Nageia nagi (Thunb.) Kuntze is widely cultivated in China for its ornamental and economic value. In August 2019, a leaf spot was observed on N. nagi plants at the campus of Jiangxi Agricultural University (28°45′56″N, 115°50′21″E). Disease incidence was about 35%, and the diseased leaf rate was above 40%. The early symptoms were small spots on the edge or tip of the leaves. The spots gradually expanded and became reddish-brown, eventually developing large irregular lesions. Leaf pieces (5 × 5 mm) from the lesion borders were surfaced sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C (Zhang et al. 2021). Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty-six isolates of Colletotrichum ssp. were obtained (isolation frequency about 82%). Three representative single-spore isolates (ZB-1, ZB-3, and ZB-7) were used for morphological studies and phylogenetic analyses. Colonies on PDA medium of the three isolates were white to gray in color with cottony mycelia. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 14.1-17.9 ×4.4-6.8 µm (15.6 ± 1.2 × 5.4 ± 0.3 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.7-9.3 × 4.6-6.9 µm (7.8 ± 0.2 × 5.6 ± 0.3 µm, n=100). Morphological features were similar to Colletotrichum siamense complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), β-tubulin 2 (TUB2), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, T1/Bt2b, CHS-79F/CHS-354R and GDF/GDR (Weir et al. 2012), respectively. Sequences of them deposited in GenBank under nos. OL826760 - OL826762 (ITS), OL830205 - OL830207 (ACT), OL830196 - OL830198 (GAPDH), OL830193 - OL830195 (TUB2), OL830199 - OL830201 (CHS-1), and OL830202 - OL830204 (CAL). A Blast search of GenBank showed that ITS, ACT, GAPDH, TUB2, CHS-1, and CAL sequences of the three isolates were identical to Colletotrichum siamense at a high level (Table 1). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed ZB-1, ZB-3, and ZB-7 in the clade of C. siamense. Based on the multi-locus phylogeny and morphology, three isolates were identified as C. siamense. The pathogenicity of three isolates was tested on six N. nagi plants (three for inoculation, three for controls), which were grown in the field. Six healthy leaves were wounded with a sterile needle and inoculated with 10 µL of conidial suspension (1 × 106 conidia/mL) per plant. Healthy leaves were inoculated with ddH2O as a control by the same method. All the inoculated leaves were covered with plastic bags to keep a high-humidity environment for 2 days. The experiment was repeated three times. All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 8 days. C. siamense was reisolated from the lesions, whereas no fungus was isolated from control leaves. Up to now, Cephleuros virescens, Pestalotiopsis longisetula, Alternaria tenuissima, A. alternate, and Phoma glomerata could infect N. nagi (Zhou et al. 2015; Zhang et al. 2016), and cause leaf spots in China. To our knowledge, this is the first report of C. siamense causing leaf spots on N. nagi worldwide. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

Phoebe bournei (Hemsl.) Yang is a typical evergreen broadleaf species widely distributed in subtropical China for its ornamental and economic value (Zhang et al. 2021). The wood of P. bournei is considered a good material for architectural decoration and furniture (Li et al. 2018). In June 2020, leaf spot symptoms were observed in Dexing (28°41′22.056″N, 115°51′52.524″E), Jiangxi province, China. Initial disease symptoms were small brown spots on the leaves. Then, the spots enlarged and coalesced into regular or irregular dark brown necrotic lesions with dark margins. Disease incidence in the field in Dexing was estimated 25%. Leaf pieces (5 × 5 mm) from the lesion borders were surface-sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C under a 14/10 h light/dark cycle for 4 days. Pure cultures were obtained by monosporic isolation, and the representative isolates, JX-N2, JX-N7, and JX-N11 were used for morphological studies and phylogenetic analyses. The colonies of three isolates grown on PDA were white, cottony, and flocculent, contained undulate edges with dense aerial mycelium on the surface. Conidia were 5-celled, clavate to fusiform, smooth, 18.7-24.6 × 5.9-8.8 μm (n = 100). The 3 median cells were dark brown to olivaceous, central cell was darker than other 2 cells, and the basal and apical cells were hyaline. All conidia developed one basal appendage (3.4-8.3 μm long; n = 100), and 2-3 apical appendages (17-30 μm long; n = 100), filiform. Morphological features were similar to Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). The internal transcribed spacer (ITS) regions, β-tubulin 2 (TUB2) and translation elongation factor 1-alpha (TEF1-α) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, T1/Bt-2b, EF1-728F/EF-2 (Maharachchikumbura et al. 2014), respectively. All sequences were deposited into GenBank (ITS, OQ355048 - OQ355050; TUB2, OQ357665 - OQ357667; TEF1-α, OQ362987 - OQ362989). A maximum likelihood and Bayesian posterior probability-based phylogenetic analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, TUB2, TEF1-α) placed JX-N2, JX-N7, and JX-N11 in the clade of N. clavispora. Based on the multi-locus phylogeny and morphology, the representative isolates were identified as N. clavispora. The pathogenicity of three isolates were tested on six 9-year-old P. bournei plants, which were grown in the field. Three leaves per plant were wounded with a sterile needle (Φ=0.5 mm) and inoculated with 20 μL conidial suspension per leaf (106 conidia/mL). Another six control plants were inoculated with sterile water. Each leaf was covered with plastic bags to keep a humidity environment for 2 days. All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 9 days. N. clavispora was reisolated from the lesions, whereas no fungus was isolated from control leaves. N. clavispora can cause leaf diseases in a variety of hosts, including Machilus thunbergii (Wang et al. 2019), Fragaria × ananassa (Shi et al. 2022), Taxus media (Li et al. 2022). However, this is the first report of N. clavispora infecting P. bournei in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

