First Report of Cercospora chevalieri Causing A Leaf Spot Disease on Amorphophallus konjac in China.

Amorphophallus konjac, commonly called voodoo lily, is a cash crop widely cultivated in southwest China (Gao et al. 2022). In August 2022, leaf spot symptoms were observed in a field (1 ha) located at Fuyuan (25.67°N; 104.25°E), Yunnan, China, resulting in substantial economic losses. Brown lesions, with an incidence ranging from 20 to 40%, typically had a whitish or gray center and were surrounded by yellow halos. Microscopic observations of the spots revealed anamorphic species Cercospora chevalieri. Conidiophores were 50-150 × 4-7 µm, cylindrical, unbranched, smooth-walled, pale brown and aggregated in dense fascicles arising from a brown stroma. The conidiogenous cells were integrated, terminal or intercalary, pale brown to brown and proliferated sympodially. The conidiogenous loci were thickened and darkened, and 2-3 µm in diam. The conidia were formed singly, obclavate-cylindrical, 90-160 × 5-7 µm, with an average of 130 × 6 µm (n = 30), 6-11 septa, thin-walled, smooth, hyaline or subhyaline, straight or curved with an obtuse apex and obconically truncate base, with thickened and darkened hilum. These morphological characteristics matched those of C. chevalieri, the causal agent of leaf spot on A. paeoniifolius (Braun et al. 2014; Saccardo et al. 1913). A conidial suspension in sterile water from lesions was used to inoculate water agar, and germinated conidia were transferred to potato dextrose agar（PDA) and incubated at 27°C for 7 days. Induction of sporulation was unsuccessful using PDA, as well as malt extract agar, potato sucrose agar and synthetic nutrient-poor agar. Two out of ten isolates were selected for molecular identification and pathogenicity assay. Genomic DNA from two pure isolates (KUNCC22-12536 and KUNCC22-12537) was extracted for PCR and amplified with primers for the internal transcribed spacers (ITS: ITS1/ITS4), calmodulin (CMD: CAL228F/CAL2Rd), translation elongation factor 1-alpha (TEF1-α: 728F/986R), actin (ACT: 512F/783R), histone H3 (HIS3: CYLH3F/CYLH3R), beta-tubulin gene (TUB2: BT-1F/BT-1R) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH: Gpd1/Gpd2), respectively (Vaghefi et al. 2021). The newly generated sequences for ITS (OP719153/OP719154), CMD(OP740904/OP740905), TEF1-α (OP740910/OP740911), ACT (OP740902/OP740903), HIS3 (OP740908/OP740909), TUB2 (OP740912/OP740913), GAPDH (OP740906/OP740907) of C. chevalieri were submitted to GenBank. So far, no sequence data of C. chevalieri were available in the GenBank database. As expected, most genes (TEF1-α, ACT, CMD, HIS, TUB2 and GAPDH) showed 91 to 95% identity to their best hits within species of the genus Cercospora. The phylogenetic tree showed that sequences retrieved from two isolates obtained from the A. konjac leaf spots clustered together within Cercospora forming a strongly supported clade. To test Koch's postulates, ten four-month-old healthy A. konjac plants grown in pots were used for a pathogenicity test in a greenhouse. One leaf of each plant was inoculated with mycelial plugs, and one leaf was inoculated with a sterile PDA plug. These plants were enclosed in plastic bags for 72 h. Only leaves inoculated with mycelium plugs produced brown lesions, which appeared after 10 to 14 days on inoculated leaves. Control plants treated with sterile PDA plugs remained asymptomatic. This experiment was repeated twice with the same results. C. chevalieri was reisolated from infected leaves and identified based on morphology and Sanger sequencing of the ITS region. To our knowledge, this is the first report of C. chevalieri causing leaf spot on A. konjac and the first report of this species from China (Braun et al. 2014), which provides key information for diagnosis and management of this disease.

Rapid Identification of Phytotoxins Produced by Glomerella cingulata Using High-Resolution Mass Spectrometry-Based Qualification, Targeted Structural Confirmation and Their Characteristics Investigation.

Glomerella cingulata is a pathogenic fungus that can cause apple Glomerella leaf spot (GLS), a new and destructive apple disease in China. Phytotoxins are important factors closely related to the disease process, but there is still no report on the phytotoxins of G. cingulata. The aim of this study was to rapidly identify the phytotoxins of this pathogen using a strategy of HRMS-based preliminary qualification, followed by targeted structure confirmation and also investigation of phytotoxicity characteristics. First, the crude toxin sample was directly analyzed by the UPLC-HRMS and GC-MS, and the data were processed to screen for possible phytotoxic compounds using MS library and the phytotoxicity-related literature. The reference standards of credible phytotoxic compounds were then subjected to targeted structure validation (signal comparison between standards and compounds in crude toxin via HPLC-DAD, UPLC-MS/MS, and GC-MS), and also the phytotoxicity assay. The results confirmed six phytotoxins produced by G. cingulata, namely 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), 2,5-bis(hydroxymethyl)furan (BHMF), 2-furoic acid (FA), 2,3-butanediol, trans-aconitic acid (TAA), and cis-aconitic acid (CAA). Of these, HMFCA and TAA exhibited greater phytotoxicity. Main characteristics: All of them were non-host-selective toxins, and toxins were synergistically phytotoxic to the host when mixed. BHMF, HMFCA, FA, TAA, and CAA could be commonly produced by all tested strains, and their phytotoxicity can be significantly inhibited or even eliminated at high temperatures or high pH. The elucidation of the phytotoxins of G. cingulata in this work could provide information on the pathogenesis and control of apple GLS.

Septoria cannabicola, a new species on Cannabis sativa from Japan.

Septoria leaf spot on hemp has re-emerged with increasing hemp cultivation worldwide. In Japan, Septoria cannabis , initially recorded as the causal pathogen in Japan, was studied with morphology based on the current criteria and detailed molecular phylogenetic analyses using seven gene loci. The robust phylogenetic data and morphology of examined specimens unveiled the existence of a new species of the genus Septoria causing leaf spot disease on Cannabis sativa .

First Report of Neofusicoccum yunnanense Causing Leaf Spot on Ivy in Yunnan Province, China.

