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Maize leaf spot occurs worldwide and affects maize production. Maize can be infected by several pathogens causing leaf spot, such as Bipolaris zeicola, Bipolaris maydis, Curvularia species, Alternaria species, etc. In the current study, 30 Epicoccum isolates recovered from symptomatic maize leaves were identified based on morphological characteristics, pathogenicity, and multilocus sequence analyses of nuLSU, ITS, tub2, and rpb2. These maize isolates were grouped into five Epicoccum species, including E. nigrum, E. layuense, E. sorghinum, E. latusicollum, and E. pneumoniae. Pathogenicity tests showed that all five Epicoccum species could produce small ellipse- and spindle-shaped spots on maize leaves. The lesion center was grayish yellow to dark gray and surrounded by a chlorotic area. Furthermore, the Epicoccum isolates exhibited high pathogenicity to 20 main maize varieties of Heilongjiang Province but showed different sensitivities to the commonly used fungicides carbendazim and tebuconazole. In addition, these Epicoccum isolates showed different production capacity of pectinase, cellulase, protease, amylase, laccase, and gelatinase, but all showed high lipase activity. This is the first report globally of E. layuense, E. latusicollum, and E. pneumoniae as causal agents of maize leaf spot. E. pneumoniae was first reported as a plant pathogen.

Maize (Zea mays L.) is a major economic crop worldwide. Maize can be infected by Alternaria species causing leaf blight that can result in significant economic losses. In this study, 168 Alternaria isolates recovered from symptomatic maize leaves were identified based on morphological characteristics, pathogenicity, and multilocus sequence analyses of the genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the internal transcribed spacer of ribosomal DNA (rDNA ITS), the RNA polymerase II second largest subunit (RPB2), and histone3 (HIS3). Maize isolates grouped to four Alternaria species including Alternaria tenuissima, A. alternata, A. burnsii, and Alternaria sp. Notably, A. tenuissima (71.4%) was the most prevalent of the four isolated species, followed by A. alternata (21.5%), Alternaria sp. (4.1%), and A. burnsii (3.0%). Pathogenicity tests showed that all four Alternaria species could produce elliptic to nearly round, or strip, lesions on leaves of maize, gray-white to dry white in the lesion centers and reddish-brown at the edges. The average disease incidence (58.47%) and average disease index (63.55) of maize leaves inoculated with A. alternata were significantly higher than levels resulting from A. tenuissima (55.28% and 58.49), Alternaria sp. (55.34% and 58.75), and A. burnsii (56% and 55). Haplotype analyses indicated that there were 14 haplotypes of A. tenuissima and five haplotypes of A. alternata in Heilongjiang Province and suggested the occurrence of a population expansion. Results of the study showed that Alternaria species associated with maize leaf blight in Heilongjiang Province are more diverse than those that have been previously reported. This is the first report globally of A. tenuissima, A. burnsii, and an unclassified Alternaria species as causal agents of leaf blight on maize.

Winter jujube originated from China and had an extremely high nutritional value. In 2021, symptomatic winter jujube fruits were collected from eight locations in Zhanhua District of Binzhou City, Shandong Province. In total, 108 fungal isolates were obtained and grouped into 11 species based on morphological characteristics and multilocus phylogenetic analysis, including Nothophoma quercina (43.52%), Fusarium lateritium (20.37%), Alternaria alternata (12.03%), F. proliferatum (7.41%), F. graminearum (4.63%), Botryosphaeria dothidea (3.70%), Fusarium sp. (2.78%), A. tenuissima (2.78%), Diaporthe eres (1.85%), Nigrospora oryzae (0.93%), and Cercospora nicotianae (0.93%). All fungal isolates obtained in this study showed aggressiveness on detached winter jujube fruits except N. oryzae and C. nicotianae isolates, of which F. proliferatum was the most virulent, while A. alternata isolates, which have been considered the major pathogen of winter jujube fruit rot, showed a relatively low-level virulence in this study. Furthermore, D. eres, F. graminearum, F. lateritium, and an unclassified Fusarium species were first reported as causal agents of winter jujube fruit rot. The typical symptoms of winter jujube fruit rot observed in this study could be distinguished into two types. N. quercina, A. alternata, A. tenuissima, Fusarium sp., D. nobilis, and F. lateritium isolates caused reddish brown to dark gray lesions on the peel, while B. dothidea, F. graminearum, and F. proliferatum isolates caused peel and pulp decay, resulting in red to reddish brown and water-soaked lesions. In addition, haplotype analysis of N. quercina isolates obtained in this study and validly published articles showed that there were 11 haplotypes worldwide; the isolates obtained in the current study were grouped into three haplotypes (Hap 1, Hap 2, and Hap 11), and two of them (Hap 2 and Hap 11) were confirmed as new haplotypes.

