Dezhen Zhang

Apple Valsa canker disease, caused by Valsa mali, is one of the most destructive diseases of apple trees in China and seriously threatens the sustainable development of the apple industry. VmSom1 acts as a core transcription factor in the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling pathway and regulates vegetative growth, development and pathogenicity of this phytopathogen. Transcriptome analysis was performed using the VmSom1 deletion mutant and the wild-type strain sdau11-175, and a significantly differentially expressed transcription factor, VmGAL4, was identified. In this study, the single-gene deletion mutant ΔVmGAL4 and the double-gene deletion mutant ΔVmSom1/VmGAL4 were constructed via homologous recombination, aiming to preliminarily explore the interaction between these two genes. Sequence analysis revealed that the VmGAL4 protein contains a conserved fungal_TF_MHR domain spanning amino acids 164 to 614. Phylogenetic analysis indicated that VmGAL4 shares the closest phylogenetic relationship with homologs from Cytospora schulzeri and Cytospora chrysosperma. Phenotypic assays demonstrated that the VmGAL4 deletion mutant exhibited markedly reduced mycelial growth rate and fewer pycnidia production. Additionally, the mutant displayed enhanced sensitivity to cell wall inhibitors and osmotic stress agents, along with significantly increased capacity to utilize various carbon and nitrogen sources and decreased pathogenicity compared with the wild-type strain. Compared with the single deletion mutant ΔVmSom1, the double mutant ΔVmSom1/VmGAL4 partially rescued the growth defects and also alleviated the reduction in pathogenicity to a certain extent. Nevertheless, conidial production remained severely inhibited in the double mutant. Collectively, VmGAL4 is involved in the regulation of vegetative growth, asexual development, cell wall integrity, osmotic stress response, carbon and nitrogen source utilization, and pathogenicity in V. mali.

Stemphylium lycopersici is one of the major pathogens causing tomato leaf spot disease and its conidial sporulation in vitro can be a challenge in the research on this fungus. With this study, eight isolates of S. lycopersici were identified by the multi‐sequence phylogenetic analysis of internal transcribed spacers 1 and 2 and intervening 5.8S nr DNA, glyceraldehyde‐3‐phosphate dehydrogenase, and calmodulin. The effect of their culture media, mycelial scraping, mycelial age, moisture content, plate sealing on the sporulation of various S. lycopersici isolates in vitro, as well as the dynamics of sporulation (one isolate only) were also evaluated. The conidiospore production assay was performed under specific conditions. Two‐day‐old mycelia were scraped and cultivated without Parafilm sealing at 15°C in continuous darkness. Under these conditions, all eight isolates produced over 100 conidiospores per mm ² . The type of culture medium and moisture level had no significant effect on the sporulation. The method derived from the study should provide sufficient conidiospores of S. lycopersici for further research in this field.

The wonder tree (Idesia polycarpa), a deciduous species in the Salicaceae family is widely distributed in China and serves both ornamental and economic purposes. In May 2024, a blight symptom was observed on approximate 60% of leaves of wonder trees located at Weifang University of Science and Technology (36°53'42.16″N, 118°47'22.4″E). Initially small brown spots appeared on the leaves, then expanded to large irregular grayish-white lesions with dark-brown edges. Ten symptomatic leaves were sampled from 10 infected trees in a 0.3 ha orchard. Fifteen sample tissues (3 × 3 mm) were sterilized with 1% sodium hypochlorite for 10 s followed by 75% ethanol for 30 s, rinsed twice in sterile distilled water and placed on a potato dextrose agar (PDA) plate. The plates were incubated at 25°C for 3 days in darkness. Ten isolates were obtained with similar morphological characteristics using the hyphal-tip method. Three isolates (S2, S5, S6) were used for the morphological characterization and molecular analysis. The colonies reached 8.5 cm in diameter on the PDA plates after 5 days incubation at 25°C。The colonies were initially white, and gradually turned to dark grey with dense aerial mycelia after five days, while the reverse side of the colonies appeared gray to black. Conidiophores were hyaline, cylindrical and smooth. Conidiogenous cells were holoblastic, hyaline, smooth, cylindrical to ampulliform and produced a single conidium at the tips. Conidia were fusiform, aseptate, straight and hyaline, 22-30 × 5-8 μm (av. of 50 conidia=26.2 × 7.3 μm). The morphological characteristics matched the description of Botryosphaeria dothidea (Crous et al., 2006; Slippers et al., 2014). To further confirm the causal fungus of leaf blight on I. polycarpa, the internal transcribed spacer (ITS)，β-tubulin (TUB2) and elongation factor 1-α (TEF1-α) genes were amplified using ITS1/ITS4, Bt2a/Bt2b and EF1-728F/EF1-986R primers, respectively (Jia et al. 2019). The sequences (GenBank accession nos. PQ252929 - PQ252931 for ITS; PQ349189 - PQ349191 for TUB2; PQ349192 - PQ349194 for TEF1-α) were aligned in GenBank showing >99% homology with the sequences of ITS (AY236949), TUB2 (AY236927) and TEF1-α (AY236898) from isolate CMW8000. The maximum-likelihood analysis was performed using concatenated sequences of ITS, TUB2 and TEF1-α. The phylogenetic analysis confirmed that all isolates clustered with B. dothidea. A pathogenicity test was performed on 20 healthy leaves of five 8-year-old I. polycarpa trees by a wound inoculation. Mycelial plugs from 5-day-old fungal colonies on PDA were inoculated to ten leaves, and ager plugs were inoculated to the same number of leaves as controls. Plastic bags were used to wrap the leaves with a wat cotton ball inside to maintain moisture conditions. After seven days, all inoculated leaves displayed lesions similar to those observed in the field, whereas controls remained asymptomatic. The pathogenicity tests were repeated three times. The fungus reisolated from the inoculated leaves were further identified as B. dothidea based on morphological characterization and molecular analyses. B. dothidea has also caused leaf blight on ornamental plants, including Mahonia fortunei (Huang et al., 2023), Parrotia subaequalis (Yang et al., 2024) and Syringa oblata (Zhi et al., 2024). This is the first report of B. dothidea causing leaf blight on I. polycarpa in China and provides a foundation for the management of the disease.

