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.

Chinese fir (Cunninghamia lanceolata) is an important timber species that has been widely cultivated in southern China. It is extensively applied in medicine, environmental monitoring, furniture, urban (e.g., street trees) and rural landscaping, windbreak forest, soil and water conservation. In January 2022, distinct leaf spot symptoms were observed in Chinese fir in Hongya Forestry (29°45′N, 103°11′E) Meishan City, Sichuan Province, China. Field surveys showed that the disease was widespread, with around 70% disease incidence. The typical symptoms initially appeared as yellowish-brown necrotic lesions on the margin of the leaves. Subsequently, lesions gradually expanded and developed into larger necrotic areas with red-brown irregular shape. The lesions later expanded throughout the leaf. Infected leaves turned dark brown and wilted, leading to seeding’s death. Diseased leaves with typical symptoms were collected for pathogen isolation and identification. Infected tissues from ten samples were cut into small pieces of 2 × 2 mm. Infected tissues were surface disinfected with 3% sodium hypochlorite and 75% ethanol for 30s and 60s, respectively, and rinsed with sterile water 3 times. They were blotted dry with autoclaved paper towels and incubated on potato dextrose agar (PDA) with streptomycin sulfate (50 μg/mL) for 5 ~ 8 days at 25°C. and 12 h light/dark period. The diameter of the colonies reached 65.7 to 75.9 mm, with a gray to black center, and white edges while the reverse sides were gray to orange. Conidia were single-celled, colorless, straight, cylindrical, bluntly rounded at both ends, Conidia dimensions varied from, 7.3 μm to 15.7 μm in length and 3.3 μm to 6.1 μm in width (n = 100). For molecular identification, the genomic DNA of isolate SM2290708, SM229070801 and SM229070802 were extracted using a fungal genomic DNA extraction kit (Beijing Solarbio Science & Technology Co., Ltd., City, China). The internal transcribed spacers of the ribosomal RNA (ITS) [ITS1/ITS4 (White et al., 1990), calmodulin (CAL) (Weir et al., 2012), β-tubulin (TUB2) (O’Donnell et al., 1997), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Templeton et al. 1992) were amplified. Sequences were deposited in GenBank (ITS: ON564877, OQ535027 and OQ535028; CAL: ON583827, OQ538101 and OQ538102; TUB2: ON583830, OQ538104 and OQ538105; and GAPDH: ON583831, OQ538108 and OQ538109). BLAST results showed that our ITS, CAL, TUB2 and GAPDH sequences were >99% identical to the corresponding sequences of Colletotrichum kahawae deposited at NCBI (GenBank JX010231, JX009642, JX010444, and JX010012). Identification was confirmed by Bayesian inference using MrBayes (Fig 2). The conidial suspension (1 × 106 conidia/ml) was used for inoculation by spraying leaves of ten 3-year-old Chinese fir plants for pathogenicity test. Fifteen leaves of each plant were inoculated. An equal number of control leaves was sprayed with sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 28°C under a 16 h/8 h photoperiod and in 73% to 79% relative humidity. After fifteen days, the symptoms observed on the inoculated plants were similar to those of the original diseased plants, but the controls remained asymptomatic. Colletotrichum kahawae was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times, which showed similar results, confirming Koch’s postulates. To our knowledge, this is the first report of brown leaf spot on C. lanceolata caused by C. kahawae in China. The results of this study provide basic information for diagnosis of the pathogen and developing prevention strategies to manage C. lanceolata leaf spot disease.