Ivy (Hedera nepalensis var. sinensis (Tobl.) Rehd) is an evergreen root-climbing vine, widely cultivated in eastern Asia because of its ornamental, environmental, and medicinal value (Wu et al. 2019). In October 2023, the leaf spot symptom of Ivy was observed in the Kunming Botanical Garden in Yunnan Province, China (25.14°N, 102.75°E), and the disease incidence was up to 38% (76 of 200 leaves). Initially, dark-brown or black small spots appeared on the leaves. As the lesions progressed, their center emerged tawny, and the black halos were expanded around the lesions. In severe cases, small spots could link together to form leaf blight even resulting in blade death. In order to obtain pure isolates, 10 diseased leaves were collected and cut into 1 mm × 1 mm pieces, surface disinfected with 75 % ethanol for 30 s, followed by 3% NaClO for 3 min, and finally washed three times with sterilized water. The pieces were placed on the potato dextrose agar (PDA) media, which was incubated at 25°C for 3 days. Individual hyphal tips from the developing fungal colonies were placed on PDA and incubated for 5 to 10 days. Six strains (6 out of 10) were obtained with same colony morphology. Conidia were hyaline, unicellular, nonseptate, ellipsoidal or fusiform, thin walled, externally smooth and ranged from 15.0 to 22.0 (avg. 18.4) µm × 5.0 to 8.0 (avg. 7.2) µm (n=30). Morphological comparison proposed that the present fungi belonged to Neofusicoccum (Zhang et al. 2021). Two isolates (GUCC23-0141 and GUCC23-0142) were selected for multi-gene phylogenetic analyses. The PCR reaction of the internal transcribed spacer region (ITS), translation elongation factor 1-α (EF1-α), and ß-tubulin genes were run using primers ITS1/ ITS4 (White et al. 1990), EF1-728F/ EF1-986R (Carbone and Kohn 1999), and Bt2a/ Bt2b (Glass and Donaldson 1995). The accession numbers of DNA sequences of GUCC23-0141 and GUCC23-0142 are ITS: PP728109 and PP728110; TUB2: PP744490 and PP744491; and TEF1-α: PP744488 and PP744489. BLAST analysis showed 100% identity for ITS and TUB2, and 98.97% for TEF1-α with the Neofusicoccum yunnanense (CSF6142). Phylogenetic analyses also supported that our isolates kept a close relationship to N. yunnanense. Three one-year plants were used for pathogenicity test, two of which were inoculated with PDA plugs containing N. yunnanense and one of which was inoculated with blank PDA plugs and used as control. For each plant, three leaves were selected to conduct the test, whose surfaces were sterilized with 75% alcohol. All the leaves were covered with cotton moistened with sterilized water on top. All plants were placed in a greenhouse with 25℃ and 75% humidity. Few small black spots were observed at the inoculation site after 3 days, which were enlarged gradually after 7 days. However, control plants remained healthy. N. yunnanense was reisolated from the diseased tissues and identified based on morphological and molecular characteristics. On basis of pathogen identification and Koch's test, we proposed the leaf spot of Ivy caused by N. yunnanense. This was the first report about N. yunnanense causing the disease of Ivy. This result provides a theoretical basis for further research into the control of the disease. As an important ornamental plant, we should pay attention to the management of this disease.

First Report of Didymella uniseptata Causing Leaf Spot on Taraxacum mongolicum in China.

Dandelion (Taraxacum mongolicum), belonging to the Asteraceae family, is one of the main associated species in the alpine meadow, and is famous for its both feeding and medicinal values (Lin et al. 2022; Wang et al. 2022). In September 2021 (vigorous growth period), a newly emerging leaf spot disease of T. mongolicum were observed on natural grassland in Ruoergai County, Aba (Ngawa) Tibetan and Qiang Autonomous Prefect, China (33°59'51'' N, 102°44'57'' E, alt. 3414 m). Leaf disease incidence was ranged from 10% to 15%. The symptoms appeared as brown to dark brown, circular or irregular, sunken spots; eventually, the infected sites of leaves formed a hole in the middle position of lesions. For isolation, 21 tissue pieces (5mm × 5mm) from 7 symptomatic leaf samples of 4 different plants were surface sterilized with 70% ethanol for 30 s and rinsed three times with sterilized distilled water. Then, these tissues were placed on potato dextrose agar (PDA) at 25°C and incubated in the dark for 2 to 7 days. Finally, six pure Didymella strains with consistent colony characteristics were obtained from hyphal tips as described by Xue et al. (2023). Colonies on PDA were brown to black with concentric circles, and abundant black pycnidia were visible; reverse similar in color. Conidia were ellipsoidal, ovoid, hyaline, 0 to 1-septate, 4.07 to 8.67 × 2.74 to 5.35 µm (average 3.60 × 6.55 µm; n = 50). Seven-week-old healthy plants were obtained by growing T. mongolicum seeds in pots (two plants per pot). The six pure Didymella strains were subsequently used to inoculate healthy plants as follows: for each strain, eight pots were spray inoculated with a mycelial suspension of about 4 × 104 CFU/ml, referring to our previous method (Xue et al. 2023). In addition, eight pots considered as non-inoculated controls were sprayed with sterilized distilled water. All pots were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse at 23 to 29°C. After incubation for 10 days, the typical symptoms consisted of brown to dark brown, sunken spots, which were similar to those previously observed in nature grassland; however, symptoms were not observed on the non-inoculated plants (controls). The same fungus was reisolated from the lesions and confirmed by the morphological and molecular methods described in this note, thus fulfilling Koch's postulates. To further identify this fungal pathogen, ITS-rDNA, and two other protein-coding genes (rpb2 and tub2) of the representative strain REG28 were amplified with primers described by Chen et al. (2022). Sequences were deposited in GenBank (PP385777 for ITS, PP781948 for rpb2, and PP781947 for tub2). A maximum likelihood (RAxML) phylogenetic tree based on the combined ITS, rpb2, and tub2 alignments showed REG28, and ex-type CGMCC 3.20069 of D. uniseptata (Chen et al. 2022) formed a subclade with 100% bootstrap support (Fig. S1). The causal agent of this disease was confirmed as D. uniseptata by the morphological, molecular, and pathogenic features described above. Recently, D. macrophylla has been reported as the first record on T. officinale in Russia (Gomzhina et al. 2020). To our knowledge, this is the first report of D. uniseptata causing leaf spots on T. mongolicum worldwide. This information will be useful for the diagnosis, detection, pathogen identification and future control of this disease on T. mongolicum in natural grassland.

Occurrence of Maize Leaf Spot Disease Caused by Leptosphaerulina australis in Yunnan, China.

