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.

Watermelon silver mottle virus (WSMoV), a potentially invasive virus, is known to reduce the yield and degrade the quality of infected crops in Cucurbitaceae and Solanaceae families, resulting in significant economic losses in limited areas of several Asian countries. WSMoV, previously detected on various crops in southern China, has now become more prevalent on watermelon and sweet pepper in the northern cities of China for the first time. A sequencing-based phylogenetic analysis has confirmed that the viral strains infecting cucumber, watermelon, and sweet pepper plants in Shandong Province are most closely related to those isolated from Guangdong, Guangxi, and Taiwan, suggesting a farther and continuous spread of WSMoV throughout China. To develop a fast, accurate, and practical protocol for WSMoV detection, we designed a set of primers from the conserved sequence of the WSMoV nucleocapsid protein (N) gene for a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). The RT-LAMP assay was performed successfully for 50 min at 61°C and exhibited a highly specific result without cross-reactions with other similar viruses and a sensitivity that is 100-fold higher than that of the traditional RT-PCR. The confirmation of 26 WSMoV suspect samples collected from various regions in Shandong through the RT-LAMP testing has demonstrated that the assay is suitable and practical for detection of WSMoV in both laboratory and field settings.

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.

Apple ring rot is one of the three major diseases of apple, which seriously affects the yield and quality of apple. At present, chemical agents are mainly used to prevent and control apple ring rot. In this study, 10 kinds of low virulence fungicides were selected. The inhibitory effects of 10 kinds of fungicides on Botryosphaeria dothidea were determined. The results indicated that 50% carbendazim and 70% thiophanate methyl had the best mycelial growth inhibition effect among 10 kinds of fungicides. The inhibitory effect of 50% carbendazim on hyphae was 91.63% under the concentration of 400 times dilution, the inhibitory effect of 70% thiophanate methyl on hyphae was 90.31% under the concentration of 500 times dilution, and there were recommended to use.

Euonymus fortunei (Turcz.) Hand.-Maz., an evergreen vine shrub, commonly used for landscaping and herbaceous medicines in China. In December 2024, leaf spot symptoms were noticed on E. fortunei plants in three parks with a 15-25% disease incidence out of 160 trees in Weifang City, Shandong Province in China (118°44'34.9''E, 36°52'17.3''N). Irregularly shaped lesions with a grayish-white center and brownish margins surrounded by a yellow halo developed on infected leaves and coalesced at later stages. To isolate the causal agent, 14 diseased leaves were randomly collected from 10 trees in different parks and a small piece (2 x 2 mm) was sliced from each lesion at the edge, surface-disinfected with 75% ethanol for 30 s, rinsed 3 times in sterilized water, placed on a potato dextrose agar (PDA), and incubated at 27°C in the dark for 3 days. Botryosphaeria sp.-like fungal colonies were isolated from 93% of the sampling plates and 3 representative fungal isolates (FFT01 to FFT03) were selected randomly for morphological and molecular characterization and hyphal-tip purified. Fungal colonies initially appeared to be white with fluffy mycelia and gradually developed a grayish-black pigment. The hyaline, unicellular, and oblong-fusiform conidia were 20.1 - 25.7 μm (mean = 23.2 ± 1.4) long and 6.3 - 9.1 μm (mean = 7.2 ± 0.8) in diameter (n = 30), similar to the morphological features of Botryosphaeria dothidea (Zhang et al. 2021). The internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (tef1) gene, and beta-tubulin (tub2) gene were amplified and sequenced using primer pairs ITS1/ITS4, EF1- 728F/EF1-986R, and Bt2a/Bt2b, respectively (White et al. 1990; Glass and Donaldson 1995; Carbone and Kohn 1999) and desposited in GenBank (Accession Nos. PV422744 to PV422746 for ITS, PV441492 to PV441494 for tef1, and PV4414485 to PV441487 for tub2). Sequences had > 99% identity with their corresponding sequences from the ex-type B. dothidea strain CMW 8000. In the maximum-likelihood phylogeny derived from the concatenated alignments, all three isolates were grouped in the B. dothidea clade. To validate the pathogenicity of FFT01 isolate, five leaves from each of three one-year-old E. fortunei seedlings were scratched with a sterilized syringe needle and then a 5 mm² mycelial PDA plug of 6-day-old culture was placed on each wounded site. Three E. fortunei seedlings inoculated with sterile PDA plugs served as controls. Each wound-inoculated seedling was enclosed in a transparent plastic bag for 3 days then kept in a growth chamber under controlled conditions (27°C, 14/10-h light/dark cycle, and 75% relative humidity). Seven days post-inoculation, grayish-white lesions surrounded by yellow halo were observed on leaves with black pycnidia developing in lesions, similar to the symptoms on the naturally infected leaves, while no lesions appeared on mock-inoculated leaves. The identity of the fungus re-isolated from symptomatic leaves was confirmed as B. dothidea based on morphology and DNA sequence. The pathogenicity test was repeated three times with similar results. Although B. dothidea has been reported as a common fungal pathogen causing leaf spot, fruit rot or stem canker on a variety of plants including plum, Kadsura coccinea, and soybean (Chen et al. 2021; Su et al. 2024; Yuan et al. 2024), this is the first report on its occurrence on E. fortunei in China. This study will provide a basis for the prevention and control of leaf spot on E. fortunei in the future.