahmad. Genetic analyses revealed high genetic diversity and clear geographic structuring. Most genetic variation occurred within populations, while significant differentiation among regions was detected. Population structure analyses indicated the presence of major genetic groups associated with geographic origin, with additional overlapping patterns suggesting gene flow among regions. Pathogenicity tests confirmed that all tested haplotypes were capable of causing charcoal canker, with isolates from western regions exhibiting greater aggressiveness than those from the southwest. Overall, the findings indicate that geographic and environmental factors are associated with variation in both the population structure and pathogenic potential of B. mediterranea, providing guidance for targeted management of oak decline in the Zagros forests.

Resilience to the interaction of multiple stress factors of drought, wildfire, and canker disease has been little studied. During the 2012 to 2016 megadrought in California, black walnut trees were observed to resprout from the base due to water stress-induced dieback. After monitoring plants for a year, wildfire burned all the plants at twoof our study sites, which was followed by vegetative resprouting. Plants also expressed symptoms of canker disease. We thus had an opportunity to assess the impacts of water stress, wildfire, growth form, and canker disease on plant performance. Water potentials and photosynthetic rates of drought-induced resprouts were monitored and compared to nearby adults that had not experienced drought-induced dieback, both inland (San Dimas, California) and at coastal exposures (Malibu, CA). We monitored the recovery of adults and drought-induced resprouts from 2017 to 2019, including fire-induced resprouts at coastal exposures following a 2018 wildfire. In addition, stem canker number and canker length were compared among irrigated and non-irrigated growth forms, including drought-induced resprouts, coppice-induced resprouts, adults, and resprouting adults. Drought-induced resprouts displayed higher water potentials and photosynthetic rates than adults, especially during dry periods, but had lower (more negative) values than fire-induced resprouts. Non-irrigated walnut trees displayed greater canker initiation and canker elongation rates than irrigated trees, with irrigated coppice-induced resprouts showing the least canker development. Resprouting can temporarily relieve water stress, but xylem water stress and canker disease agents feed off one another. Repeat stresses of drought, fire and disease compound to reduce the resilience of individuals and populations of walnut.

Climate refugia endemics have persisted through past environmental shifts, but their responses to modern severe weather remain poorly understood. Torreya taxifolia (Taxaceae), a critically endangered conifer endemic to steephead ravines of Florida and Georgia, has declined for decades due to fungal canker diseases. Hurricane Michael, a Category 5 storm in 2018, caused extensive forest damage in its refugial habitat. We investigated how natural variation in disturbance was linked to tree health and microclimatic conditions relevant to disease. We monitored the health and habitat of 40 wild T. taxifolia individuals for 4 years after the hurricane by measuring branch growth and mortality and temperature and light around each T. taxifolia. We assessed the abundance and area of fungal cankers. Hurricane disturbance was characterized using complementary metrics of forest structural change (basal area) and the size-weighted abundance of fallen and standing trees. Tree fall was associated with increased branch growth when quantified with summed diameter at breast height (DBH) metrics, but not with basal area metrics. Neither metric of tree fall was significantly associated with branch mortality. Local temperatures were positively associated with canker abundance, though not with total canker area, and were not directly linked to measures of forest change. Hurricane disturbance was associated with short-term after-storm growth responses among surviving T. taxifolia individuals, while warmer local conditions were linked to increased canker disease symptoms. Together, these results suggest that torreya growth was more closely associated with neighborhood-scale disturbance intensity than with broad reductions in stand-level biomass.

Rugonectria rugulosa is a member of the family Nectriaceae associated with trunk canker diseases in woody plants, including avocado (Persea americana cv. Hass). Here, we report the 53 Mb draft genome assembly of a Colombian isolate, providing a genomic resource to investigate the biology and pathogenic potential of this species.