Maize (Zea mays) is vital as a staple food and livestock feed crop. Yunnan is one of the main maize-producing provinces in China (National Bureau of Statistics, 2022). While corn production in Yunnan is lower than the national average, the development of drought-tolerant varieties has contributed to improving productivity. In August 2021, a new leaf spot disease on maize was observed in Lancang, Yunnan (22°26'38.11"N to 22°48'38.68"N, 99°48'15.13"E to 99°59'20.03"E), causing serious damages to maize production with incidence up to 76.19 %. Initially, small light yellow lesions were seen scattered on diseased maize leaves, round or polygon, measuring 0.3 to 2.0 cm in diameter. In the intermediate phase, these lesions sank, ruptured, and turned white with dark brown borders. In severe cases, they merged into large irregular patches, reaching up to 10 cm, leading to complete leaf necrosis. Small black ascomata were seen on the lesions. Tissue sections reveal perithecium embedded in leaves, measuring 94~145 µm in diameter. Symptomatic tissues were sterilized in 1.5% NaClO for 60s, and washed twice withsterile purified water, then plated on potato dextrose agar (PDA) at 25℃, 90% relative humidity (RH), and a 12-hour light cycle. 6 isolates were obtained from 2 diseased maize cultivars. In 20 days, the colony reached the edge of the PDA plate, the center darkening from white, featuring white aerial mycelium on top and black on the reverse side. Brown ascomata, solitary or clustered, measured 80.1~176.7 × 55.57~138.9 µm. The ellipsoid to oblong ascospores were 17.9~39.7 × 10.9~14.1 µm, and the bitunicate, thick-walled asci were 90.1~133.3 × 26.6~33.5 µm. The genomic DNA was extracted using the Chelex-100 method (Möller et al. 1992). For molecular identification, the ITS, LSU, and ß-tubulin (Tub2) genes were amplified using primer pairs ITS1/ITS4 (White et al. 1990), LR0R/LR5 (Vilgalys et al. 1990) and Btub2Fd/Btub4Rd (Woudenberg et al. 2009), respectively. Sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd. The sequenced loci (GenBank accession nos.: LSU, OL687348-53; ITS, OL617009-10, and OL664058-61; Tub2, OL741678-83) of the isolates exhibited 100%/ 99%/ 100% similarities with L. australis genes: LSU, MH868885; ITS, KF381084; Tub2, GU237541, respectively. Using MEGA 11.0, phylogenetic trees were constructed using the maximum-likelihood algorithm on concatenated sequences of LSU, ITS, and Tub2 for isolates LCMB1 to 6. The isolates clustered with two L. australis strains with 100 % bootstrap support (1,000 replicates). The results were consistent with the Bayesian Inference tree. The pathogenicity test used strain LCMB4 on six healthy maize plants during the heading period under natural conditions. Three leaves pre-plant were wounded with sterile sandpaper and sprayed with conidial suspension (106 spores ml-1, diluted in sterilized water) in the greenhouse at 28℃, 90% RH, and a 12-hour light cycle, with sterilized distilled water used for control. Inoculated leaves developed symptoms consistent with the described after 10 days, while control leaves remained symptomless. The same pathogen was re-isolated from the infected leaves, fulfilling Koch's postulates. Previously, L. australis has been isolated from turfgrass (Mitkowski et al. 2004), Alfalfa (Zhang et al. 2021), soil (Li et al. 2018), and Paris polyphylla var. chinensis (Fu et al. 2019), but not from maize. This is the first report of L. australis causing leaf spot on maize globally.

Genomic characterization of bZIP gene family and patterns of gene regulation on Cercospora beticola Sacc resistance in sugar beet (Beta vulgaris L.).

Sugar beet (Beta vulgaris L.) is one of the most important sugar crops, accounting for nearly 30% of the world's annual sugar production. And it is mainly distributed in the northwestern, northern, and northeastern regions of China. However, Cercospora leaf spot (CLS) is the most serious and destructive foliar disease during the cultivation of sugar beet. In plants, the bZIP gene family is one of important family of transcription factors that regulate many biological processes, including cell and tissue differentiation, pathogen defense, light response, and abiotic stress signaling. Although the bZIP gene family has been mentioned in previous studies as playing a crucial role in plant defense against diseases, there has been no comprehensive study or functional analysis of the bZIP gene family in sugar beet with respect to biotic stresses. In this study, we performed a genome-wide analysis of bZIP family genes (BvbZIPs) in sugar beet to investigate their phylogenetic relationships, gene structure and chromosomal localization. At the same time, we observed the stomatal and cell ultrastructure of sugar beet leaf surface during the period of infestation by Cercospora beticola Sacc (C. beticola). And identified the genes with significant differential expression in the bZIP gene family of sugar beet by qRT-PCR. Finally we determined the concentrations of SA and JA and verified the associated genes by qRT-PCR. The results showed that 48 genes were identified and gene expression analysis indicated that 6 BvbZIPs were significantly differential expressed in C. beticola infection. It is speculated that these BvbZIPs are candidate genes for regulating the response of sugar beet to CLS infection. Meanwhile, the observation stomata of sugar beet leaves infected with C. beticola revealed that there were also differences in the surface stomata of the leaves at different periods of infection. In addition, we further confirmed that the protein encoded by the SA signaling pathway-related gene BVRB_9g222570 in high-resistant varieties was PR1, which is closely related to systemic acquired resistance. One of the protein interaction modes of JA signal transduction pathway is the response of MYC2 transcription factor caused by JAZ protein degradation, and there is a molecular interaction between JA signal transduction pathway and auxin. Despite previous reports on abiotic stresses in sugar beet, this study provides very useful information for further research on the role of the sugar beet bZIP gene family in sugar beet through experiments. The above research findings can promote the development of sugar beet disease resistance breeding.

First report of Diaporthe tulliensis causing leaf spot on Millettia speciosa in China.

