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Postharvest fruit rot caused by pathogens is a serious problem in the pear industry. This study investigated the fungal diversity and main pathogens and identified a new pathogen in the stored 'Huangguan' pear (Pyrus bretschneideri Rehd.), the dominant pear variety in northern China. We sampled 20 refrigeration houses from five main producing regions in Hebei Province and used Illumina sequencing technology to detect the fungal composition. Alternaria (56.3%) was the most abundant fungus, followed by Penicillium (9.2%) and Monilinia (6.2%). We also isolated and identified nine strains of Alternaria and four strains of Penicillium. Moreover, we observed a new postharvest fruit disease in 'Huangguan' pear caused by Stemphylium eturmiunum, which was confirmed by phylogenetic analysis by combining the sequences of three conserved genes, including internal transcribed spacer, gapdh, and calmodulin. This study marks the first documentation of S. eturmiunum causing fruit rot in 'Huangguan' pears, offering valuable insights for identifying and controlling this newly identified postharvest disease.

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Pecan [Carya illinoinensis (Wangenh.) K. Koch] is an important nut tree species, which has been widely planted in Jiangsu and Anhui Provinces of China in recent years (Mo et al. 2018). In May 2022, a new leaf spot disease was observed on both young and old leaves of pecan trees in the Luhe area, Nanjing, Jiangsu Province. Approximately 30% of pecan trees suffered from the disease, which affected the growth of young trees and nut production to cause economic loss. Initially, the leaf spots were grayish black and round. Then, disease spots enlarged and joined together, forming irregular lesions with uneven edges. In the last stage, the leaflets were withered. To isolate the pathogen, three symptomatic leaves were collected from each of three different pecan trees. Leaf sections (3 to 4 mm) were excised from the margin of spots, surface sterilized in 75% alcohol for 30 s, then sterilized in 1.5% NaClO for 90 s. After rinsing three times with sterile distilled water, leaf sections were placed on potato dextrose agar (PDA) medium and incubated at 25 °C in a dark environment for 5 days. Pure cultures were obtained by monosporic isolation. A total of 20 isolates were obtained, and 12 isolates were identified as Stemphylium sp. with the same morphological features and ITS sequences. A representative isolate, named LH3-3, was selected for further study. Colonies on PDA were light yellow with dense mycelium and were brownish yellow on the reverse side. Conidia were 16.3 to 34.4 × 8.1 to 16.3 µm) (n=35), muriform, brown, with transverse and longitudinal septa, lightly deformed at the transverse septa. Ascomata were not observed. The morphological characteristics were consistent with the description of Stemphylium eturmiunum (Simmons 2001). The internal transcribed spacer region (ITS) and portions of genes for calmodulin (cmdA) and glyceraldehyde-3-phosphate dehydrogenase (gpd) were amplified and sequenced with the primers ITS1/ITS4 (White et al. 1990), CALDF1/CALDR2 (Xu et al. 2022) and GPD-F/R (Xie et al. 2019), respectively. Sequences were deposited in GenBank under accessions OP482492 (ITS), OP495734 (cmdA), and OP495735 (gpd). BLAST analysis showed that the sequences had 99.67-100% homology to ITS (525/525 bp) of S. eturmiunum strain ST14 (MH843733), cmdA (694/694 bp) of strain CBS122124 (KU850832), and gpd (299/300 bp) of isolate UMSe0030 (MK336876). MEGA 7.0 was used to construct a phylogenetic analysis based on concatenated sequences of ITS, cmdA, and gpd using the neighbor-joining method. The results showed that LH3-3 clustered on the branch of S. eturmiunum, and the support rate was 100%. A spore suspension in sterile water was made from strain LH3-3 grown on PDA, and adjusted to 1×106 spores/mL with a hemocytometer. To test pathogenicity, 20 µl drops of the spore suspension were placed on the left sides of four healthy detached leaflets of mature pecan trees and leaves of three 3-month-old seedlings. The right side of each leaflet was inoculated with 20 µl drops of sterile distilled water as the control. All inoculated seedlings and detached leaflets were covered with a transparent plastic bag and cultured in a greenhouse at 25 °C, 80% relative humidity, and a 12 h light cycle until symptom appeared. The experiment was repeated three times. After 7 days of inoculation, grayish black lesions appeared on all inoculation sites with the spore suspension but not in the controls. The leaf spot symptoms were similar to those observed in orchards. The same fungus, identified by morphological characteristics and sequencing of ITS, cmdA, and gpd, was re-isolated from the diseased spots of the inoculated leaflets to complete Koch's postulates. S. eturmiunum has been reported to infect garlic (Dumin et al. 2022) and tomato (Prencipee et al. 2021), but this is the first report of S. eturmiunum causing leaf spot of C. illinoinensis. This study provides a basis for further study on the biology, epidemiology, and management of the disease.