Bacterial canker disease (BCD), mainly caused by Pseudomonas syringae pv. syringae (Pss) and P. amygdali pv. morsprunorum (Pam), threatens the Pacific Northwest (PNW) cherry industry. One major cause of young tree death in the PNW is heading-cut infections by Pseudomonas in newly established orchards. While copper has long been used for cut-wound protection, copper resistance in pseudomonads is widespread in the PNW. This study evaluated alternative chemicals and cultural strategies for management of cut-wound infections. Lime sulfur (LS) exhibited concentration- and time-dependent bacteriostatic activity in vitro, with 5-10% LS efficiently reducing Pss and Pam populations within 6h. Clove oil at concentrations equal to or above 0.0625% completely eliminated both pathogens within 30 s. To evaluate the efficacy of alternative chemicals, field trials in 2024 revealed that kasugamycin and oxytetracycline significantly reduced canker development when heading-cuts were performed under hot, dry weather conditions as compared with copper- and latex-paint-based treatments. When heading-cuts were done under cool, humid conditions, oxytetracycline, Actigard, and kasugamycin exhibited better cut-wound protection in terms of canker length. Canker development was more rapid and occurred at a higher frequency in trees pruned under cool, humid conditions compared to those pruned during hot, dry conditions. In 2025 field trials, kasugamycin (Kasumin) again suppressed canker progression, while addition of Vacciplant or additional chemical treatment offered no benefit. Furthermore, when plant tissues were removed at least 12.7 cm below visible cankers, no pathogen can be recovered. Our results indicate that chemical application and weather conditions during heading cut might be crucial in preventing cut-wound pseudomonads infection.

ABSTRACTThe taxonomic boundaries of Ceratocystis species related to C. manginecans have remained contentious due to limited morphological variation, interfertility in laboratory mating studies, and the application of different species concepts. However, recent studies have highlighted significant differences among species recently reduced to synonymy with C. manginecans, including host associations and biological traits, all with important implications for disease management. We examined the phylogenetic relationships, genetic diversity and population structure of isolates considered broadly as Ceratocystis manginecans, specifically those treated as C. eucalypticola and C. manginecans in various previous studies. The study included a comprehensive dataset of isolates from multiple hosts and regions, including both historical and recent outbreaks of Ceratocystis canker and wilt disease. Multi‐locus phylogenetic analyses, supported by genotyping at 16 SSR loci, resolved two genetically distinct lineages: Lineage 1 (previously treated as C. eucalypticola) and Lineage 2 (previously treated as C. manginecans). These lineages showed structured genetic differentiation across geographic regions and host associations, consistent with previously reported differences in aggressiveness and infection biology. Detailed morphological comparisons also provided support for the two groupings. Although the results revealed the existence of two lineages among isolates currently treated as C. manginecans, discordance among loci and signals of admixture in a subset of isolates were consistent with evidence for mixed ancestry and/or incomplete lineage sorting. This suggests incomplete reproductive isolation that is contributing to current taxonomic ambiguity and confusion. The results support treating the isolates in the two groups as distinct lineages (Lineage 1 and 2) within C. manginecans, recognising their differences while maintaining a conservative taxonomic approach and thus facilitating important quarantine considerations and future studies focused on disease management.

Cytospora is a species-rich and globally distributed genus in Diaporthales, characterized by hyaline, aseptate, and allantoid ascospores and conidia. Many species in this genus are recognized as important plant pathogens that cause branch cankers on a wide range of hosts. Recent taxonomic revisions, integrating morphological and molecular phylogenetic data, have clarified the generic limits of Cytospora, resulting in the recognition of three groups and eight types in the teleomorph and three groups and thirteen types in the anamorph, as well as ten species complexes. In this study, canker diseases on Populusszechuanica var. tibetica and Salixlasiogyne were investigated in Xizang, China. Species identification was conducted using a polyphasic approach combining morphological characteristics and multi-locus phylogenetic analyses based on ITS, act, rpb2, tef1, and tub2 sequences. Consequently, two new species, Cytosporachayuensis from the C.leucostoma species complex and C.sinopopuli from the C.kantschavelii species complex, are introduced herein. This study contributes to knowledge of Cytospora species diversity and provides foundational data for the management of poplar and willow canker diseases.

Whole genome analysis of Diplodia bulgarica associated with apple canker disease provides new insights into its virulence and host-pathogen interactions