Panicle hydrangea (Hydrangea paniculata), belonging to the Saxifragaceae family (Wu et al. 2001), is an ornamental flowering plant which is native to China and Japan. In July 2021, brown leaf spots (diameter ranged from 3 to 5 mm) were observed on panicle hydrangea plants in a 0.1 ha field of Northeast Agriculture University (126.72°E, 45.74°N), Heilongjiang Province. The incidence was approximately 30%. Small circular or irregular brown spots initially appeared on the older leaves, and these lesions gradually expanded with time. In some serious cases, lesions joint together and caused leaf wilting. To identify the causal agent, ten symptomatic leaves from different panicle hydrangea plants were collected, and surface sterilized with 70% ethanol for 30 s, followed by 0.5% NaClO treatment for 4 min, and then rinsed with sterile water three times. Tissues between healthy and necrotic area were cut into 5×5 mm pieces after air drying on sterile filter paper. The pieces were placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 mg/liter) and incubated at 25°C for 5 days. Fifteen pure isolates were obtained using the hyphal tip technique and cultured on PDA for 7 days at 25°C for morphological and molecular identification. Colonies of all isolates were dark olivaceous with white margin. Conidiophores were septate, singly arising, and light brown, with a size range of 10.2-60.1 × 1.4-5.6 µm (n=50). Conidia were obclavate to obpyriform, brown to dark brown, and in a size range of 15.0-25.2 × 5.1-15.3 µm (n=50). To further identify these isolates, four different genomic DNA regions including ribosomal DNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (rpb2), and histone 3 (HIS3) were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), RPB2-5F2/RPB2-7R (Sung et al. 2007), and H3-1a/H3-1b (Xu et al. 2022) for three representative isolates (DXQ2-2, DXQ2-3, and DXQ2-4), respectively. The sequences of ITS, GADPH, rpb2, and HIS3 for these three isolates were identical and only sequences of DXQ2-2 were deposited in GenBank (GenBank accession nos. OL305828, OL333601, OL333602, and OL436242). These sequences showed 100%, 100%, 100%, and 99.61% identity with A. tenuissima BJ-CX-1 (GenBank accession nos. MK683974, MK683784, MK684069, and MK683879), respectively. Based on morphological features and DNA sequences analyses, these isolates were identified as Alternaria tenuissima (Simmons 2007). To fulfill Koch's postulates, ten healthy and surface disinfected leaves of one panicle hydrangea plant grown in pot were sprayed with a conidial suspension (1×106 conidia/mL) of isolate DXQ2-2. Meanwhile, ten surface disinfected leaves of another panicle hydrangea plant grown in pot sprayed with sterilize water served as the control. All plants were maintained in a greenhouse at 25℃ and 70% relative humidity. Five days after inoculation, leaves inoculated with conidial suspension showed leaf spot symptoms that were similar to those observed in the field, whereas no symptom was observed on the control leaves. The experiment was conducted twice. The Alternaria tenuissima isolate was successfully re-isolated from the symptomatic leaves and confirmed based on above morphological and molecular methods. To our knowledge, this is the first report of leaf spot on panicle hydrangea caused by A. tenuissima. Leaf spot has a negative effect on the aesthetic value of panicle hydrangea, so further investigation and management is needed to control this disease.

Leaf spot is a serious disease in the growth and development of muskmelon, which can affect its quality and yield. Over the past years, Malianzhuang Muskmelon Base, the main muskmelon producing area in Shandong Province, China, has been seriously affected by leaf spot. Since 2018, symptomatic leaves were collected from 11 production areas of this base to determine the pathogens of muskmelon foliar diseases. Two-hundred fungal strains were isolated and 10 genera and 17 species were identified based on morphological characteristics and multilocus phylogenetic analysis (ITS, GADPH, RPB2, HIS3, EF-1α, and LSU). The most frequently isolated species from each sampling area was Alternaria tenuissima with 77 strains, followed by A. alternata. Pathogenicity experiments showed that A. alternata, A. tenuissima, Fusarium neocosmosporiellum (formerly Neocosmospora vasinfecta), F. acuminatum, Exserohilum rostratum, Bipolaris sorokiniana, and Stagonosporopsis cucurbitacearum (formerly Didymella bryoniae) could cause symptoms highly similar to those of infected leaves observed under natural conditions in the field. Therefore, these fungal isolates are considered to be the primary pathogens causing muskmelon leaf spot, and A. tenuissima and A. alternata were the most common and virulent pathogens in this study. In addition, this is the first study of F. neocosmosporiellum, F. acuminatum, E. rostratum, and B. sorokiniana as pathogens associated to muskmelon leaf spot in China as well as the world.