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Welsh onion (Allium fistulosum L. ) is native to Southwest China (Tomoo and Shinji 2012), with a cultivated area of about 550,000 ha and a production of 20 million tons in 2020, and widely cultivated throughout the world as a condiment. During April 2020, a Stemphylium-like disease occurred on a three-hectare field in Nanchang County (28°33'N, 115°57'E) was first observed and then monitored in Ningdu (26°23'N, 115°59'E) and Leping (28°50'N, 117°18'E), Jiangxi Province, China. The disease incidence was estimated to be nearly 30% in a survey of 300 plants. Disease symptoms were oval or irregular lesions at the center of outer leaves. The lesions were light brown at the edges and black-violet in the middle. Previous studies reported that the disease was mainly caused by S. vesicarium (Misawa et al. 2012). Besides, S. botryosum (Misawa et al. 2011) and S. solani (Dumin et al. 2021) can also cause this disease. To investigate the causal agent of the disease in Jiangxi, pieces of tissue from the margins of the symptomatic leaves were surface sterilized with 75% ethanol for 15 s, rinsed in sterile water three times, and placed on Potato Dextrose Agar (PDA) followed by incubation at 25°C in the dark for 5 days. Colonies were initially yellowish-brown and gradually turned into reddish-brown after 2 weeks. Conidia were solitary, oblong or ellipsoid, mid to deep brown (22-45×14-25 µm) with transverse and longitudinal septa (Fig. S1). Morphological characteristics were consistent with S. eturmiunum reported by Simmons (Simmons 2001). For molecular identification, genomic DNA was extracted from two representative isolates. ITS and gapdh genes of the isolates were amplified with primers ITS1/ITS4 (White et al. 1990) and gdp1/gdp2 (Berbee et al. 1999). BLASTn analysis showed that ITS sequences (OM846570, OM846571) of the two isolates both > 99% identity with S. eturmiunum strain CBS 109845 (MH862841) (Vu et al. 2018); and also had 100% identity for gapdh gene sequences (OM867337, OM867338) with S. eturmiunum strain CBS 109845 (KU850689) (Woudenberg et al. 2017). Phylogenetic tree based on ITS and gapdh gene sequences were constructed using a maximum-likelihood method with 1000 bootstraps indicated that the two isolates were clustered with S. eturmiunum (Fig. S2). Based on morphological characteristics and molecular analysis, the fungus was identified as S. eturmiunum. A pathogenicity test was performed on healthy welsh onion leaves to fulfill Koch's postulates. Twenty leaves were inoculated with a spore suspension of 1×104 conidia/mL followed by incubation at 25℃ (relative humidity > 90%) in the greenhouse. Negative controls were inoculated with sterile water. The experiment was repeated three times. Purple brown lesions were observed on all inoculated leaves at 7 days post-inoculation that similar to those observed in the field, and control remained symptomless. S. eturmiunum was re-isolated from the diseased leaves. It has reported that S. eturmiunum can infect tomato, garlic, sweet cherry and many other crops and cause different diseases. To the best of our knowledge, this is the first report of S. eturmiunum can cause Stemphylium leaf blight on welsh onion in China. Appropriate strategies should be developed to manage this disease.