Stemphylium lycopersici is the causal pathogen of the devastating tomato gray leaf spot with a wide range of alternative plant hosts. To mitigate its potential endemic in facility-cultivated tomatoes, novel disease control strategies should be attempted to minimize the use of chemical fungicides. In this study, we identified a metalloprotease from S. lycopersici genome and designated it as SlMEP1, as it appears to be a typical zinc metalloproteinase containing a WLM (WSS1-like metalloprotease) domain and a characteristic HEXXH motif, which we determined by analyzing its transcriptional profile and enzymatic functions. The transcription level of SlMEP1 increased greatly during the fungal invasion of tomato leaves. The deletion of the SlMEP1 gene from S. lycopersici hindered its mycelial growth and reduced its pathogenicity. An assessment of the functional dissection indicated that SlMEP1 induced cell collapse and inhibited the expression of the host chitinases, which consequently made tomato cells more susceptible to S. lycopersici and other pathogenic fungi.

Hawthorn (Crataegus pinnatifida) is an important economic fruit and Chinese medicinal plant, which is widely distributed in the northern China. In early July 2024, a fruit rot disease was observed on the young fruits of hawthorn in a park of Shouguang, Shandong Province, China (36°53'42.16″N, 118°47'22.4″E). Forty plants were surveyed (from approximately 120 trees), which had 15 to 30% of fruits infected. Diseased fruits displayed brown, sunken, approximately circular lesions. Small sections (3-4 mm) cut from the edges between the diseased and healthy tissue from 20 diseased fruits were surface sterilized by using 75% ethanol for 30 s, and rinsed thrice with sterile distilled water. The diseased tissues were then dried and plated onto potato dextrose agar (PDA) and incubated at 25°C in the dark. Cultures were purified by hyphal tipping. In total, 12 colonies were isolated from the necrotic tissues, and all of them showed similar morphological characteristics. The colonies were initially white, gradually turning gray-green to dark gray after 5 days. Conidia were hyaline, aseptate, ellipsoidal to obovoid, 20.1-29.3 × 5.0-8.3 μm, with an average of 24.2 × 6.6 μm (n = 30). These morphological traits suggested that the pathogen shares similarities with the Botryosphaeria dothidea (Zhang, et al., 2021). For accurate identification, three representative isolates (SGSZ-01 to SGSZ-03) were selected for molecular identification by amplifying and sequencing the internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF1-α), and β-tubulin (TUB2) with the primers ITS1/ITS4, EF1-728F/986R, and Bt2a/Bt2b (Carbone and Kohn 1999; Jia et al. 2019), respectively. GenBank accession numbers were PQ197601, PQ586368, and PQ586369 for ITS, PQ201924, PQ588676, and PQ588677 for TEF1-α, and PQ201925, PQ588678, and PQ588679 for TUB2, and gene sequencing showed 99.2 to 100% identity with the ex-type strain of B. dothidea (CMW 8000). Results from the maximum likelihood phylogenetic analysis, based on the three concatenated gene sequences, placed our isolates in a clade with B. dothidea. The pathogenicity of SGSZ-01 was conducted using a wound inoculation method on 11-week-old fruits of 10-year-old hawthorn trees both on living plants and detached fruits. All fruits were wounded with a sterilized needle and inoculated by placing 5 mm² agar plugs of 5-day-old cultures grown on PDA. Control fruits were treated with agar plugs. All the attached fruits were enclosed in a plastic bag with a wet cotton ball for 5 days. The same number of detached inoculated fruits were placed in Petri dishes on a piece of wet filter paper and incubated at 25°C. The pathogenicity test was conducted three times independently. Necrotic lesions (4.5 ± 0.3 mm in diameter) resembling natural infections were observed on fruits 7 days after inoculation, and no symptoms appeared on the control fruits. The pathogen was reisolated from the lesions and was identified by the methods described above. No pathogens were isolated from control fruits. Therefore, the pathogen of hawthorn fruit rot in China is B. dothidea. B. dothidea has previously been reported causing fruit rot on mango, plum, pomegranate fruit, and apple in China (Feng et al. 2023; Gu et al. 2020; Tang et al. 2012; Yuan et al. 2024). To our knowledge, this is the first report of B. dothidea causing fruit rot disease on C. pinnatifida. The information on identification of this fungus may be helpful to the control and prevention of the disease.

