Taxus chinensis var. mairei is the endemic, endangered, and first-class protected tree species in China. This species is considered as an important resource plant because it can produce Taxol which is an effective medicinal compound against various cancers (Zhang et al., 2010). Stem blight was observed in two plant nurseries in Ya’an (102°44′E,30°42′N), Sichuan province in April 2021. The symptoms first appeared as round brown spots on the stem. As the disease progressed, the damaged area gradually expanded into an oval or irregular shape, which was dark brown. About 800 square meters of planting area were investigated and the disease incidence was up to approximately 64.8%. Twenty obviously symptomatic stems which exhibited the same symptoms as above were collected from 5 different trees in the nursery. To isolate the pathogen, the symptom margin was cut into small blocks (5 x 5 mm), and the blocks were surface sterilized in 75% ethanol for 90 s and 3% NaClO solution for 60 s . Finally incubated on Potato Dextrose Agar (PDA) at 28℃ for 5 days. Ten pure cultures were isolated by transferring hyphal and the three strains (HDS06, HDS07 and HDS08) were selected as representative isolates for further study. Initially, colonies on the PDA of three isolates were white and cotton-like, and then gradually turned gray-black from the center. After 21 days, conidia were produced and were smooth-walled, single-celled, black, oblate, or spherical, measuring 9.3 to 13.6 × 10.1 to 14.5 μm in size (n = 50). Conidia were present at the tip of conidiophores on hyaline vesicles. These morphological features were generally consistent with those of N. musae (Wang et al., 2017). To validate the identification, DNA were extracted from the three isolates, followed by the amplification of transcribed spacer region of rDNA (ITS), the translation elongation factor EF-1 (TEF-1), and the Beta-tubulin (TUB2) sequences with the respective primer pairs ITS1/ITS4 (White et al., 1990), EF-728F/EF-986R (Vieira et al., 2014) and Bt2a/Bt2b (O'Donnell et al., 1997) .The sequences were deposited in GenBank with the accession numbers ON965533, OP028064, OP028068, OP060349, OP060353, OP060354, OP060350, OP060351 and OP060352, respectively. Phylogenetic analysis of combined ITS, TUB2, and TEF genes using the Mrbayes inference method showed that the three isolates clustered with Nigrospora musae as a distinct clade (Fig. 2). Combine with morphological characteristics and phylogenetic analysis, three isolates were identified as N. musae. 30 2-year-old healthy potted plants of T. chinensis were used for pathogenicity test. 25 of these plants were inoculated by injecting 10 μL of the conidia suspension (1 × 106 conidia/mL) into stems and then wrap around the seal to moisturize. The remaining 5 plants were injected with the same amount of sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 25°C and 80% relative humidity. After 2 weeks, the inoculated stems developed lesions similar to those observed in the field, whereas controls were asymptomatic. N. musae was re-isolated from the infected stem and identified by both morphological characteristics and DNA sequence analysis. The experiments repeated three times showed similar results. As far as we know, this is the first report of N. musae causing T. chinensis stem blight in the world. The identification of N. musae could provide a certain theoretical basis for field management and further research of T. chinensis.

Prunus sibirica L. (Siberian apricot) is a member of the Rosaceae family and an ecologically important tree species in China (Buer et al., 2022). Shot hole symptoms on the leaves were observed in five Siberian apricot groves in Chengdu (103.81 E, 30.97 N), Sichuan province in July 2020. The symptoms first appeared as small purplish-brown spots with yellow rings around them. As the disease progressed, the damaged area (diameter 1.5-3.0 cm) became necrotic and fell off. The disease incidence was about 60% and the disease index was 28.6 of leaves in the grove. in most severe cases. Fifteen symptomatic leaves were collected from 5 different trees in an orchard. Pathogen isolation was performed from symptomatic leaf tissue (5 × 5 mm) though surface disinfection (in 70% ethanol and 2% NaClO) and incubation on Potato Dextrose Agar (PDA) at 28℃ for 3 days. Overall 10 isolates with similar colony morphology were obtained from the 15 infected tissue pieces, and three representative isolates (XCK 2-4) were selected for further study. Colonies of the isolates on PDA were initially cottony, pale white to grayish-green with abundant aerial hyphae and produced conidial masses after 7 days. Conidiogenous cells were clavate and aggregated in acervuli. Conidia were smooth-walled, single-celled, straight, and slightly obtusely rounded at both ends, 12.8 to 18.7 × 4.3 to 5.7 μm in size (Fig. 1). The morphological characteristics of the three isolates were consistent with the description of species in the Colletotrichum gloeosporioides complex. DNA was amplified using the following primers pairs for the internal transcribed spacer (ITS) region of rDNA and partial sequences of beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and translation elongation factor (TEF-1), respectively: ITS1/ITS4, T1/Bt2b, GDF/GDR, CHS-F/CHS-R, and EF-F/EF-R (Vieira et al., 2014). Accession numbers (MW228049, MW284974, MW284976, MW284975 and MW284977, respectively) were obtained afterepositing all the resulting sequences in GenBank. Nucleotide blast showed 99 to 100% identities with Colletotrichum fructicola (GenBank accessions nos. MZ961683, MW284974, MN525881, MN525860, MF627961). Phylogenetic analysis of combined ITS-TUB-GAPDH genes using the Mrbayes inference method showed that the three isolates clustered with three reference isolates of C. fructicola as a distinct clade (Fig. 2). To verify Koch’s postulates, ten 3-year-old healthy potted plants of P. sibirica were inoculated by spraying a conidial suspension (6 × 105 conidia/mL) of isolate XCK2 on both sides of leaves, and the control leaves were sprayed with sterile water. Then, all treatments were placed in a moist environment (25±2°C, 80% relative humidity, natural light). The inoculated plants showed typical symptoms of plants with natural infections, while the controls remained asymptomatic after 14 days. The pathogen C. fructicola was re-isolated from all inoculated plants, and the culture and fungus characteristics were the same as those of the original isolate. Colletotrichum fructicola was not isolated from the control plants. The results indicated that C. fructicola is the causal agent of the disease. Colletotrichum fructicola was reported as a leaf pathogen on Camellia chrysantha in China (Zhao et al., 2021). This is the first report of C. fructicola causing P. sibirica leaf shot-hole in the world. The identification of C. fructicola could provide relevant information for applying management strategies and research on the Siberian apricot disease.

Alnus cremastogyne Burk, a broad-leaved tree endemic to south-western China, has both ecological and economic value. The tree is widely used in furniture, timber, windbreaks and sand fixation, and soil and water conservation (Tariq et al. 2018). In December 2020, a new leaf spot disease was discovered on A. cremastogyne in two plant nurseries in Bazhong City (31°15′ to 32°45N, 106°21′ to 107°45′E), with 77.53% disease incidence. Among the infected trees, 69.54% of the leaves were covered with symptoms of the disease. The typical symptoms initially appeared as irregular brown necrotic lesions, while some lesions were surrounded by a light yellow halo. As the disease progressed, the number of necrotic lesions increased, and lesions gradually expanded and coalesced (Fig. 1). Finally, the disease caused the leaves of A. cremastogyne to wither, curl, die, and fall off. Ten symptomatic leaves were collected from 5 different trees in the two plant nurseries. The leaves with symptoms of leaf spot disease were collected and cut from the junction between the diseased and the healthy tissues. The infected tissues from 10 samples were cut into small 2.5 × 2.5 mm pieces. Infected tissues was sterilized in 3% NaClO solution for 60 s followed by 75% ethanol for 90 s, rinsed three times in sterile water, blot-dried with autoclaved paper towels, and then cultured on potato dextrose agar (PDA) at 25℃ for 4 to 8 days in 12 h/12 h light/dark conditions. After 8 days, the colony diameter reached 71.2 to 79.8 mm. The colonies were initially light pink, and then turned white with pale orange beneath. The conidia were single-celled, aseptate, colorless, cylindrical, straight, bluntly rounded at both ends, and measured 11.6 to 15.9 × 4.3 to 6.1 μm (n = 100). These morphological characteristics were consistent with the description of Colletotrichum gloeosporioides (Pan et al. 2021). For molecular identification, the genomic DNA of a representative isolate, QM202012, was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The internal transcribed spacer (ITS), actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified with primers ITS1/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone & Kohn, 1999) and GDF/GDR (Templeton et al. 1992), respectively. Sequences were deposited in GenBank (ITS: OL744612, ACT: OL763390, and GAPDH: OL799166). BLAST results indicated that the ITS, ACT, and GAPDH sequences showed >99% identity with C. gloeosporioides sequences in NCBI (GenBank NR160754, MG561657, and KP145407). Identification was confirmed by Bayesian inference using Mr Bayer (Fig 2) A conidial suspension (1 × 106 conidia/ml) was used to test pathogenicity on the leaves of 4-year-old A. cremastogyne plants (10 plants). Fifteen leaves of each plant (10 pots in total) were inoculated with the spore suspension on the leaves. The same number of control leaves was sprayed with sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 25°C under 16 h/8 h photoperiod and 67 to 78% relative humidity. The symptoms observed on the inoculated plants were similar to those of the original diseased plants, with 100% of the inoculated plants being infested with brown leaf spots, but the controls remained symptom-free. C. gloeosporioides was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times, showing similar results each time, confirming Koch’s postulates. To our knowledge, this is the first report of leaf spot on A. cremastogyne caused by C. gloeosporioides in China. This finding indicates that C. gloeosporioides may become a serious threat to A. cremastogyne production in Bazhong City and helps to further examine and prevent leaf spot disease in A. cremastogyne growing areas in Bazhong City.