First report of Alternaria alternata causing leaf spot on Toona ciliate in China.

Toona ciliate is an excellent timber and ornamental tree cultivated in China (Li et al. 2018). In May 2018, a leaf spot disease was observed on the foliage of T. ciliate in Nanchang city, Jiangxi province. Disease incidence averaged approximately 40%. Initial symptoms were small, brown spots with yellow halos, then the spots gradually enlarged and coalesced to form large lesions. To identify the pathogen, thirty pieces (5 × 5 mm) from the lesion margins were surface sterilized in 70% ethanol (30 s), then in 3% NaOCl (1 min), and finally rinsed three times with sterile water. The pieces were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation. Fourteen strains with similar morphological characters were isolated, and three representative isolates (MT-2, MT-5, MT-8) were used for morphological and molecular characterization. The colonies on PDA were gray to brown after 7 days. Ovoid or elliptical conidia were brown to light-brown in color with a short beak, 1-5 diaphragms, and 0-3 mediastinum. The diameter of these conidia were thick (18.2-47.4×7.9-15.1 µm, n= 100). The morphological characteristics of three isolates matched those of Alternaria sp. with straight or curved primary conidiophores with obclavate, long ellipsoid conidia (Woudenberg et al. 2013). The internal transcribed spacer (ITS) regions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), small subunit (SSU), large subunit (LSU), RNA polymerase second largest subunit (RPB2), translation elongation factor 1-alpha (TEF1) (Woudenberg et al. 2013) and Alternaria major allergen gene (Alt a 1) (Woudenberg et al. 2014) were amplified by using the following primer pairs ITS1/ITS4, GPD-1/GPD-2, NS1/NS4, LR0R/LR05, RPB2-5F2/fRPB2-7cR, EF1-728F/EF1-986R and Alt-f/Alt-r, respectively. The sequences were deposited in GenBank (ITS: ON459540, ON459541, ON459542; GAPDH: ON427936, ON427937, ON427938; SSU: ON422107, ON422108, ON422109; LSU: ON422110, ON422111, ON422112; RPB2: ON427939, ON427940, ON427941; TEF1: ON427933, ON427934, ON427935; Alt a 1: ON427942, ON427943, ON427944). A maximum likelihood and Bayesian posterior probability-based analyses using IQ-tree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, GAPDH, SSU, LSU, RPB2, TEF1, Alt a 1) placed three isolates in the clade of Alternaria alternata (Fr.) Keissl. The three isolates were identified as A. alternata based on morphological and molecular characteristics. For pathogenicity tests, 10 T. ciliate plants (two leaves each, n=20) grown outdoors were pin-pricked with a sterile needle and inoculated with a drop of spore suspension (106 conidia per mL) in July. Another 20 healthy leaves were inoculated with sterile water as the control. All the inoculated leaves were wrapped with plastic bags to keep them moist for 2 days. The pathogenicity tests were repeated twice. The resulting symptoms were similar to those on the original infected plants, whereas the control leaves remained asymptomatic for 10 days after inoculation. The same fungus was re-isolated from the lesions, confirming Koch's postulates. The pathogen was previously reported to cause leaf spots on Aquilegia flabellata (Garibaldi et al. 2022), Chrysanthemum morifolium (Luo et al. 2022), Liriodendron chinense × tulipifera (Jin et al. 2021) and so on. To our knowledge, this is the first report of A. alternata associated with leaf spot disease on T. ciliate in China. This disease may potentially decrease the value of ornamental T. ciliate plants under favorable conditions and proper management strategies should be applied.

First report of Neopestalotiopsis clavispora causing leaf spot on Liquidambar formosana in China.