Caliciopsis pinea is the ascomycete plant pathogen that causes Caliciopsis canker disease on North American Pinus strobus (eastern white pine). Infections result in downgrading of lumber because of canker formation and overall loss of vigor in P. strobus, which is a critical cover species throughout its native range. C. pinea is challenging to distinguish from closely related species (some of which can coinfect P. strobus) based on macroscopic and microscopic observations alone. C. pinea and other Caliciopsis species that infect conifers all have sign development that is indistinguishable to the naked eye and often indistinguishable using microscopy and exhaustive measurement. Additionally, symptoms may develop on a host without fruiting bodies, making molecular techniques necessary for detection and diagnosis. Improved sensitivity and specificity of diagnostic and detection tools are critical for the early identification of and response to C. pinea infections. Therefore, this study aimed to develop a quantitative PCR assay for the detection, diagnosis, and quantification of C. pinea from infected plant material. This assay targets the internal transcribed spacer region of C. pinea, and specificity was confirmed on a panel of 51 target and nontarget isolates from throughout the P. strobus native range. The limit of detection for the assay was determined to be 10 fg of C. pinea DNA. The technology was transferred out to be tested in another laboratory, as well as another thermocycling platform to ensure reproducibility of results. This study reports the most sensitive and specific assay for C. pinea detection for our current understanding of species diversity within the genus.

Canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) poses a major threat to cultivated kiwifruit, and utilization of wild relatives are key to improve resistance. However, comprehensive comparative genomic analyses between cultivated kiwifruit and their wild relatives with enhanced resistance to Psa remain limited. Here we generate chromosome-scale genome assemblies for eleven wild Actinidia eriantha accessions and one interspecific hybrid between Actinidia eriantha and cultivated Actinidia chinensis var. chinensis. Integrating these with twelve previously released genomes including three Actinidia eriantha and nine Actinidia chinensis var. chinensis, we construct a reference-unbiased graph-based pangenome. These datasets reveal extensive genomic variation, including 31,790,044 SNPs, 13,512,079 InDels and 623,478 structural variations, and provide a landscape of structural variations within and between the two species. Leveraging these datasets, we identify a wild allele showing allele-specific expression, AeNLR25-1, which enhances Psa resistance in cultivated kiwifruit. Genetic and molecular analyses demonstrate that a transposable element-induced structural variation in the AeNLR25-1 promoter introduces a species-specific WRKY binding site, conferring enhanced defense against Psa. Pangenome across cultivated species and wild relatives provides a theoretical framework for accelerating kiwifruit genetic improvement through pangenome-enabled identification of favorable wild alleles.

ABSTRACTWoody canker diseases cause devastating timber losses worldwide. Canker fungi exploit cell wall‐degrading enzymes (CWDEs) to promote plant infection, yet the underlying mechanism of the transcriptional regulation of CWDEs remains largely unknown. Using RNA‐seq analysis and biochemical assays, we show that the poplar pathogen Cytospora chrysosperma upregulates CWDEs during canker progression, especially increased secretion of laccases. The histone lysine methyltransferase Kmt3 (CcKmt3) is induced by C. chrysosperma infection. Loss of CcKmt3 leads to a genome‐wide reduction in H3K36me3 methylation. The ΔCckmt3 strain presented hyphal growth defects, reduced production of hydrolytic enzymes, and impaired virulence. By combining ChIP‐seq with RNA‐seq, we demonstrated that CcKmt3 controls two hydrolases, CcLac11 and CcPme5, for plant cell degradation and virulence in C. chrysosperma. Furthermore, we showed that CcKmt3 affects hyphal growth by regulating the fungal cell wall integrity pathway. Taken together, this study demonstrates that CcKmt3 regulates the transcriptional expression of genes related to CWDEs by modulating H3K36me3 levels. This regulation influences enzyme activity, ultimately impacting the degradation of the plant cell wall and the pathogenicity processes of the poplar canker fungus.

Canker and dieback diseases caused by fungal pathogens represent an increasing threat to woody plants in both urban and forest environments, where sustainable management options are urgently needed. In this study, the biocontrol potential of Bacillus strain N1 was investigated against Neofusicoccum parvum and Gnomoniopsis smithogilvyi, causal agents of canker diseases on Eucalyptus globulus and Castanea sativa, respectively. The whole-genome sequence confirmed the taxonomic identification of strain N1 as B. velezensis, showing high average nucleotide identity and digital DNA–DNA hybridization values with reference strains. AntiSMASH analysis revealed the presence of multiple biosynthetic gene clusters associated with the production of antimicrobial secondary metabolites, including polyketides, non-ribosomal peptides, and lipopeptides, reflecting strain N1’s genomic potential to produce compounds that may contribute to its antifungal activity. Moreover, B. velezensis strain N1 significantly inhibited the growth of N. parvum and G. smithogilvyi and showed a biocontrol efficacy on detached eucalyptus and chestnut shoots. In both preventive and curative treatments and pathosystems, the application of B. velezensis N1 resulted in a significant reduction in the length of necrotic lesions, compared to pathogen-only controls, while no phytotoxic effects were observed on treated shoots. Overall, this study supported B. velezensis N1 as a promising candidate for the sustainable control of canker-associated pathogens in woody plants.