Millettia speciosa Champ, renowned for its diverse applications in traditional medicine, is extensively cultivated in the Guangxi region of China, spanning roughly 5,973 hectares. In July 2021, a plantation in Yulin, Guangxi, China (22°64'N; 110°29'E), exhibited severe leaf spot disease on M. speciosa. Notably, a 46,690 square meters area had over 40% leaf spot incidence. Initially, symptoms appeared as small, circular, pale-yellow lesions on the leaves, then turned into irregular, dark brown spots with yellow halos, leading to the wilt and defoliation of leaves. To identify the responsible pathogen, a total of five symptomatic leaves were collected and sterilized systematically. Small tissue segments (5×5 mm) from lesion peripheries were aseptically excised, then surface sterilized with 75% ethanol for 10 s, and 1% sodium hypochlorite (NaClO) for 3 min. Following this, the sterilized tissues were triple-rinsed with sterile water and cultured on potato dextrose agar (PDA) at 28 °C in the dark for 7 d. A total of seven isolates were obtained through single-spore isolation, and one representative isolate, N2-3, was selected for further analysis. After 7 d of incubation, colonies displayed flat, white, and extensively branched aerial hyphae. Over time, the reverse side of the colony changed from white to yellowish-white. The pycnidia were black with conidial droplets ranging from cream to pale yellow exuding from their ostioles. The α-conidia were one-celled, hyaline, ovoid to cylindrical, typically with one or two droplets, 2.6 to 5.9 ×1.4 to 3.9 µm (n=50). These morphological traits align with those of the genus Diaporthe, as reported by Li et al. (2022) and Crous et al. (2015). To identify the species, isolate N2-3 underwent sequencing of the internal transcribed spacer (ITS), ß-tubulin (BT), and translation elongation factor 1 alpha (EF1-α) sections (Huang et al. 2021). Obtained sequences of ITS, BT and EF1-α (Genebank accessions nos. OR600532, OR662169 and OR662168) displayed a 99% similarity to Diaporthe tulliensis (Genebank accessions nos. OP219651, ON932382, OL412437, respectively). Based on the concatenated ITS, BT and EF1-α, a neighbor-joining phylogenetic analyses using MEGA7.0 clustered with D. tulliensis. Therefore, the fungus was identified as D. tulliensis (teleomorph name) based on morphological and molecular features. A pathogenicity test was conducted on 1-year-old M. speciosa seedlings by gently abrading healthy leaves with sterilized toothpicks to create superficial wounds. Wounded leaves were then inoculated with 5 mm diameter mycelial plugs, while control seedlings received PDA plugs. Three leaves per plant and five plants per treatment were selected for assessment. All seedlings were kept in a controlled greenhouse (12/12h light/dark, 25 ± 2 °C, 90% humidity). After 7 d, the inoculated leaves showed symptoms like those in the field, while control plants remained healthy. The fungus was consistently reisolated from the infected leaves, satisfying Koch's postulates. Notably, D. tulliensis has caused Boston ivy leaf spot, bodhi tree leaf spot, cacao pod rot, and jasmine stem canker (Huang et al. 2021; Li et al. 2022; Serrato-Diaz et al. 2022; Hsu et al. 2023). This discovery is significant as it marks the first report of Diaporthe tulliensis causing leaf spot on Millettia speciossa in China, which has direct implications for the development of diagnostic tools and research into potential disease management strategies.

First Report of Fusarium kyushuense Causing Leaf Spot on Sweet Persimmon in China.

Sweet persimmon (Diospyros kaki L.) is a fruit of significant nutritional and commercial value in Asia. In summer 2023, leaf spots were observed affecting 20 to 30% of sweet persimmon trees in a commercial orchard located in Gongcheng City, Guangxi, China. Initially, the infected leaves exhibited sparse light brown spots on their upper surface, which subsequently evolved into brown circular to irregular lesions encircled by a yellow halo. Eventually, these lesions became densely distributed across the leaves leading to insufficient nutrient accumulation in the fruit. To isolate the pathogen, diseased leaves were cut into small pieces (5×5 mm), disinfected with 75% ethanol for 15 seconds, followed by 1% NaClO for 1minute, rinsed three times with sterile water, and then transferred onto potato dextrose agar (PDA) plates. The plates were then incubated in darkness for 3 days at 25°C. Pure cultures were obtained using the hyphal-tip method and single-spore isolation. On PDA, the colonies initially appeared fluffy and white after 24 hours, turning yellowish or red after 3 days. Macroconidia (average length of 26.1 µm in length × 4.3 µm in width, n = 50) exhibited dorsiventral curvature and were hyaline, with 3 to 5 septa. Microconidia (average length of 9.45 µm in length × 3.4 µm in width, n = 50) were hyaline, aseptate, and oval. Two representative isolates, Gxfky1 and Gxfky2, were selected for further molecular analyses. Their internal transcribed spacer (ITS) region rDNA gene were amplified via PCR and sanger sequenced (GenBank Accession Nos. PP506475, PP506593) using the primer pair ITS1/ITS4 (White et al. 1990), showing more than 99% sequence identity with Fusarium kyushuense type-material strain NRRL3509 (NR_152943) according to BLASTn analysis in NCBI. To further confirm the identity of the isolates, four gene sequences were amplified: RPB1 (PP532864, PP532865), RPB2 (PP532866, PP532867), TEF1 (PP580505, PP580506), and TUB2 (PP532862, PP532863), using the F5/G2R, 5f2/11ar, EF1/EF2, and T1/T2 primer sets, respectively (O'Donnell et al., 1997; O'Donnell et al., 2010). A multi-locus maximum likelihood phylogenetic analysis revealed that Gxfky1 and Gxfky2 clustered with strains F. kyushuense with 100% bootstrap support. Pathogenicity tests using Gxfky1 and Gxfky2 were conducted on leaves of two-year-old sweet persimmon plants using non-wound inoculation. Specifically, 5-mm mycelial plugs and sterile agar plugs were placed on six leaves and secured with cling film, with six plugs each for the inoculation treatment and negative control, respectively. They were then incubated in a greenhouse at room temperature (25 ± 2°C) with a relative humidity of 70 to 80%. After 5 days, the same symptoms on naturally infected plants were observed on leaves inoculated with mycelium, while no symptoms were observed on the controls. The same fungus were reisolated from the inoculated leaves and identified based on morphology and the TEF1 gene sequence, thus fulfilling Koch's postulates. Fusarium kyushuense has previously been reported to cause diseases in various plant species, including maize (Cao et al., 2021), rice (Wang et al., 2024), and tobacco (Wang et al., 2013). To our knowledge, this is the first report of F. kyushuense causing leaf spot on sweet persimmon in China, which expands the known host range of this pathogen.

First Report of Grovesinia moricola Causing Zonate Leaf Spot on Hernandia nymphaeifolia in Taiwan.

