Calonectria pseudoreteaudii is an important causal agent of Eucalyptus leaf blight in southern China. This pathogen causes Eucalyptus tree disease across numerous regions in southern China. In addition to diseased leaves, C. pseudoreteaudii has occasionally been isolated from soil in Eucalyptus plantations. The aim of this study was to clarify whether C. pseudoreteaudii causing Eucalyptus leaf blight in China is mainly clonally reproduced and to determine the potential spreading mechanism of C. pseudoreteaudii between diseased leaves and soil. To this end, 10 polymorphic microsatellite markers were analyzed to detect the genetic diversity of 97 C. pseudoreteaudii isolates from diseased leaves and soil in a Eucalyptus plantation in Guangxi Zhuang Autonomous Region, southern China. The analysis showed that the genetic diversity of the isolates from both the diseased leaves and soil was high. However, the gene and genotype diversity of the C. pseudoreteaudii isolates from diseased leaves were higher than those of the isolates from the soil. Moreover, all genotypes detected in the isolates from the soil were also found in the isolates from the diseased leaves. Structural analyses did not show clear population structures related to the population substrates of the diseased leaves or soil, and molecular variance analyses indicated that no significant genetic differentiation existed between the diseased leaf and soil populations. These results suggest that C. pseudoreteaudii in soil spreads from diseased leaves, and that an asexual cycle is the primary reproductive mode in both diseased leaf and soil populations. This is the first study on the genetic diversity and population structure of C. pseudoreteaudii. The high genetic diversity and spread pathways of this pathogen may pose challenges in controlling the disease. C. pseudoreteaudii from both diseased leaves and soils in Eucalyptus plantations needs to be carefully monitored for disease control and management.

The genus Calonectria includes many aggressive plant-pathogenic species with a worldwide distribution. Calonectria leaf blight is one of the most prominent diseases of Eucalyptus trees in Southeast Asian and South American plantations. Inoculation trials to evaluate pathogenicity of Calonectria spp. typically use conidial suspensions but this is not possible for species that do not sporulate sufficiently in culture. Calonectria pseudoreteaudii is one of the species that is most aggressive to Eucalyptus in China but most isolates fail to produce conidia in culture, requiring an alternative procedure for artificial inoculation. This study compared inoculations utilizing conidial and hyphal fragment suspensions. Two Eucalyptus genotypes were used, and these were inoculated with different concentrations of hyphal fragments or conidia of three C. pseudoreteaudii isolates. Three days after inoculation, the treated Eucalyptus plants displayed similar disease symptoms, regardless of whether they had been inoculated with conidia or hyphal fragments. This was consistent for all C. pseudoreteaudii isolates and also the different Eucalyptus genotypes. The results demonstrate that hyphal fragment suspensions can be used to provide a reliable indication of C. pseudoreteaudii isolate pathogenicity when conidia are not available for inoculation studies.

Calonectria eucalypti is a destructive fungal pathogen causing Eucalyptus leaf blight in China and Indonesia. Despite its ecological and economic impact, the molecular mechanisms underlying its pathogenicity remain largely unexplored. We assembled a high-quality 62.1 Mb genome of C. eucalypti comprising 19 contigs and 13,112 predicted genes, including 1,006 carbohydrate-active enzymes (CAZymes), 351 candidate effectors, and 90 secondary metabolite biosynthetic gene clusters (SMGCs). Comparative genomics revealed 60 expanded gene families and eight species-specific clusters in C. eucalypti, predominantly enriched in virulence-associated functions such as transferase activity, sporulation, and host adhesion. Time-course transcriptome profiling during infection of Eucalyptus urophylla × E. tereticornis EC254 at 12, 24, 48, and 72 h post-inoculation (hpi) identified 708 CAZyme genes, 188 putative effectors, and 68 SMGCs as differentially expressed, exhibiting stage-specific patterns related to fungal penetration, host cell wall degradation, immune suppression, and nutrient acquisition. Notably, glycoside hydrolases (GHs) were the most abundantly induced CAZyme family, suggesting a central role in degrading host cell wall components. Prioritized gene candidates, such as GH28 polygalacturonase A08104 and effectors A12658 and A12266, showed strong homology to known virulence factors and high expression during host colonization. Moreover, most SMGCs were located in transposable element (TE)-rich regions, supporting a “two-speed” genome architecture that may facilitates adaptive evolution and pathogenic innovation. This study provides the first integrative genome and infection-stage transcriptome map of C. eucalypti, revealing a coordinated, stage-specific deployment of pathogenicity-related gene clusters. These findings provide a foundational resource for understanding fungal virulence and developing targeted strategies to manage Eucalyptus leaf blight.