Abstract Plant elicitor peptides (Peps) are conserved damage-associated molecular patterns (DAMPs) that regulate plant immunity and growth, but their roles in citrus, a major economically important fruit crop, remain underexplored. A pangenome analysis of 15 Aurantioideae species identified 17 PRECURSOR OF PEP ( PROPEP ) genes encoding 22-amino-acid mature Peps with conserved receptor-binding motifs. Expression analysis demonstrated that Citrus sinensis PROPEP ( CsPROPEP ) and its receptor CsPEPR were significantly modulated by infection with Xanthomonas citri subsp. citri ( Xcc ), the causal pathogen for citrus canker disease and by phytohormones, including jasmonic acid (JA) and salicylic acid (SA). Supplementing the regeneration medium with 0.01 nM CsPep boosted shoot regeneration efficiency during genetic transformation by 2.43- to 3.21-fold in Citrus sinensis . Likewise, adding CsPep to the Agrobacterium rhizogenes infection solution enhanced root development in stem cuttings, increasing lateral root numbers by 2.23- to 2.98-fold and maximum root length by 1.74- to 2.59-fold. This growth-promoting effect was conserved across other citrus species, including pummelo and lemon. Furthermore, both exogenous application of CsPep and transient overexpression of CsPROPEP activated citrus immune responses, conferring enhanced resistance to citrus canker. These findings highlight the dual role of CsPep in activating immune defenses and promoting regeneration, bridging a critical gap in DAMP peptide signaling in citrus. Our findings offer a peptide-based strategy for sustainable canker management and improved genetic transformation efficiency.

Canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) is a severe bacterial infection threatening global kiwifruit production. Psa causes lignin degradation, cell wall rupture, leaf wilting, and canker formation on branches and trunks, often leading to plant death. The plant cell wall serves as a structural barrier against pathogens, with its thickness, composition, and cell density influencing disease resistance. Comparative studies between resistant germplasms Actinidia eriantha "Maohuaxiong" (A. eriantha 'MHX') and Actinidia latifolia "Kuoye" (A. latifolia 'KY') and susceptible cultivars Actinidia chinensis "Hongyang" (A. chinensis 'HY') and "Donghong" (DH) indicate that the resistant lines developed smaller lesions and slower disease progression after Psa infection, compared with susceptible cultivars. Histological and biochemical analyses revealed that "MHX" and "KY" had denser mesophyll cells and higher lignin deposition. Transcriptomic analysis and transient overexpression screening identified AcLFYL1 as a positive regulator of Psa resistance. AcLFYL1 overexpression increased cell density, lignin content, and disease resistance, while RNAi silencing produced the opposite phenotypes. Yeast one-hybrid, dual-luciferase reporter, and ChIP-qPCR assays confirmed that AcLFYL1 directly activates AcCSE, a key gene in lignin biosynthesis. Consistent with this, overexpression of AcCSE similarly increased cell density and lignin content and improved Psa resistance, whereas knockdown of AcCSE in both wild-type (WT) and AcLFYL1 overexpression lines significantly reduced lignin accumulation and compromised disease resistance. These findings demonstrate that AcLFYL1 enhances resistance by promoting lignin biosynthesis and increasing mesophyll cell density through direct regulation of AcCSE, offering valuable genetic targets for breeding Psa-resistant kiwifruit varieties.