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The Gtr1 protein was a member of the RagA subfamily of the Ras-like small GTPase superfamily and involved in phosphate acquisition, ribosome biogenesis and epigenetic control of gene expression in yeast. However, Gtr1 regulation sexual or asexual development in filamentous fungi is barely accepted. In the study, SeGtr1, identified from Stemphylium eturmiunum, could manipulate mycelial growth, nuclear distribution of mycelium and the morphology of conidia in Segtr1 silenced strains compared with its overexpression transformants, while the sexual activity of Segtr1 silenced strains were unchanged. SeASF1, a H3/H4 chaperone, participated in nucleosome assembly/disassembly, DNA replication and transcriptional regulation. Our experiments showed that deletion Seasf1 mutants produced the hyphal fusion and abnormal conidia. Notably, we characterized that Segtr1 was down-regulated in Se∆asf1 mutants and Seasf1 was also down-regulated in SiSegtr1 strains. We further confirmed that SeGtr1 interacted with SeASF1 or SeH4 in vivo and vitro, respectively. Thus, SeGtr1 can cooperate with SeASF1 to modulate asexual development in Stemphylium eturmiunum.

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Garlic leaf blight caused by Stemphylium eturmiunum was first reported in Jiangsu Province in China. The dicarboximide fungicide (DCF) procymidone is reported to possess broad-spectrum action in inhibiting filamentous fungi and is widely used to control leaf disease of various plants. Of 41 Stemphylium eturmiunum isolates collected in this study from commercial garlic farms in Pizhou and Dafeng counties of Jiangsu Province, eight isolates were resistant to procymidone. The following three phenotypes were categorized according to in vitro responses to DCFs: sensitive, low resistance to iprodione and procymidone, and high resistance to all iprodione and procymidone. The fitness of all resistant isolates was decreased in accordance with data on mycelial growth, conidiation, and virulence. After treatment with 10 µg/ml of procymidone for 4 h, mycelial intracellular glycerol concentrations of resistant isolates were significantly lower than those of sensitive isolates. Positive cross-resistance was observed between dicarboximides and phenylpyrroles, but there was no cross-resistance between dicarboximides and fluazinam or difenoconazole in the two resistant phenotypes. Nucleotide sequence alignment of two-component histidine kinase genes from sensitive and resistant isolates indicated that amino acid mutations were located at the histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein and at the phosphatase domain of the N-terminal region and the response regulator domain of the C-terminal region. To our knowledge, this is the first report of DCF resistance in Stemphylium eturmiunum, and these findings will help establish a rational strategy to manage DCF-resistant populations of Stemphylium eturmiunum in the field.

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The formation of sexual fruiting bodies for plant pathogenic fungi is a key strategy to propagate their progenies upon environmental stresses. Stemphylium eturmiunum is an opportunistic plant pathogen fungus causing blight in onion. This self-fertilizing filamentous ascomycete persists in the soil by forming pseudothecia, the sexual fruiting body which helps the fungus survive in harsh environments. However, the regulatory mechanism of pseudothecial formation remains unknown. To uncover the mechanism for pseudothecial formation so as to find a practical measure to control the propagation of this onion pathogen, we tentatively used DNA methyltransferase inhibitor 5-azacytidine (5-AC) to treat S. eturmiunum. 5-AC treatment silenced the gene-encoding monoacylglycerol lipase (magl) concomitant with the presence of the inheritable fluffy phenotype and defectiveness in pseudothecial development. Moreover, the silence of magl also resulted in a reduction of arachidonic acid (AA) formation from 27 ± 3.1 µg/g to 9.5 ± 1.5 µg/g. To correlate the biosynthesis of AA and pseudothecial formation, we created magl knockdown and overexpression strains. Knockdown of magl reduced AA to 11 ± 2.4 µg/g, which subsequently disabled pseudothecial formation. In parallel, overexpression of magl increased AA to 37 ± 3.4 µg/g, which also impaired pseudothecial formation. Furthermore, exogenous addition of AA to the culture of magl-silenced or magl knockdown strains rescued the pseudothecial formation but failed in the gpr1 knockdown strain of S. eturmiunum, which implicates the involvement of AA in signal transduction via a putative G protein-coupled receptor 1. Thus, AA at a cellular level of 27 ± 3.1 µg/g is essential for sexual development of S. eturmiunum. Disturbance in the biosynthesis of AA by up- and down-regulating the expression of magl disables the pseudothecial development. The specific requirement for AA in pseudothecial development by S. eturmiunum provides a hint to curb this onion pathogen: to impede pseudothecial formation by application of AA.

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