Trunk canker poses a major threat to the production of Chinese hickory tree (Carya cathayensis Sarg.), which is primarily determined by Botryosphaeriaceae. In our previous work, we identified Botryosphaeria dothidea as the predominant pathogen of this disease. However, it is still unclear about corresponding gene families and mechanisms associated with B. dothidea’s pathogenicity on Chinese hickory tree. Here, we present a comparative analysis of high-quality genome assemblies of Botryosphaeria dothidea and other isolated pathogens, showing highly syntenic relationships between B. dothidea and its closely related species and the conservative evolution of the Botryosphaeriaceae family. Higher GC contents were found in the genomes of B. dothidea and three other isolated pathogens (Botryshaeria cortices, Botryshaeria fabicerciana, and Botryshaeria qingyuanensis) compared to Macrophomina phaseolina, Neofusicoccum parvum, Diplodia corticola, and Lasiodiplodia theobromae. An investigation of genes specific to or expanded in B. dothidea revealed that one secreted glucanase, one orsellinic acid biosynthesis enzyme, and two MFS transporters positively regulated B. dothidea’s pathogenicity. We also observed an overrepresentation of viral integrase like gene and heterokaryon incompatibility proteins in the B. dothidea’s genome. In addition, we observed one LRR-domain-containing protein and two Sec-domain-containing proteins (Sec_1 and Sec_7) that underwent positive selection. This study will help to understand B. dothidea’s pathogenicity and potential influence on the infection of Chinese hickory, which will help in the development of disease control and ensure the security of Chinese hickory production.

Lasiodiplodia theobromae, as one of the causative agents associated with Chinese hickory trunk cankers, has caused huge economic losses to the Chinese hickory industry. Although the biological characteristics of this pathogen and the occurrence pattern of this disease have been well studied, few studies have addressed the related mechanisms due to the poor molecular and genetic study basis of this fungus. In this study, we sequenced and assembled L. theobromae strain LTTK16-3, isolated from a Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Phylogenetic analysis and comparative genomics analysis presented crucial cues in the prediction of LTTK16-3, which shared similar regulatory mechanisms of transcription, DNA replication, and DNA damage response with the other four Chinese hickory trunk canker-associated Botryosphaeria strains including, Botryosphaeria dothidea , Botryosphaeria fabicerciana , Botryosphaeria qingyuanensis, and Botryosphaeria corticis . Moreover, it contained 18 strain-specific protein clusters (not conserved in the other L. theobromae strains, AM2As and CITRA15), with potential roles in specific host-pathogen interactions during the Chinese hickory infection. Additionally, an efficient system for L. theobromae protoplast preparation and polyethylene glycol (PEG) -mediated genetic transformation was firstly established as the foundation for its future mechanisms study. Collectively, the high-quality genome data and the efficient transformation system of L. theobromae here set up the possibility of targeted molecular improvements for Chinese hickory canker control. IMPORTANCE: Fungi with disparate genomic features are physiologically diverse, possessing species-specific survival strategies and environmental adaptation mechanisms. The high-quality genome data and related molecular genetic studies are the basis for revealing the mechanisms behind the physiological traits that are responsible for their environmental fitness. In this study, we sequenced and assembled the LTTK16-3 strain, the genome of Lasiodiplodia theobromae first obtained from a diseased Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Further phylogenetic analysis and comparative genomics analysis provide crucial cues in the prediction of the proteins with potential roles in specific host-pathogen interactions during the Chinese hickory infection. An efficient PEG-mediated genetic transformation system of L. theobromae was established as the foundation for the future mechanisms exploration. The above genetic information and tools set up valuable clues to study L. theobromae pathogenesis and assist in Chinese hickory canker control.

Saffron corm rot (SCR), the most serious disease affecting saffron, has been confirmed to be caused by Fusarium oxysporum in previous studies. Compared to other fungal species, F. oxysporum exhibits host specialization, a special phenomenon associated with the secreted in xylem (SIX) genes. This study examined the pathogenicity specialization of F. oxysporum isolated from saffron corms with SCR disease. The results showed that this F. oxysporum strain was strongly pathogenic to saffron corms, causing SCR; weakly pathogenic to the corms of freesia, which is in the Iridaceae family along with saffron; and not pathogenic to watermelon, melon, and tomato. Other formae speciales of F. oxysporum were not pathogenic to saffron corms. This suggests that F. oxysporum saffron strains exhibit obvious pathogenicity specialization for Iridaceae spp. Subsequently, the F. oxysporum saffron strain (XHH35) genome was sequenced, and a comparative genomics study of XHH35 and three other formae speciales was conducted using OrthoVenn3. XHH35 contained 90 specific genes absent in the other three formae speciales. These genes are involved in certain key biological processes and molecular functions. Based on BLAST homology searching, the F. oxysporum saffron strain (XHH35) genome was predicted to contain seven SIX genes (SIX 4, SIX 6, SIX 7, SIX 10, SIX 11, SIX 12, and SIX 14) highly homologous to F. oxysporum f. sp. lycopersici, which was verified using polymerase chain reaction (PCR) amplification. The corresponding individual phylogenetic tree indicated that the F. oxysporum saffron strain (XHH35) showed a separate branch with different formae speciales. This study is the first-ever report of F. oxysporum f. sp. crocus, a new forma specialis. Based on the specificity of its SIX genes, the SIX 10 gene was selected to further establish a rapid identification technique for F. oxysporum f. sp. crocus, which will be useful in future research.