Fusarium spp. are among the most important plant pathogens in the world. A survey on maize leaf blight was carried out in Heilongjiang province from 2019 to 2021. Based on morphological characteristics and a phylogenetic analysis on translation elongation factor (tef1) and second-largest subunit of RNA polymerase II (rpb2) genes, 146 Fusarium isolates were obtained and grouped into 14 Fusarium species, including F. ipomoeae (20.5%), F. compactum (17.1%), F. sporotrichioides (9.59%), F. graminearum (9.59%), F. citri (8.9%), F. asiaticum (6.85%), F. verticillioides (6.85%), F. acuminatum (5.48%), F. glycines (5.48%), F. temperatum (2.74%), F. armeniacum (2.74%), Fusarium sp. (2.05%), F. flagelliforme (1.4%), and F. annulatum (0.68%). The Fusarium incarnatum-equiseti species complex (FIESC, including F. ipomoeae, F. compactum, F. citri, and F. flagelliforme) was the most prevalent, indicating an evolving occurrence of the Fusarium species causing maize leaf blight. The typical symptoms observed on the maize leaves were oval to long strip lesions, with a gray to dark gray or brownish red coloration in the center and a chlorotic area at the edges. Based on the tef1 gene, seven haplotypes of FIESC were identified in Heilongjiang province, suggesting a population expansion. This is the first report of F. ipomoeae, F. compactum, F. flagelliforme, F. citri, F. sporotrichioides, F. graminearum, F. asiaticum, F. acuminatum, F. glycines, F. temperatum, F. armeniacum, Fusarium sp., and F. annulatum causing maize leaf blight in Heilongjiang province, China. The current research is informative for managing disease, exploring the phylogenetic relationship among Fusarium species, and clarifying the diversity of Fusarium species associated with maize leaf blight.

Maize (Zea mays L.) is the most important crop in Heilongjiang province. In July 2021, maize stalk rot was observed on approximately 10% of maize in a 2.4 ha field of Xiangfang District, Harbin City (N45°44'23″, E126°43'19″). Infected plants showed softening of the stalks at the lower internodes, and the pith tissue was disintegrated and brown to reddish. Fifteen symptomatic plants were collected from the field. The discolored stalk pith tissues were cut into small pieces (4 × 2 mm), superficially disinfected with 1% NaClO for 3 min, 70% ethanol for 10 s, and then washed three times with sterile distilled water. The disinfected tissues were placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 mg/L) and incubated at 25°C for 1 week. Twenty-one cultures were obtained using hyphal tip technology and cultured on PDA for 7 days at 25°C for morphological and molecular analyses. The mycelia of the cultures were initially white but became grayish with time, and reddish-brown diffusible pigments were produced. A dark green discoloration was produced on malt extract agar (MEA) using the NaOH spot test (REF). Pycnidia were brown, predominantly spheroidal, and measured 80.1 to 130.2 × 110.5 to 220.6 μm. Conidia were ellipse, aseptate, and in a size range of 4.3 to 6.8 × 2.1 to 3.2 μm. The isolates were initially identified as Epicoccum latusicollum based on morphological features (Chen et al. 2017). To confirm the identity of E. latusicollum, primers TUB2Fd/TUB4Rd, LR0R/LR5, ITS1/ITS4 and RPB2-5F2/fRPB2-7cR (Valenzuela-Lopez et al. 2018) were used to amplify beta tubulin (tub2), nuclear large subunit rDNA (LSU), internal transcribed spacer (ITS), and RPB2 genes, respectively for the representative isolate JF3. These sequences were deposited in GenBank (GenBank accession no. OK490498, OK445527, OK483136, and OK490497) and had 100% (276/276 bp), 100% (842/842 bp), 100% (501/501 bp), and 100% (589/589 bp) nucleotide identity with E. latusicollum isolate GZDS2018BXT010 (GenBank accession no. MK516208, MK516207, MK516206, and MK852278). To fulfill Koch's postulates, pathogenicity tests for all isolates were performed by individually inoculating surface-disinfected stalks of five healthy maize plants (10-leaf stage) between the 2nd and 3rd stem nodes with 20 μL conidial suspension at a concentration of 106 conidia/mL as described by Zhang et al. (2016). Five other healthy surface-disinfected maize plants inoculated with sterile distilled water served as control. All plants were kept at 25 ± 0.5°C in a greenhouse with a photoperiod of 12 h and approximately 90% relative humidity. After 10 days, all inoculated plants showed symptoms that were similar to those of the infected maize plants observed in the field, whereas the control plants were asymptomatic. The Epicoccum isoaltes were re-isolated from symptomatic plants, and species identification was performed using the morphological and molecular methods described above. To our knowledge, this is the first report of E. latusicollum causing maize stalk rot in China, and this report will assist with monitoring distribution of the disease and developing management recommendations.