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Celtis sinensis Pers., a deciduous tree, is widely cultivated in China for its ornamental value (Yang et al. 2022). In July 2020, leaf spot symptoms were observed on Ce. sinensis plants at the campus of Jiangxi Agricultural University (28°45′56″N, 115°50′21″E) in Nanchang city, Jiangxi province, China. The disease incidence was estimated to be above 15%. The early symptoms were small spots on the edge or tip of the leaves. The spots gradually expanded and became grayish brown, eventually developing large irregular lesions. Leaf pieces (5 × 5 mm) from the lesion borders were surfaced and sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation, and the representative isolates, JPS-4, JPS-9, and JPS-13 were used for morphological studies and phylogenetic analyses. Colonies on PDA medium of the three isolates were white to gray with cottony mycelia and grayish-white on the undersides of the culture. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 14.3-18.2 ×4.3-6.9 µm (15.8 ± 1.1 × 5.3 ± 0.4 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.6-9.4 × 4.5-6.8 µm (7.6 ± 0.1 × 5.4 ± 0.2 µm, n=100). Morphological features were similar to Colletotrichum gloeosporioides species complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), β-tubulin 2 (TUB2), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, T1/Bt2b, CHS-79F/CHS-354R and GDF/GDR (Weir et al. 2012), respectively. All sequences were deposited into GenBank (ITS, ON207804 - ON207806; ACT, ON239113 - ON239115; GAPDH, ON239122 - ON239124; TUB2, ON239125 - ON239127; CHS-1, ON239119 - ON239121; CAL, ON239116 - ON239118). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed JPS-4, JPS-9, and JPS-13 in the clade of C. siamense. Based on the multi-locus phylogeny and morphology, three isolates were identified as C. siamense. To confirm pathogenicity, nine 6-year-old Ce. sinensis plants (three leaves each, n=27) grown outdoors were pin-pricked with a sterile needle and inoculated with 100 μL spore suspension per leaf (106 conidia per mL). Another 27 healthy leaves were inoculated with sterile water as the control. All the inoculated leaves were covered with plastic bags to keep a high-humidity environment for 2 days. The experiment was repeated three times. All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 8 days. Colletotrichum siamense was reisolated from the lesions, whereas no fungus was isolated from control leaves. Colletotrichum siamense can cause leaf diseases in a variety of hosts, including Allamanda cathartica (Huang et al. 2022), Osmanthus fragrans (Liu et al. 2022), and Crinum asiaticum (Khoo et al. 2022). To our knowledge, this is the first report of C. siamense causing leaf spots on Ce. sinensis worldwide. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

Photinia bodinieri Lévl. is an evergreen broadleaf species widely cultivated in subtropical China as an ornamental value (Zhang et al. 2018). In July 2021, leaf spot symptoms were observed on the campus of Jiangxi Agricultural University (28°45′56″N, 115°50′21″E), Jiangxi province, China. The spots were circular to irregular, gray in the center, and dark brown on the lesion margin. The disease incidence was estimated 15%. Leaf pieces (5 × 5 mm) from the lesion borders were surface-sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C in the dark. Pure cultures were obtained by monosporic isolation, and the representative isolates, SN-3, SN-7, and SN-11 were used for morphological studies and phylogenetic analyses. The colonies of three isolates grown on PDA were white, cottony, and exhibited flocculent, contained undulate edges with dense aerial mycelium on the surface. Conidia were 5-celled, clavate to fusiform, smooth, 18.2-24.3 × 5.5-8.4 μm (n = 100). The 3 median cells were dark brown to olivaceous, central cell was darker than other 2 cells, and the basal and apical cells were hyaline. Conidia developed filiform appendages; one basal appendage (3.3-8.2 μm long; n = 100), and 2-3 apical appendages (16-29 μm long; n = 100). Morphological features were similar to Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). Portions of internal transcribed spacer (ITS) regions, β-tubulin 2 (TUB2) and translation elongation factor 1-alpha (TEF1-α) genes were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, T1/Bt-2b, EF1-728F/EF-2 (Maharachchikumbura et al. 2014), respectively. All sequences were deposited into GenBank (ITS, OQ572345 - OQ572347; TUB2, OQ597847 - OQ597849; TEF1-α, OQ597844 - OQ597846). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed SN-3, SN-7, and SN-11 in the clade of N. clavispora. Based on the multi-locus phylogeny and morphology, three isolates were identified as N. clavispora. Pathogenicity of the three isolates was verified on nine disease-free 7-year-old Photinia bodinieri plants, which were grown in the field. Two healthy leaves per plant were wounded with two pricks using a sterile needle (Φ=0.5 mm) and inoculated with 20 μL conidial suspension per leaf (106 conidia/mL). Another nine control plants were inoculated with sterile water. 36 leaves were used for the pathogenicity test of three isolates. All leaves were covered with plastic bags to maintain a humid environment for 2 days. The inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic after 10 days. The fungi were consistently reisolated only from the inoculated and symptomatic leaves, fulfilling Koch’s postulates. N. clavispora can cause leaf diseases in a variety of hosts, including Kadsura coccinea (Xie et al. 2018), Photinia serratifolia (Yang et al. 2018), Camellia chrysantha (Zhao et al. 2020). Photinia spp. is an excellent landscape gardening plant, threatened with grey blight (Pestalotiopsis microspore) (Ye et al. 2022), anthracnose (Colletotrichum sp.) (Guan et al. 2013). However, this is the first report of N. clavispora infecting Photinia bodinieri in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.