Botryosphaeria dothidea , the primary causal agent of trunk cankers in Chinese hickory ( Carya cathayensis ), threatens a multimillion-dollar industry in China. Understanding the fundamental biological mechanisms underlying B. dothidea ’s stress adaption and pathogenesis is critical for developing novel control strategies against canker disease. Heat shock factors (Hsfs) are evolutionarily conserved regulators of stress responses and pathogen virulence, yet their roles in woody plant pathogenic fungi remain largely unexplored. This study investigated the functional contributions of Hsfs in B. dothidea , focusing on their involvement in stress adaptation, reproduction, and pathogenesis. A total of two Hsf orthologs, BdHsf1 and BdHsf2, were identified in B. dothidea , and BdHsf2 was found to be indispensable for fungal viability. Deletion of BdHSF1 accelerated hyphal growth, abolished asexual fruiting body formation, and increased sensitivity to oxidative stress, extreme pH, osmotic pressure, cell wall disruptors, and quinone outside inhibitors (QoIs) and dicarboximide fungicides, but not to thermal stress or benzimidazole and DMI fungicides. Unlike in other fungi, BdHsf1 is dispensable for thermal stress response and virulence though BdHsf1 was upregulated during the infection. Additionally, BdHsf1 critically governs oxidative defense through nuclear translocation and domain-specific mechanisms. Further transcriptomic analysis underscores the broad influence of BdHsf1 on antioxidant, cellular metabolism, signaling, and chaperone pathways, which correlates well with phenotypic deficiency observed in BdHSF1 deletion mutant. Collectively, these results provide the first comprehensive functional characterization of Hsfs in B. dothidea , which advances our understanding of Hsf evolution and function in phytopathogens and provides potential targets for managing canker diseases.

Chinese hickory (Carya cathayensis Sarg.) is an economically important tree species widely cultivated in southeastern China, where trunk canker disease caused by Botryosphaeria dothidea poses a serious threat to tree health and production. Pectin-degrading enzymes are important virulence-associated factors that facilitate fungal colonization and host tissue maceration, but their evolutionary diversification and functional roles in B. dothidea during woody host infection remain poorly understood. Comparative genomic analysis revealed lineage-specific variation in the GH28 glycoside hydrolase family among the examined Botryosphaeriaceae species, with B. dothidea exhibiting an expanded GH28 repertoire relative to the analyzed species. Expression analysis and functional assays revealed that BdGH28_3 showed the highest transcript abundance during infection stage and contributed to the full virulence of B. dothidea. A predicted protein–protein interaction (PPI) network suggested potential associations between BdGH28_3 and other pectinolytic enzymes, including polygalacturonases, pectin lyases, and pectinesterases. Collectively, these findings identify GH28 diversification as a distinctive feature of the B. dothidea genome and establish BdGH28_3 as a virulence-associated member, providing a foundation for investigating GH28-mediated pathogenicity in woody hosts.

Lasiodiplodia theobromae, as one of the causing agents associated with Chinese hickory trunk cankers, has caused huge economic losses to the Chinese hickory industry due to its extremely strong pathogenicity. Though the biological characteristics of this pathogen and the occurrence pattern of this disease have been reported, few studies have focused on investigating the mechanisms responsible for L. theobromae survival strategies and pathogenicity. The high-quality genome data and the efficient transformation system are the basis for researching above mechanism events. In this study we sequenced and assembled L. theobromae strain LTTK16-3, and established the first protoplasmic preparation method and polyethylene glycol (PEG) -mediated genetic transformation system for L. theobromae. These genetic information and transformation methods established the foundation for the future mechanisms study of L. theobromae and set up the possibility of targeted molecular improvements for Chinese hickory canker control.