Liquidambar formosana Hance, a deciduous tree, is widely cultivated in China for its ornamental and afforestation value (Yin et al. 2021). In July 2019, leaf spot symptoms were observed with 20 to 30% disease incidence in Li shan forest farm (27°19'27.2″N, 115°32'51.08″E) in Ji'an city, Jiangxi province, China. Initial disease symptoms were small spots, which enlarged and circular to irregular, gray in the center, and dark brown to black circular on the lesion margin. 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 (Si et al. 2022). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation, and the representative isolates, FX-2, FX-5, and FX-9 were used for morphological studies and phylogenetic analyses. The colonies of three isolates on PDA grew fast, covering the entire plate with white cottony mycelia with black acervuli after 8 to 10 days. Conidia were 5-celled, clavate to fusiform, smooth, 19.6-24.2 × 6.2-8.5 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 (3.5-8.2 µm long; n = 100), and 2-3 apical appendages (18-31 µm long; n = 100), filiform. Morphological features were similar to Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). The internal transcribed spacer (ITS) regions, ß-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, ON622512- ON622514; TUB2, ON676532 - ON676534; TEF1-α, ON676529 - ON676531). 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 FX-2, FX-5, and FX-9 in the clade of N. clavispora. Based on the multi-locus phylogeny and morphology, three isolates were identified as N. clavispora. To confirm pathogenicity, 10 healthy 2-year-old seedlings, and 5 leaves per seedling were wounded with a sterile needle (Φ=0.5 mm) and inoculated with 200 µL conidial suspension per leaf(106 conidia/mL). Ten control plants were inoculated with ddH2O. All the inoculated leaves were covered with plastic bags and kept in a greenhouse at 26 ± 2 °C and RH 70%. All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 8 days. N. clavispora was reisolated from the lesions, whereas no fungus was isolated from control leaves. N. clavispora can cuase leaf diseases in a variety of hosts, including × Taxodiomeria peizhongii (Zhang et al. 2022), Macadamia integrifolia (Qiu et al. 2020), Dendrobium officinale (Cao et al. 2022). N. cocoes, N. chrysea, Pestalotiopsis neglecta and P. neolitseae were also reported to infect L. formosana (Fan et al. 2021). However, this is the first report of N. clavispora infecting L. formosana in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

First report of Colletotrichum siamense causing leaf spot on Celtis sinensis in China.

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), ß-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.

First report of Neopestalotiopsis clavispora causing leaf spot on Photinia bodinieri in China.

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, ß-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.

First report of Colletotrichum siamense causing leaf spot on Macropanax rosthornii in China.

Macropanax rosthornii (Harms) C. Y. Wu ex Hoo is an evergreen broadleaf species cultivated in subtropical China as an ornamental (Liang et al. 2015). In August 2020, leaf spot symptoms were observed on the campus of Jiangxi Agricultural University (28°45'56″N, 115°50'21″E), Jiangxi province, China. The early symptoms were small spots on the edge or tip of the leaves. The spots gradually expanded and became grayish brown with reddish egdes, eventually developing large irregular lesions. The disease incidence was estimated at 45%. Leaf pieces (5 × 5 mm) from the lesion borders were surface disinfested in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water (Li et al. 2023). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C in the dark. Three representative single-spore isolates (DS-2, DS-3, and DS-5) were used for morphological studies and phylogenetic analyses. Colonies on PDA of the three isolates were white-to-gray with cottony mycelia. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 14.3-18.1 ×4.3-6.9 µm (15.8 ± 1.1 × 5.3 ± 0.2 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.6-9.4 × 4.5-6.9 µm (7.7 ± 0.3 × 5.5 ± 0.2 µm, n=100). Morphological features were similar to the Colletotrichum gloeosporioides species complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, calmodulin (CAL), actin (ACT), ß-tubulin 2 (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and chitin synthase (CHS-1) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, CL1/CL2, ACT-512F/ACT-783R, T1/Bt2b, GDF/GDR and CHS-79F/CHS-354R (Weir et al. 2012), respectively. Sequences were deposited in GenBank under nos. OL895315 - OL895316 (ITS), OL830190 - OL830192 (ACT), OL830181 - OL830183 (GAPDH), OL830178 - OL830180 (TUB2), OL830184 - OL830186 (CHS-1), and OL830187 - OL830189 (CAL). 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 DS-2, DS-3, and DS-5 in the clade of C. siamense. Based on the multi-locus phylogeny and morphology, three isolates were identified as C. siamense. Pathogenicity of the three isolates was verified on six 5-year-old Macropanax rosthornii plants, which were grown in the field. Three healthy leaves per plant were wounded using a sterile needle (Φ=0.5 mm) and inoculated with a 20-µL conidial suspension per leaf (106 conidia/mL). Another six control plants were treated with sterile water. Eighteen leaves were used for the pathogenicity test of three isolates. All leaves were covered with plastic bags to maintain humidity for 2 days. The inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic after 8 days. The fungi were consistently reisolated only from the inoculated and symptomatic leaves, fulfilling Koch's postulates. C. siamense can cause leaf diseases in a variety of hosts, including Liriodendron chinense × tulipifera (Zhu et al. 2019), Salix matsudana (Zhang et al. 2021), Carya illinoinensis (Zhuo et al. 2023). However, this is the first report of C. siamense infecting Macropanax rosthornii in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

First report of Colletotrichum siamense causing leaf spot on Machilus pauhoi in China.