IntroductionCitrus canker disease, caused by the pathogen Xanthomonas citri subsp. citri (Xcc) poses a substantial challenge for citrus production due to the limited efficacy of chemical control and increasing pathogen resistance.MethodsIn this study, we isolated an endophytic bacterial strain, Endophyte S2, from the phyllosphere of citrus plants in Yunnan Province, China. We evaluated its efficacy both as a biocontrol agent and as a modulator of the citrus leaf microbiome.Results and DiscussionIn vitro antagonism assays revealed that Endophyte S2 achieved the highest inhibition rate (68.2%) against Xcc among all tested isolates. Molecular identification based on 16S rRNA gene sequencing classified S2 as Pseudomonas parafulva SAPEU-01. In greenhouse trials, citrus plants with Xcc infestation were treated with SAPEU-01, and phyllosphere samples were collected before treatment and one month after, and analyzed by Illumina MiSeq sequencing. Post-treatment, α-diversity (richness and evenness) increased significantly, and β-diversity (PCoA, Bray–Curtis) showed a clear separation of microbial community structure, with reduced intra-group variability. Taxonomic shifts included the enrichment of Proteobacteria (particularly Pseudomonadaceae and Sphingomonadaceae), as well as genera such as Pseudomonas, Sphingomonas, and Methylobacterium, concomitant with a marked decline in Xanthomonadaceae (including X. citri) and opportunistic taxa such as Escherichia coli O157:H7 and Klebsiella aerogenes. Beneficial taxa, including Leuconostoc tardus, Sphingomonas, and Curtobacterium luteum, also increased. These results suggest Pseudomonas parafulva SAPEU-01 not only suppresses the pathogen but also restructures the phyllosphere microbiome toward greater stability and potential resilience.

After the turn of the 20th century and the change in cultural practices to achieve high production, canker diseases of stone fruits and nut crops, caused by various fungal pathogen species, have become major problems, significantly impacting tree growth and fruit production in California. This study involved multi-year and multi-location field experiments to determine the efficacy of fungicide thiophanate-methyl (Topsin MTM) on reduction of canker development in prune, almond, and avocado orchards. Treatments mainly included applications of Topsin M on pruning wounds and on main trunks and crotches of young trees. Different measurements to determine the efficacy included canker severity, canker length, incidence of shoots, trunks or tree crotches showing canker symptoms, as well as the proportion of samples showing changes in canker situation before and after treatments. The overall results demonstrated that with one exception, Topsin M significantly reduced canker development compared with the untreated control for all the experiments in all the three tree crops. The study strongly confirms the efficacy of thiophanate-methyl applications in these three tree crops to reduce risk of canker development and its usefulness in designing canker control strategies.

The Botryosphaeriaceae family comprises a large and diverse group of fungi that can behave as endophytes, saprophytes, or pathogens, causing dieback and canker diseases worldwide. These fungi can persist in asymptomatic plants, with symptoms often triggered under host stress. This study employed a high-throughput sequencing (HTS) metabarcoding approach to investigate Botryosphaeriaceae populations in symptomatic and asymptomatic almond, avocado and blueberry plants, and to assess other fungi that may act as disease triggers, symptom enhancers or potential biocontrol agents. Botryosphaeriaceae species were detected in 100%, 93%, and 80% of symptomatic almond, avocado, and blueberry plants, respectively, and in 60%, 73% and 66% of asymptomatic plants. The predominant genera were Neofusicoccum, Botryosphaeria, Diplodia and Lasiodiplodia, with significant host-dependent variation in relative abundance. Multiple Botryosphaeriaceae infections predominated over single infections in symptomatic and asymptomatic plants, particularly in avocado. High diversity of total fungal and Botryosphaeriaceae communities was observed across all crops, with greater richness and evenness in avocado. Beta diversity analyses revealed highly significant differences among the fungal microbiomes of the three crops. Several non-Botryosphaeriaceae wood-colonizing fungi, including Cylindrocarpon, Cytospora, Diaporthe, Fusarium and Phaeoacremonium were also detected in symptomatic almond samples, suggesting possible synergistic interactions, whereas potential biocontrol genera such as Trichoderma and Clonostachys were detected exclusively in some symptomatic almond plants. This work contributes to a better understanding of the fungal etiology of canker and dieback diseases in almond, avocado, and blueberry. Furthermore, the high concordance between HTS and multiplex qPCR supports their complementary use to improve plant disease diagnosis, particularly for early preventive detection.