Hernandia nymphaeifolia (C. Presl) Kubitzki, a native tree of Taiwan, is a sea drift plant (Yang and Lu 1996). It is a salt- and wind-tolerant tree (Bezona et al. 2009) and was selected for the afforestation of badlands in coastal areas of Taiwan. In December 2022, all H. nymphaeifolia seedlings at a nursery in Wu-Lai, Taiwan were diseased and wilted with a similar progression. The initial symptom was small zonate white or gray lesions with water-soaked periphery on leaves. Then, expansion and fusion of leaf spots which caused leaf blight and defoliation were observed. Seedlings eventually wilted. Sporophores found on the host were generally hypophyllous, solitary, erect, and easily detachable. The upper portion of the sporophore was considered an individual conidium and consisted of a pyramidal head that was fusiform to ventricose, 206.3 to 501.8 µm (average: 378.0 ± 75.3 µm) long, and 63.6 to 104.5 µm (average: 85.0 ± 16.2 µm) wide at the broadest point (n=30). Branches within the pyramidal head were short, compact, and di- or trichotomously branched. The central stipe was hyaline, broad, septate, tapering toward an acute apex, and sometimes constricted at the basal septum. Sclerotia were gray or black, spherical, and 1.0 to 2.5 mm (n=10) in diameter and observed on older lesions. The fungus was isolated from infected tissue and sporophores and maintained on potato dextrose agar (PDA) at 20°C in darkness. Sclerotia were produced on PDA after 4 to 5 weeks and were irregular or spherical, but no sporophore was developed. The fungus was identified as Grovesinia moricola (I. Hino) Redhead based on morphological characteristics (Tomoko et al. 2006). Three DNA samples was obtained from the cultures isolated from the diseased leaf, sporophores and sclerotia. They were then amplified by PCR with primers for the internal transcribed spacer region (ITS; primers ITS5/ITS4) and the large subunit nuclear ribosomal RNA gene (LSU; primers LR0R/LR5) (Cho et al. 2017), and then sequenced respectively. The sequences were deposited into GenBank with accession nos. PP727191 to PP727193 and PP748518 to PP748520. BLAST analysis of the three isolates showed 100% identity to the sequences of G. moricola from Taiwan (OP550202, OP550203) for the ITS region and 99.9% identity to the sequence of G. moricola from the USA (MW013804) for the LSU rRNA gene. The specimens (FS2022-140) and the culture (Asco-0109) in this study were deposited into the herbarium of Taiwan Forestry Research Institute in Taiwan. Koch's postulates were performed by inoculating four 8-month-old, asymptomatic, potted H. nymphaeifolia plants; every plant was inoculated with sporophores from infected leaves on the upper surface of each of five leaves. Four uninoculated plants were kept in separate pots and served as controls. All plants were covered with transparent plastic bags individually and incubated in a growth chamber at 18 to 20°C with 8 h of light. Similar leaf spots and sporophores were observed after 2 to 4 days and 10 days on every inoculated plant but not on uninoculated plants. The pathogen with a similar colony on PDA was reisolated from the leaf spots of the inoculated plants. Molecular identification of the reisolated pathogen by the above method was carried out. The sequences showed 99.9% identity to the sequence of G. moricola, and were deposited into GenBank with accession nos. PQ157896 to PQ157897 (ITS region) and PQ157701 to PQ157702 (LSU rRNA gene). The pathogenicity test was repeated once. G. moricola is known to cause severe defoliation on woody and annual plants, including at least 73 host species and 36 families distributed in the eastern United States and Japan (Trolinger et al. 1978). This is the first report of G. moricola on H. nymphaeifolia in the world. Control of the disease would play an important role in maintaining healthy seedlings for the afforestation in Taiwan.

Discovery of Two Novel Phthalide Phytotoxins from the Wheat Tan Spot Fungal Pathogen Pyrenophora tritici-repentis.

The Dothideomycete fungal pathogen Pyrenophora tritici-repentis (Ptr) is the causal agent of the tan spot disease of wheat. The proteinaceous necrotrophic effectors ToxA and ToxB are well characterized. A nonproteinaceous effector called ToxC has also been partially characterized. Ptr produces a number of other small molecular weight compounds, but these remain poorly characterized. In this study, two novel compounds, designated ToxE1 and ToxE2, capable of inducing chlorotic symptoms on wheat leaves in a cultivar-specific manner, were purified from Ptr liquid cultures. There is no evidence that these compounds correspond to ToxC. Most isolates produced ToxE1, ToxE2, or both, and both compounds were detected in infected wheat leaves. The structures of both analogues were elucidated by NMR spectroscopy and comprise a phthalide core structure with an amide moiety. We postulate that these compounds have a general phytotoxic effect and may have an ancillary role in disease development.

First Report of Leaf Spot Caused by Alternaria alternata on Chard (Beta vulgaris var. cicla L.) in China.

Chard (Beta vulgaris var. cicla L.) is popular vegetable in China. In June 2023, a leaf spot disease was observed on Chard plants in Hunan Province (27°46'10.99″N, 112°05'52.80″E), China. The disease incidence was 30% in a surveyed of about 500 plants. Symptoms began as many light brown round- to polygon-shaped spots on chard leaves, then developed and enlarged into grayish-white lesions, with the edge of the spots brown to dark brown. A total of 10 symptomatic samples were randomly collected. To identify the pathogen, symptomatic tissues (0.5 × 0.5 cm) from the lesion margin surface were sterilized with 75% ethanol for 30 s and 2% NaClO for 1 min, rinsed 3 times with sterile water, air dried. The sterile pieces were placed on potato dextrose agar (PDA) and incubated at 25°C. A total of nine isolates were obtained. Fungal colonies cultured on potato carrot agar (PCA) were almost the same as each other, and two representative isolates (TC0, TC10) were used for further identification. On PCA, the fungal hyphae were initially white and finally gray-brown with flocculent aerial mycelia. Conidia were solitary or in chains, with various shapes, mostly subglobose, the size was 13.2 to 28.0 µm long and 5.8 to 13.0 µm wide (n = 30). The cultural and morphological characteristics of isolates were similar to those of Alternaria sp (Simmons et al. 2007). For molecular identification, four loci, ITS (White et al. 1990), RPB2 (O'Donnell, 2022), H3 (Zheng et al. 2015), and GAPDH (Berbee et al. 1999), were sequenced from two representative isolates (TC0, TC10). Compared with a reference isolate, Alternaria alternata strain CBS 107.27, GenBank accession nos. KP124300.1 (ITS), KP124768.1 (RPB2), KP124157.1 (GAPDH). The ITS, RPB2, and GAPDH sequences of TC0 and TC10 showed 99% (502 of 504 bp ), 100% (753 of 753 bp), and 99% (560 of 561 bp) similarity, respectively. Compared with a reference isolate, A. alternata isolate 21-5, GenBank accession no. MN840996.1 (H3), H3 sequences of TC0 and TC10 showed 99% (399 of 401 bp) similarity. The sequences of two isolates (TC0, TC10) were deposited in GenBank with accession numbers PP837733.1, PP565404.1(ITS), PP839298.1, PP573905.1(RPB2), PP839299.1, PP573904.1 (GAPDH), and PP839297.1, PP573903.1(H3). Phylogenetic trees were constructed using the sequences and showed that isolates (TC0, TC10) were in the same clade with A. alternata strains. TC0 and TC10 were identified as A. alternata based on the morphological characteristics and molecular phylogeny. Pathogenicity testing was conducted on six-month-old healthy plants, (cv. Green Stalk), three plants were inoculated by spraying spore solution (1 × 106 conidia/mL), and three plants were sprayed with sterile water as a control. The pathogenicity test was performed 3 times. Plants were maintained at 28°C and >80% RH. Plants showed symptoms after 30 days, symptoms were observed similar to those of the original infected plants, control plants were asymptomatic. The fungus was reisolated, confirmed as A. alternata based on conidial characteristics, no pathogenic fungus was isolated from the control plants. A. alternata has previously been reported on beet (also Beta vulgaris) in China (Tai, F. L. 1979; Zhuang, W. Y. 2005). To our knowledge, this is the first report of leaf spot caused by A. alternata on chard in China. This result may expand the etiological study of A. alternata and the control strategy of Chard leaf spot.