Machilus pauhoi Kaneh. is an excellent evergreen broad-leaved tree species widely grown in China for its ornamental and economic value (He et al. 2022). In September 2021, a leaf spot was observed on M. pauhoi plants on Guantian forest farm (27°06'15.6″N, 114°34'20.72″E) in ji' an city, Jiangxi province, China. The disease incidence was estimated to be above 20%. The symptoms began as brown irregular spots, then the spots gradually expand over time, with a gray-to-brown center and dark brown-to-black edges. Small infected tissues (3 to 5 mm2) were surface-sterilized in 70% ethanol for 30 s and 2% NaClO for 60 s, and rinsed three times with sterile water (Ju et al. 2021). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty-two isolates of Colletotrichum ssp. were obtained (isolation frequency about 78%). Three representative single-spore isolates (PN-1, PN-4, and PN-9) were used for morphological studies and phylogenetic analyses. Colonies on the PDA 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 11.4-16.8 ×4.1-5.5 µm (13.2 ± 1.0 × 4.4 ± 0.3 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.2-8.8 × 4.1-6.2 µm (6.7 ± 0.2 × 5.1 ± 0.3 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), beta-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. All sequences were deposited into GenBank (ITS, ON176154 - ON176156; ACT, ON185554 - ON185556; GAPDH, ON185563 - ON185565; TUB2, ON185566 - ON185568; CHS-1, ON185560 - ON185562; CAL, ON185557 - ON185559). 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 PN-1, PN-4, and PN-9 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 nine M. pauhoi plants, which were grown in the field. Healthy leaves were wounded with a sterile needle and inoculated with 10 µL of spore suspension (106 conidia/mL). The spore suspension of each isolate was inoculated onto six leaves. Another three plants inoculated with ddH2O served as the control (Wan et al. 2022). All the inoculated leaves were covered with plastic bags to keep them moist for 2 days (relative humidity > 80%). All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 7 days. C. siamense was reisolated from the lesions, whereas no fungus was isolated from control leaves. Up to now, Pestalotiopsis chamaeropis, Corynespora cassiicola and Arthrinium arundinis could infect M. pauhoi plants (Zhang et al. 2021), and cause leaf spots in China. To our knowledge, this is the first report of C. siamense causing leaf spots on M. pauhoi. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

First report of anthracnose on Acer fabric caused by Colletotrichum siamense in China.

Acer fabri Hance, an evergreen tree, is widely cultivated in China for its ornamental value (Lin. 2020). In July 2020, a leaf spot disease, with an incidence of Approximately 48% (12 out of 25), was observed on A. fabri plants (almost 9-year-old) at the campus of Jiangxi Agricultural University (28°45'56″N, 115°50'21″E). On average, 30% of the leaves per individual tree were affected. Small spots initially formed along the edge or tip of the leaves and gradually expanded into dark brown spots, and eventually the diseased leaves withered. 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 (Wan et al. 2020). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation, and the representative isolates, LFY-1, LFY-5, and LFY-8 were used for morphological studies and phylogenetic analyses. Colonies on PDA 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 12.8-17.4 ×4.3-5.7 µm (14.3 ± 1.1 × 4.6 ± 0.4 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.6-9.3 × 4.7-6.6 µm (7.4 ± 0.3 × 5.5 ± 0.4 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), beta-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. All sequences were deposited into GenBank (ITS, OL818322- OL818324; ACT, OL830175 - OL830177; GAPDH, OL830166 - OL830168; TUB2, OL830163 - OL830165; CHS-1, OL830169 - OL830171; CAL, OL830172 - OL830174). 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 LFY-1, LFY-5, and LFY-8 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 A. fabri plants, which were grown in the field. Healthy leaves were wounded with a sterile needle and inoculated with 10 µL of spore suspension (106 conidia/mL). The spore suspension of each isolate was inoculated onto five leaves. Another three plants inoculated with ddH2O served as the control (Si et al. 2019). All the inoculated leaves were covered with plastic bags to keep a high-humidity for 2 days. 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. The pathogen was previously reported to cause anthracnose on Kadsura coccinea (Jiang et al. 2022), Carica papaya (Zhang et al. 2021), Michelia alba (Qin et al. 2021). This study is the first to report C. siamense causing anthracnose on A. fabric. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

First report of Colletotrichum siamense causing leaf spot on Manglietia decidua in the world.