Tomato chlorosis virus (ToCV), transmitted by the whitefly, Bemisia tabaci (Gennadius; Hemiptera: Aleyrodidae) has been continuously emerging on tomato plants and causing a significant economic loss throughout China. In the current study, RNA-Seq analysis was used to explore the gene expression profiles of B. tabaci Mediterranean (MED) that fed on both ToCV-infected and -uninfected tomato plants for 6, 12, 24, and 48 hours, respectively. The results revealed that dynamic changes occurred in the gene expressions of whiteflies at different time intervals after they acquired the virus. A total of 1709, 461, 4548, and 1748 differentially expressed genes (DEGs) were identified after a 6, 12, 24, and 48 hours feeding interval for the viral acquisition, respectively. The least number of expressed genes appeared in whiteflies with the 12 hours feeding treatment, and the largest numbers of those found in those with 24 hours feeding treatment. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that B. tabaci MED responded to ToCV acquisition through altering its nerve system development, fertility, detoxification, glucose metabolism, and immune function before it lost its ability to transmit the virus. The number of DEGs, degree of differential gene expressions, expression level of the same gene, involved biological processes, and metabolic functions in whiteflies post the 12 hours feeding, and viral acquisition were different from those from other three feeding treatments, which could be a significant finding suggesting an effective control of B. tabaci MED should be done less than 12 hours after whiteflies started feeding on ToCV-infected tomatoes. Our results further provided a clarified understanding in how B. tabaci was protected from viral acquisitions through comparison of the differential profile of gene expressions in whiteflies feeding on plants that were infected by semipersistent viruses.

Tomato (Solanum lycopersicum) gray leaf spot disease is a predominant foliar disease of tomato in China that is caused mainly by the necrotrophic fungal pathogen Stemphylium lycopersici. Little is known regarding the pathogenic mechanisms of this broad-host-range pathogen. In this study, a comparative transcriptomic analysis was performed and more genetic information on the pathogenicity determinants of S. lycopersici during the infection process in tomato were obtained. Through an RNA sequencing (RNA-seq) analysis, 1,642 and 1,875 genes upregulated during the early infection and necrotrophic phases, respectively, were identified and significantly enriched in 44 and 24 pathways, respectively. The induction of genes associated with pectin degradation, adhesion, and colonization was notable during the early infection phase, whereas during the necrotrophic phase, some structural molecule activity-related genes were prominently induced. Additionally, some genes involved in signal regulation or encoding hemicellulose- and cellulose-degrading enzymes and extracellular proteases were commonly upregulated during pathogenesis. Overall, we present some putative key genes and processes that may be crucial for S. lycopersici pathogenesis. The abilities to adhere and colonize a host surface, effectively damage host cell walls, regulate signal transduction to manage infection, and survive in a hostile plant environment are proposed as important factors for the pathogenesis of S. lycopersici in tomato. The functional characterization of these genes provides an invaluable resource for analyses of this important pathosystem between S. lycopersici and tomato, and it may facilitate the generation of control strategies against this devastating disease.