Objectives: Lasiodiplodia pseudotheobromae is an important fungal pathogen associated with die-back, canker and shoot blight in many plant hosts with a wide geographic distribution. The aim of our study was to provide high-quality genome assemblies and sequence annotation resources of L. pseudotheobromae, to facilitate future studies on the systematics, population genetics and genomics of the fungal pathogen L. pseudotheobromae. Data description: High-quality genomes of five L. pseudotheobromae isolates were sequenced based on Oxford Nanopore technology (ONT) and Illumina HiSeq sequencing platform. The total size of each assembly ranged from 43 Mb to 43.86 Mb and over 11,000 protein-coding genes were predicted from each genome. The proteins of predicted genes were annotated using multiple public databases, among the annotated protein-coding genes, more than 4,300 genes were predicted as potential virulence genes by the Pathogen Host Interactions (PHI) database. Moreover, the genome comparative analysis among L. pseudotheobromae and other closely related species revealed that 7,408 gene clusters were shared among them and 152 gene clusters unique to L. pseudotheobromae. This genome and associated datasets provided here will serve as a useful resource for further analyses of this fungal pathogen species.

Calonectria leaf blight is one of the most destructive diseases affecting Eucalyptus plantations worldwide. Although Guangxi holds the largest area of Eucalyptus plantations in China, the species diversity and geographic distribution of Calonectria associated with this disease remain poorly understood. To address this gap, a systematic field survey was carried out across 19 Eucalyptus plantations in Guangxi in 2025, planted with E. urophylla × E. grandis or other commercial hybrids. A total of 497 Calonectria isolates were obtained from symptomatic plant tissues, and eight species belonging to three species complexes were identified based on multi-locus phylogenetic analyses. These included C. queenslandica (83.3%), C. pseudoreteaudii (9.7%), C. reteaudii (4.6%) in the C. reteaudii species complex, C. aciculata (0.2%), C. eucalypti (0.8%), C. honghensis (0.2%) in the C. colhounii species complex, C. hongkongensis (0.8%), C. ilicicola (0.4%) in the C. kyotensis species complex. Calonectria species composition varied among the three studied clusters, with seven species detected in the West, three in the South and two in the East. Calonectria queenslandica was present and dominant in all three clusters, indicating that it is the main causal agent of Calonectria leaf blight outbreaks across the province. MaxEnt modeling identified annual mean temperature (Bio1, contribution 66.8%) as the most influential bioclimatic variable associated with the predicted distribution of C. queenslandica, with high suitability habitats concentrated in central-southern Guangxi, central-eastern Guangdong, and southern Fujian. Precipitation of warmest quarter contributed 12.2% but had zero permutation importance, indicating no independent effect, though rainfall and humidity may still influence disease development. Pathogenicity tests confirmed that all species were pathogenic on E. urophylla × E. tereticornis trees, with significant variation in aggressiveness both between and within species. This study provides a comprehensive assessment of Calonectria diversity and distribution in Guangxi Eucalyptus plantations. The findings extend the known geographic ranges of Calonectria and offer a scientific basis for developing sustainable management strategies for the disease.