Maize (Zea mays L.) is one of the major crops in China. In July 2020, leaf blight was observed on approximately 18% of maize plants at the 852 Farm, Heilongjiang province. Symptoms appeared as yellow necrotic lesions on leaf tips and margins, which later expanded to the entire. The disease was first observed on the lower leaves of the plants and then progressed up the plant. Thirty symptomatic leaves were collected in 2020, and tissue samples between healthy and necrotic area (4 × 2 mm) were surface disinfected with 1% NaOCl for 3 min, 70% ethanol for 10 s, and washed three times with sterile water. Disinfected tissues were placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 mg/liter) and incubated at 25°C for 1 week. Cultures were purified using the hyphal-tip technique for morphological and molecular analyses. Morphological characteristics were observed on 1-week-old PDA cultures grown at 25°C. Mycelium changed from cream colored to pale pink or reddish, and the back of plate turned pink with time. Hyphae were hyaline, 2 to 7 μm wide, with clamps at primary septa. Hyaline basidiospores were sphere to ellipsoid, and ranged from 8 to 10 × 6 to 9 μm. Based on the presence of clamp connections and morphological features, the fungus was preliminarily identified as Limonomyces roseipellis (Stalpers et al. 1982). To confirm the identity of L. roseipellis, primers ITS1/ITS4 (White et al. 1990), MS1/MS2 (White et al. 1990), and LR0R/LR5 (Vilgalys and Hester 1990) were used to amplify the internal transcribed spacer (ITS) region, the partial mitochondrial small subunit rDNA (mtSSU) and nuclear large subunit rDNA (nuLSU), respectively. These sequences were deposited in GenBank (GenBank accession no.s MW067756, MW322806 and MW386178). The ITS sequence had 99.55% nucleotide identity (660 bp/668 bp) with L. roseipellis isolate EF82 (GenBank accession no. MK918632). The mtSSU sequence was 99.69% identical (634 bp/636 bp) to that of L. roseipellis strain SY-LQG101 (Genbank accession no. KF824718). The nuLSU sequence was 99.14% identical (924 bp/932 bp) to that of L. roseipellis (GenBank accession no. EU622844). A single basidiospore was isolated and cultured on PDA for pathogenicity testing. To fulfill Koch's postulates, ten healthy, surface-disinfected maize plants grown in pots (four to five leaves stage) were sprayed with basidiospore suspension (1×106 spores/ml); another ten healthy surface-disinfected maize plants sprayed with distilled water to serve as controls. Plants were sealed in plastic bags immediately after inoculation and maintained at 90% relative humidity in a mist chamber for 24 h at 25°C with a 12-h light cycle (Nicoli et al. 2016). Plants were moved and maintained in the greenhouse and observed for disease development. The experiment was conducted twice. Leaf blight symptoms appeared on all inoculated plants 3 to 5 days postinoculation and were consistent with symptoms observed in the field. No disease symptoms were observed on control plants. The pathogen was reisolated from diseased plants, and species identification was confirmed by the morphological and molecular method described. L. roseipellis has been reported to infect Cynodon dactylon, Lolium perenne and Festuca arundinacea, respectively. To our knowledge, this is the first report of the identification of L. roseipellis as a pathogen of maize in China, and this report will assist with monitoring distribution of the disease to assist with developing management recommendations.