First report of wheat leaf spot disease caused by Curvularia inaequalis in China.

Wheat (Triticum aestivum) is an economically important crop widely cultivated in China. In August 2022, brown oval leaf spots with yellow halos were observed on approximately 10% wheat seedlings over an area of about 1 hectare in Xining City, Qinghai Province, which adversely affected wheat growth and production. Six diseased leaves were collected from the field in Huangyuan county (101°69' E, 37°04' N). The 0.5 cm × 0.5 cm pieces were cut from the border between healthy and diseased regions of the sampled leaves, surface sterilized for 10 s in 75% ethanol, followed by a 1% NaClO for 90 s, and rinsed three times with distilled sterile water. The pieces of leaf tissue were dried with sterile tissue, and plated on potato dextrose agar (PDA) amended with streptomycin (0.02 g/L) and ampicillin sulfate (0.05 g/L) to eliminate bacterial contamination. The dishes were placed in an incubator at 25°C for 72 h in dark. Three isolates, WGC201, WGC202 and WGC203, were obtained by a single-spore culture method. Fungal colonies on PDA media were dark green (Fig. 1A and 1B). Conidiophores were septate and geniculate terminals, while conidia exhibited straight or slightly curved forms with four transverse septa, the central cell being notably longer and wider than the others. The size of such conidia were 27.34 µm to 40.62 µm× 11.61 µm to 15.97 µm (number = 50) (av. 32.71 µm× 13.11 µm) (Fig. 1C and 1D) (Moubasher et al. 2010). The internal transcribed spacer (ITS) region of nuclear ribosomal DNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified and sequenced using universal primers ITS1/ITS4 and GPDF/GPDR (White et al. 1990; Berbee et al. 1999). DNA sequences were deposited into the NCBI database (ITS, PP789629, PP801333, and PP801574; GAPDH, PP849124, PP849125, and PP849126). Phylogenetic analysis with a neighbor-joining method based on the concatenated sequences of ITS and GAPDH genes showed that the three isolates clustered within a C. inaequalis branch (Fig. 2). Based on morphological and molecular identification, the fungal isolates were identified as C. inaequalis. The pathogenicity test was conducted in a greenhouse at 25°C using a spore suspension method and three isolates were used. Conidia were produced on PDA media (25℃) for 14 days. Plates were washed with sterilized distilled water and filtered with cheese cloth. Conidial suspension was adjusted to a concentration of 1×107 conidia/mL. Fifteen healthy seedlings of a wheat cultivar Xiaoyan-6 at a 3-4 leaf stage were inoculated by evenly spraying a 100mL spore suspension. Plants inoculated with sterile water served as a control. All plants were covered with plastic bags for 3 days. At 7 days after inoculation, all pathogen-inoculated plants showed similar symptoms (brown leaf oval spots with yellow halos) with those observed in the field, while all plants inoculated with sterile water showed no symptoms (Fig. 1E and 1F). The pathogen was reisolated from the symptomatic leaves and proved to be C. inaequalis. Morphological, molecular and pathogenic results indicated that C. inaequalis is the pathogen causing wheat leaf disease in China. The results are consistent with a previous report in Azerbaijan (Özer et al. 2020). To our knowledge, this is the first report of C. inaequalis causing spot disease on wheat in China. The occurrence, spread and economic importance to different wheat cultivars of the emerging disease in China will be further investigated and evaluated.

Recent advances in the population biology and management of maize foliar fungal pathogens Exserohilum turcicum, Cercospora zeina and Bipolaris maydis in Africa.

Maize is the most widely cultivated and major security crop in sub-Saharan Africa. Three foliar diseases threaten maize production on the continent, namely northern leaf blight, gray leaf spot, and southern corn leaf blight. These are caused by the fungi Exserohilum turcicum, Cercospora zeina, and Bipolaris maydis, respectively. Yield losses of more than 10% can occur if these pathogens are diagnosed inaccurately or managed ineffectively. Here, we review recent advances in understanding the population biology and management of the three pathogens, which are present in Africa and thrive under similar environmental conditions during a single growing season. To effectively manage these pathogens, there is an increasing adoption of breeding for resistance at the small-scale level combined with cultural practices. Fungicide usage in African cropping systems is limited due to high costs and avoidance of chemical control. Currently, there is limited knowledge available on the population biology and genetics of these pathogens in Africa. The evolutionary potential of these pathogens to overcome host resistance has not been fully established. There is a need to conduct large-scale sampling of isolates to study their diversity and trace their migration patterns across the continent.

First Report of Nigrospora oryzae Causing Leaf Spot on Peanut (Arachis hypogaea L.) in the Republic of Korea.