Eucalyptus cinerea (Myrtaceae), commonly known as Argyle apple or Eucalyptus, is an evergreen shrub or small tree native to Australia. It was introduced to China in the 1980s (Silva et al. 2011) and is valued for pulp production and as ornamental foliage. In April 2025, a stem canker disease was observed on 2- to 10-year-old E. cinerea in forests and nurseries in Kunming, Songming, and Pu’er City, Yunnan Province, China. Symptoms included dry, sunken, rough, and cracked tissues on the main stems. Cracks often developed between diseased and healthy tissues, with translucent gummy exudates, a condition exacerbated after rainfall. The disease incidence exceeded 41% across four surveyed forest areas. Total 40 symptomatic tissues were randomly collected from four tree nurseries. Single-spore isolates were obtained from necrotic cortical tissue at the canker margin following Choi et al. (1999), using a sterile microneedle to transfer individual conidia to PDA amended with kanamycin. Seven morphologically similar isolates (YAGF01-YAGF07) were obtained. On potato dextrose agar (PDA), colonies were initially white, turning grey, with short, thick aerial hyphae and irregular margins. Conidiogenous cells were hyaline, aseptate, and narrowly fusiform. Conidia were hyaline, aseptate, subcylindrical, measuring 11.2-15.3×3.4-5.1 μm (n=50), consistent with Botryosphaeria species (Crous et al. 2006). B. dothidea was successfully isolated from 37 of 40 symptomatic tissues (92.5%), with consistent morphology and sequence identity across all isolates. For molecular identification, the internal transcribed spacer (ITS), translation elongation factor 1- alpha (ef1-α) gene, and the beta-tubulin 2 (β-tub2) gene were amplified and sequenced (ITS accessions PX452877-PX452883; ef1-α accessions PX507525-PX507531; β-tub2 accessions PX507532-PX507538) using published primers (White et al. 1990; Ignazio and Linda, 1999; Glass and Donaldson 1995). BLASTn analysis revealed 99-100% identity to the ex-type strain of B. dothidea CWM8000. A phylogenetic tree constructed by Maximum Parsimony (MP) and Maximum Likelihood (ML) methods placed the seven isolates in a clade with B. dothidea with high bootstrap support (94%/95%) and were distinguished clearly from other species. Pathogenicity tests were performed using isolate YAGF01. Stems of six healthy 1-year-old E. cinerea saplings were surface sterilized with 75% ethanol and wounded with a sterile scalpel. A 3-mm mycelial plug of YAGF 01 from a 5-day-old PDA culture was applied to the wound. Control plants were inoculated with sterile agar plugs. All plants were incubated at 25°C with a 12 h photoperiod and 80% humidity. The entire assay was independently repeated three times. Within 5 days, all inoculated stems developed stem canker symptoms identical to those observed in the field, while controls remained asymptomatic. B. dothidea was re-isolated from all symptomatic tissues and confirmed by morphology and sequencing, thus fulfilling Koch's postulates. No fungus was isolated from controls. B. dothidea has been reported to cause stem canker on various hosts, including Cyclocarya paliurus, soybean, and apple(Ilyukhin et al. 2022; Chen et al. 2021; Zheng et al. 2020). To our knowledge, this is the first report of B. dothidea causing stem canker on E. cinerea in China. Given the high disease incidence observed, this pathogen poses a potential threat to the cultivation and economic value of E. cinerea in the region.

Suppression of pine pitch canker disease and promotion of seedling growth in Japanese red pine using a newly isolated strain of Bacillus subtilis

The outbreak of poplar canker caused by Botryosphaeria dothidea poses a severe threat to poplar growth. Its resistance mechanisms are closely linked to the regulation of plant secondary metabolism and transcription factor-mediated defense pathways. However, as a plant-specific regulatory factor family, the functional mechanisms of the WRKY transcription factor (TF) family in poplar resistance to B. dothidea remain unclear. This study systematically elucidated the evolutionary characteristics of the WRKY gene family in Populus trichocarpa and their roles in disease resistance regulation in P. davidiana × P. alba var. Pyramidalis (Pdpap) through integrated genome-wide identification and molecular functional validation. Using BLASTp and Hidden Markov Model screening, 102 PtrWRKYs were identified. Phylogenetic analysis classified them into seven subfamilies based on Arabidopsis thaliana classification criteria. Functional diversification of this family was driven by plasticity in motif combinations, segmental duplication events, and subfamily-specific cis-regulatory elements. PdpapWRKY11, selected via RNA-seq and gene family analysis, significantly enhanced resistance to B. dothidea in transgenic Pdpap lines. Using the Pdpap–B. dothidea interaction system as a model, we further propose that PdpapWRKY11 may activate key phenylpropanoid pathway genes (PdpapPAL and PdpapCAD), promoting lignin accumulation and thereby enhancing pathogen resistance. This research provides foundational insights into WRKY TF functions in poplar and establishes a theoretical basis for improving disease resistance for controlling canker disease.