The haloacid dehalogenase (HAD) superfamily represents a large group of enzymes across diverse taxa. However, the characteristics and functional roles of HAD members in the destructive apple canker pathogen, Valsa mali strain Vm1 (Vm1), remain poorly understood, particularly regarding their expression during infection. In this study, the full-length cDNA sequence of the VmHAD gene from Vm1 was cloned using rapid amplification of cDNA ends (RACE) technology, and its bioinformatic properties, subcellular localization, and expression patterns during infection were characterized. The VmHAD cDNA was 1044 bp in length, containing a complete open reading frame (ORF) of 798 bp that encodes a 265 amino acid protein with a conserved HAD-like domain. Phylogenetic analysis revealed that VmHAD shares the highest similarity with the (S)-2-haloacid dehalogenase (accession no. KUI70710.1) from Cytospora mali 03-8, belonging to the L-2-haloacid dehalogenase family within the HAD hydrolase superfamily. Subcellular localization analysis using a transient expression system in Nicotiana benthamiana indicated that VmHAD is distributed in both the nucleus and cytoplasm. Expression profiling demonstrated that VmHAD was significantly upregulated during the infection of detached apple branches by Vm1, with relative expression levels increasing 3.13-, 4.25-, and 3.98-fold at 3, 5, and 7 days post-inoculation, respectively, compared with day 1, whereas no expression was detected in the uninoculated control. These findings identify VmHAD as a novel HAD family member in Vm1 and suggest that it plays a potential role in the infection process and pathogenicity. This work provides new insights into the molecular mechanisms underlying V. mali pathogenicity and contributes to the development of effective strategies for disease management.

The cereal cyst nematode, Heterodera avena, is responsible for substantial economic losses in the global production of wheat, barley, and other cereal crops. Extracellular enzymes, particularly those from the glycoside hydrolase 18 (GH18) family, such as chitinases secreted by Trichoderma spp., play a crucial role in nematode control. However, the genome-wide analysis of Trichoderma longibrachiatum T6 (T6) GH18 family genes in controlling of H. avenae remains unexplored. Through phylogenetic analysis and bioinformatics tools, we identified and conducted a detailed analysis of 18 GH18 genes distributed across 13 chromosomes. The analysis encompassed gene structure, evolutionary development, protein characteristics, and gene expression profiles following T6 parasitism on H. avenae, as determined by RT-qPCR. Our results indicate that 18 GH18 members in T6 were clustered into three major groups (A, B, and C), which comprise seven subgroups. Each subgroup exhibits highly conserved catalytic domains, motifs, and gene structures, while the cis-acting elements demonstrate extensive responsiveness to hormones, stress-related signals, and light. These members are significantly enriched in the chitin catabolic process, extracellular region, and chitinase activity (GO functional enrichment), and they are involved in amino sugar and nucleotide sugar metabolism (KEGG pathway enrichment). Additionally, 13 members formed an interaction network, enhancing chitin degradation efficiency through synergistic effects. Interestingly, 18 members of the GH18 family genes were expressed after T6 parasitism on H. avenae cysts. Notably, GH18-3 (Group B) and GH18-16 (Group A) were significantly upregulated, with average increases of 3.21-fold and 3.10-fold, respectively, from 12 to 96 h after parasitism while compared to the control group. Meanwhile, we found that the GH18-3 and GH18-16 proteins exhibit the highest homology with key enzymes responsible for antifungal activity in T. harzianum, demonstrating dual biocontrol potential in both antifungal activity and nematode control. Overall, these results indicate that the GH18 family has undergone functional diversification during evolution, with each member assuming specific biological roles in T6 effect on nematodes. This study provides a theoretical foundation for identifying novel nematicidal genes from T6 and cultivating highly efficient biocontrol strains through transgenic engineering, which holds significant practical implications for advancing the biocontrol of plant-parasitic nematodes (PPNs).