Peanut (Arachis hypogaea L.) is one of the most profitable upland crops, yielding 10,711 tonnes in an area of 4,062 ha in the Republic of Korea (Ministry of Agriculture, Food and Rural Affairs 2023). In September 2023, dark gray spots surrounded by yellow halos were observed on the peanut leaves over an area of 880 m2 at the National Institute of Crop Science (35°50'31.4"N 127°02'41.0"E), with a disease incidence up to 80%. Early symptoms appeared as small, brown, circular or irregular spots that enlarged and were surrounded by chlorotic halos. Leaf cuttings (5 mm x 5 mm) from five symptomatic plants were surface-sterilized with 70% EtOH for 1 min, followed by 1% NaClO for 1 min, and rinsed 3 times with sterile water. The pieces were placed on Potato Dextrose Agar (PDA) and incubated at 25 °C in the dark for 3 days. Three isolates obtained by single-spore isolation were designated as F23025, F23026, and F23027. Two isolates, F23025 and F23026 were deposited in the Korean Agricultural Culture Collection (https://genebank.rda.go.kr) under the accession numbers 410722 and 410723. Fungal colonies were initially white and turned sooty gray after 5 days. Conidia were unicellular, brown to black, and spherical or sub-spherical with 6.8 µm to 14.3 µm (mean = 11.1 µm ± 1.8, n = 50). The morphology of the three isolates was identical and showed the same characteristics as Nigrospora oryzae (Ellis 1971; Hudson 1963). For molecular identification, the Internal Transcribed Spacer (ITS) region (GenBank accession PP388306 and PP574448), beta tubulin (PP397027 and PP580108), and translation elongation factor 1- ɑ (PP397028 and PP580109) of isolates F23025 and F23026 were amplified and sequenced with primers of ITS5/ITS4, Bt2a/Bt2b, EF1-727F/EF2, respectively and showed high identity of 99.62% (530 bp/532 bp), 100% (384/384), and 99.79% (475/476) with N. oryzae strain LC2693 (GenBank accessions KX985994, KY019471, and KY019299, respectively). Multilocus sequence analysis showed isolates F23025 and F23026 were on the same clade with N. oryzae strain LC2693. To determine the pathogenicity to peanut, a conidial suspension (1 x 106 conidia/mL) was sprayed onto leaves of five 3-week-old plants 'Sewon' grown in pots, while sterile distilled water was sprayed onto two plants used as negative control. Sprayed plants were placed in a dew chamber at 25℃ for two days and grown in a growth chamber at 25℃ and 80% of relative humidity with a 16L:8D cycle. Two weeks later, dark spots with chlorotic halos appeared only on leaves sprayed with conidia, and no symptoms on leaves sprayed with sterile distilled water. The pathogenicity test was repeated three times, and each time the pathogen was re-isolated and identified by ITS sequence, thus fulfilling Koch's postulates. Nigrospora species are cosmopolitan, and some species have a wide host range as plant pathogens. Recently, two species of the genus Nigrospora, N. sphaerica and N. aurantiaca, were reported to cause peanut leaf blight in China (Liu et al. 2020; He et al. 2023). To the best of our knowledge, this is the first report of N. oryzae causing leaf spot to A. hypogaea L. in the Republic of Korea. As identifying new pathogens and registering fungicides to control them are important for the continued cultivation of peanut, this report will help in that endeavor.

First Report of Curvularia trifolii Causing Leaf Spot on Zanthoxylum bungeanum in China.

Chinese prickly ash (Zanthoxylum bungeanum Maxim.), which is a Rutaceae plant as shrubs or small trees and indigenous to China, is widely grown in this country for its flavor, as well as its high economic and ecological value. So far, in China, the cultivated area and yield of Chinese prickly ash rank first in the world. In June 2023, a leaf spot disease with approximately 30% incidence was observed on Z. bungeanum in Zhenfeng County (25°44'21.38″ N, 105°56'47.15″ E, 1,083 m), Guizhou Province, China. Foliar symptoms appeared as irregularly shaped lesions, yellowish-brown with dark brown margins surrounded by yellow halos, which enlarged, resulting in the lesions dropping from the leaves and leaving holes. To isolate and identify the pathogen, symptomatic leaves were taken and cut into 5 mm × 5 mm pieces, surface sterilized with 2% NaClO for 3 min, 75% ethanol for 30 s, rinsed three times with sterile water, and incubated on PDA at 28°C. Ten isolates with identical morphology were obtained. After one week of incubation at 28℃, the colonies on PDA were brown, reverse dark brown, fluffy, reaching 7.0-7.5 cm in diameter. Conidia were straight or slightly curved, narrowly ellipsoidal or fusiform, 1-3 but mostly 3 septate, light or dark brown, with the middle cells usually darker than the terminal cells, smooth, 20.5-31.0 × 9.0-19.0 µm (x̄ = 26.0 × 14.0 µm, n = 30). The morphological features matched the description of Curvularia trifolii (Kauffman) Boedijn (Ellis 1971; Falloon 1976). Additionally, the internal transcribed spacer (ITS), large subunit (LSU) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes were amplified by PCR with primers ITS5/ITS4 (White et al. 1990), LROR/LR5 (Vilgalys & Hester 1990) and GPD1/GPD2 (Berbee et al. 1999), and the ITS, LSU and gapdh sequences of the isolate GUCC 23-321 (PP837870, PP837881, PP855474) were deposited in GenBank. The BLAST showed 98.5% (ITS, HG779023, 598/709 bp), 99.87% (LSU, HG779077, 779/858 bp), and 97.79% (gapdh, HG779124, 543/498 bp) identities with C. trifolii (CBS 173.55). Furthermore, the phylogenetic tree of ML analysis based on the combined sequence data of ITS, LSU and gapdh revealed that GUCC 23-321 clustered with C. trifolii. Both morphology and phylogenetic analyses supported the identification of GUCC 23-321 as C. trifolii. Pathogenicity tests were carried out twice according to Koch's postulates. Five healthy 2-year-old Chinese prickly ash plants were sprayed with a conidial suspension (1 × 106 conidia/mL) of the isolate GUCC 23-321, while the controls (five other plants) were sprayed with sterile water. All plants were maintained in a greenhouse at 28°C, 80% relative humidity. After 8 days, the inoculated plants developed leaf spots similar to those showed in the field, but control plants were asymptomatic. Re-isolation of pathogenic fungi from the leaf lesions of the inoculated plants and according to molecular analysis and morphology, the fungi were identified as C. trifolii, fulfilling Koch's postulates. C. trifolii is a common fungal phytopathogen that has been reported to infect a variety of plants and cause leaf spot disease, such as Trifolium alexandrinum (Khadka 2016) and Nicotiana tabacum (Chen et al. 2017). This is the first worldwide report of C. trifolii causing Z. bungeanum leaf spot. The report will be beneficial for accurately diagnosing this disease, and proposing specific control measures.

Time of arrival during plant disease progression and humidity additively influence Salmonella enterica colonization of lettuce.