Manglietia decidua Q. Y. Zheng is a deciduous broad-leaved plant and native to Jiangxi province, China. It is cultivated for its timber and urban landscaping (Xiong et al., 2014). In September 2019, a new foliar disease was observed on approx. 25% of 121 M. decidua trees in Jiangxi Agricultural University (N28°45'56″, E115°50'21″), Nanchang city, Jiangxi Province, China. The disease site belongs to the subtropical monsoon humid climate, with rainfall (1,600-1,700 mm) and red soil region. Initially, infection appeared on the leaf margins or tips as water-soaked, irregular lesions, then expanded to the center, developed into large black-brown, irregular necrotic lesions. Finally, the lesions fall off the leaves. To identify the pathogen, 15 diseased leaves were collected from 5 trees (3 leaves per tree) randomly. Small pieces (5 × 5 mm) cut from the lesion margins were surfaced sterilized (70% ethanol for 30 s, 3% NaOCl for 1 min, rinsed 3 times with sterile water), and placed on potato dextrose agar (PDA) at 25 °C. Among the isolated fungi, Colletotrichum-like colonies were about 91%, and 18 monoconidial isolates were obtained. Isolates HML-1, HML-4, and HML-7 were selected and preserved for further studies. Colonies on PDA were white, cottony, and grayish-white on the reverse side. Setae absent. Acervuli were brown, circular. Conidiophores were clear, septate, non-branching or branching at the base, conidiogenous cells were enteroblastic, phialidic, colorless, cylindrical, ampulliform. Conidia were elliptical, single-celled, straight, hyaline, and measured 13.3-17.9 × 4.3-5.7 µm (14.8 ± 1.2 × 4.8 ± 0.4 µm, n = 100). Appressoria were oval to irregular, dark brown, and ranged from 5.3-9.1 × 4.4-6.3 µm (7.2 ± 0.3 × 5.1 ± 0.2 µm, n=100). Morphological characteristics matched the description of Colletotrichum gloeosporioides sensu lato (Weir et al. 2012). The internal transcribed spacer regions (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), chitin synthase (CHS-1), calmodulin (CAL), and beta-tubulin 2 (TUB2) were sequenced (Weir et al., 2012), and deposited in GenBank (ITS: OL757565-OL757567; ACT: OL627398-OL627400; CHS-1: OL757358-OL757360; GAPDH: OL757361-OL757363; CAL: OL757355-OL757357; TUB2: OL757364-OL757366). Six loci were concatenated, and the aligned sequences (2056 bp) were 99.9%, 99.8% homologous to C. siamense ICMP 18574 and ex-type ICMP18578, respectively. In the maximum-likelihood phylogenetic tree, the highest log likehihood was -9259.74, and 3 isolates were in the C. siamense clade. Based on the phylogeny and morphology, 3 isolates were identified as C. siamense. The pathogenicity of 3 isolates was tested on 12 M. decidua plants (variety: Yi lin ke) grown in the field. Healthy leaves were wounded slightly with a needle (Φ=0.5 mm) and inoculated with 10 µL of spore suspension (106 conidia/mL). Controls were treated with ddH2O (Si et al. 2021). All the treated leaves were covered with plastic bags to keep a high-humidity environment for 2 days. The experiments were repeated twice. Within 9 days, all the inoculated points showed similar symptoms to those observed in the field, whereas controls were asymptomatic. The same isolate was re-isolated from the lesions, whereas no fungus was isolated from control leaves. Manglietia decidua is an ancient and endangered plant, threatened with southern blight (Sclerotium rolfsii) (Yi et al. 2021a), root rot (Calonectria ilicicola) (Yi et al. 2021b). This is the first report of the newly emerging disease caused by C. siamense in the world. The potential threat should be evaluated for conservation in the future. This study provided crucial information for epidemiological studies and appropriate control strategies.