The interplay between plant hosts, phytopathogenic bacteria, and enteric human pathogens in the phyllosphere has consequences for human health. Salmonella enterica has been known to take advantage of phytobacterial infection to increase its success on plants, but there is little knowledge of additional factors that may influence the relationship between enteric pathogens and plant disease. In this study, we investigated the role of humidity and the extent of plant disease progression on S. enterica colonization of plants. We found that high humidity was necessary for the replication of S. enterica on diseased lettuce, but not required for S. enterica ingress into the UV-protected apoplast. Additionally, the Xanthomonas hortorum pv. vitians (hereafter, X. vitians)-infected lettuce host was found to be a relatively hostile environment for S. enterica when it arrived prior to the development of watersoaking or following necrosis onset, supporting the existence of an ideal window during X. vitians infection progress that maximizes S. enterica survival. In vitro growth studies in sucrose media suggest that X. vitians may allow S. enterica to benefit from cross-feeding during plant infection. Overall, this study emphasizes the role of phytobacterial disease as a driver of S. enterica success in the phyllosphere, demonstrates how the time of arrival during disease progress can influence S. enterica's fate in the apoplast, and highlights the potential for humidity to transform an infected apoplast into a growth-promoting environment for bacterial colonizers. IMPORTANCE: Bacterial leaf spot of lettuce caused by Xanthomonas hortorum pv. vitians is a common threat to leafy green production. The global impact caused by phytopathogens, including X. vitians, is likely to increase with climate change. We found that even under a scenario where increased humidity did not enhance plant disease, high humidity had a substantial effect on facilitating Salmonella enterica growth on Xanthomonas-infected plants. High humidity climates may directly contribute to the survival of human enteric pathogens in crop fields or indirectly affect bacterial survival via changes to the phyllosphere brought on by phytopathogen disease.

Dense Convolutional Neural Network-Based Deep Learning Pipeline for Pre-Identification of Circular Leaf Spot Disease of Diospyros kaki Leaves Using Optical Coherence Tomography.

Circular leaf spot (CLS) disease poses a significant threat to persimmon cultivation, leading to substantial harvest reductions. Existing visual and destructive inspection methods suffer from subjectivity, limited accuracy, and considerable time consumption. This study presents an automated pre-identification method of the disease through a deep learning (DL) based pipeline integrated with optical coherence tomography (OCT), thereby addressing the highlighted issues with the existing methods. The investigation yielded promising outcomes by employing transfer learning with pre-trained DL models, specifically DenseNet-121 and VGG-16. The DenseNet-121 model excels in differentiating among three stages of CLS disease (healthy (H), apparently healthy (or healthy-infected (HI)), and infected (I)). The model achieved precision values of 0.7823 for class-H, 0.9005 for class-HI, and 0.7027 for class-I, supported by recall values of 0.8953 for class-HI and 0.8387 for class-I. Moreover, the performance of CLS detection was enhanced by a supplemental quality inspection model utilizing VGG-16, which attained an accuracy of 98.99% in discriminating between low-detail and high-detail images. Moreover, this study employed a combination of LAMP and A-scan for the dataset labeling process, significantly enhancing the accuracy of the models. Overall, this study underscores the potential of DL techniques integrated with OCT to enhance disease identification processes in agricultural settings, particularly in persimmon cultivation, by offering efficient and objective pre-identification of CLS and enabling early intervention and management strategies.

Dipicolinic acid reduces Epicoccum sorghinum symptoms on maize and inhibits tenuazonic acid biosynthesis.

BACKGROUND: Epicoccum sorghinum is a pathogenic fungus that causes leaf spot in a wide range of plants, including maize, and synthesizes the mycotoxin tenuazonic acid (TEA), which is carcinogenic. Despite the relevant economic and yield losses caused by E. sorghinum worldwide, methods for the control of this pathogen are lacking. RESULTS: In this work, the efficacy of Bacillus-produced dipicolinic acid (DPA) for control of E. sorghinum was evaluated using in vitro and in vivo assays, and compared with the efficacy of three commercial fungicides, including carbendazim, prochloraz, and thiram. DPA inhibited E. sorghinum mycelial growth, and conidia germination, and produced important alterations in E. sorghinum hyphae. Interestingly, 10 mM DPA reduced TEA biosynthesis by 86.6%. Although DPA rapidly degraded on maize leaves, 10 mM DPA showed higher preventive (67.4% lesion length inhibition) and inhibitory (89.5% lesion length inhibition) efficacies for the control of E. sorghinum on maize leaves compared to the commercial fungicides. CONCLUSIONS: Collectively, this study presents the first method for the control of E. sorghinum on maize and demonstrates that DPA application is a suitable approach to inhibit E. sorghinum symptoms in plants and TEA biosynthesis. © 2024 Society of Chemical Industry.

Biocontrol potential and growth-promoting effect of endophytic fungus Talaromyces muroii SD1-4 against potato leaf spot disease caused by Alternaria alternata.

BACKGROUND: Alternaria alternata is the primary pathogen of potato leaf spot disease, resulting in significant potato yield losses globally. Endophytic microorganism-based biological control, especially using microorganisms from host plants, has emerged as a promising and eco-friendly approach for managing plant diseases. Therefore, this study aimed to isolate, identify and characterize the endophytic fungi from healthy potato leaves which had great antifungal activity to the potato leaf spot pathogen of A. alternata in vitro and in vivo. RESULTS: An endophytic fungal strain SD1-4 was isolated from healthy potato leaves and was identified as Talaromyces muroii through morphological and sequencing analysis. The strain SD1-4 exhibited potent antifungal activity against the potato leaf spot pathogen A. alternata Lill, with a hyphal inhibition rate of 69.19%. Microscopic and scanning electron microscope observations revealed that the strain SD1-4 grew parallel to, coiled around, shrunk and deformed the mycelia of A. alternata Lill. Additionally, the enzyme activities of chitinase and ß-1, 3-glucanase significantly increased in the hyphae of A. alternata Lill when co-cultured with the strain SD1-4, indicating severe impairment of the cell wall function of A. alternata Lill. Furthermore, the mycelial growth and conidial germination of A. alternata Lill were significantly suppressed by the aseptic filtrate of the strain SD1-4, with inhibition rates of 79.00% and 80.67%, respectively. Decrease of leaf spot disease index from 78.36 to 37.03 was also observed in potato plants treated with the strain SD1-4, along with the significantly increased plant growth characters including plant height, root length, fresh weight, dry weight, chlorophyll content and photosynthetic rate of potato seedlings. CONCLUSION: The endophyte fungus of T. muroii SD1-4 isolated from healthy potato leaves in the present study showed high biocontrol potential against potato leaf spot disease caused by A. alternata via direct parasitism or antifungal metabolites, and had positive roles in promoting potato plant growth.