Prunus serrulate Lindl is widely cultivated in urban areas of China. It is mainly used for wood cultivation and urban landscaping. In May 2021, new leaf spot disease was observed in Chengdu city (30°42′ to 30°45′N, 103°51′ to 104°7′E), with 69.3% disease incidence, which could inhibit leaf growth and reduce their biomass. A planting area of more than 1000 square meters was investigated. The diseased leaves were mostly concentrated in the lower position of plants, where the humidity was higher. The disease infected P. serrulata leaves and occurred in the field from March to October, with the highest incidence in early May. The typical symptoms initially appeared as brown necrotic lesions on the margin of the leaves. The lesions then enlarged gradually and developed into reddish brown spots, eventually coalescing into large irregular, necrotic lesions with dark brown margins. Finally, the diseased leaves withered and died. Conidiomata were not formed on the diseased tissue. Ten symptomatic leaves were collected from 5 different trees in the planting area. Infected tissues from ten samples were cut into small pieces of 3 × 3 mm. The infected tissues were surface-sterilized by 3% sodium hypochlorite and 75% ethanol respectively for 30s and 60s, and rinsed three times in sterile water. Then they were blot-dried with autoclaved paper towels and cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/mL), and incubated at 25°C for 4 to 8 days. After culturing for 8 days at 25℃ and 12 h/12 h light/dark on PDA, the colony diameter reached 67.5 to 78.6 mm. The colonies were initially white, cottony, then became light pink to misty rose at the center, and the reverse side of the colony turned dark red to red and had pale yellowish borders. The conidia were straight, smooth-walled, colorless, fusiform with acute ends, measuring 8.2 to 16.7 × 3.1 to 5.9 μm in size (n = 100). For molecular identification, the genomic DNA of the representative isolate RBWY202105 was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The internal transcribed spacer (ITS) [ITS1/ITS4 (White et al., 1990)], histone3 (HIS3) [CYLH3F/CYLH3R (Crous et al. 2004)], chitin synthase (CHS-1) [CHS-79F/CHS-345R (Carbone & Kohn, 1999)], actin (ACT) [ACT512F/ACT (Carbone & Kohn, 1999)], β-tubulin (TUB2) [BT2A/BT2B (O’Donnell et al., 1997)], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [GDF/GDR (Templeton et al. 1992)] were amplified. Sequences were deposited in GenBank (ITS: ON000436, HIS3: ON014581, CHS-1: ON014579, ACT: ON014583, TUB2: ON014582, and GAPDH: ON014580). BLAST results indicated that the ITS, HIS3, CHS-1, ACT, TUB2 and GAPDH sequences showed >99% identity with Colletotrichum fioriniae (Marcelino & Gouli) R.G. Shivas & Y.P sequences at NCBI (GenBank MW497230 (561/582), MT740312 (415/415), KU736865 (258/258), MK680659 (246/246), MK967342 (757/757), and MW656269 (263/263)). The conidial suspension (1 × 106 conidia/ml) was used for inoculation by spraying leaves of ten 4-year-old P. serrulata plants for pathogenicity test. Fifteen leaves of each plant were inoculated with spore suspensions on the leaves (600 μl per leaf). The same amount 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 a 16 h/8 h photoperiod and 67 to 78% relative humidity. After ten days, the symptoms observed on the inoculated plants were similar to those of the original diseased plants, but the controls remained asymptomatic. Colletotrichum fioriniae was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis (The ITS, HIS3, TUB2, CHS-1, GAPDH and ACT genes). The pathogenicity test was repeated thrice, which showed similar results, confirming Koch’s postulates. To our knowledge, this is the first report of brown leaf spot on P. serrulata caused by C. fioriniae in China. The identification of C. fioriniae could provide relevant information for taking management strategies and further research on the Prunus serrulata disease.

Jacaranda mimosifolia D. Don is widely cultivated in southwest China (Yunnan, Sichuan, and other regions). It is widely applied in papermaking, medicine, environmental monitoring, timber, urban and rural afforestation, and soil and water conservation. In October 2020, a new brown leaf spot disease of J. mimosifolia was discovered in Xichang City (27°49′ to 27°56′N, 102°16′ to 102°11′E), with approximately 66.23% disease incidence. Firstly, the typical symptoms showed deep yellow necrotic lesions in the center or on the margin of the leaves. Gradually, the necrotic lesions expanded and developed into brown spots. Under humid conditions, the edges of necrotic lesions turned dark brown progressively. Finally, the leaves withered, died, and fell off. Infected tissues from ten samples were cut into small pieces of 2.5 × 2.5 mm. The surfaces of infected tissues were sterilized for 30 s in 3% sodium hypochlorite, 60 s in 75% ethanol, and rinsed three times in sterile water. They were then blot-dried with autoclaved paper towels and cultured on potato dextrose agar (PDA) at 25℃ for 3 to 8 days. After culturing for 8 days at 25℃ and 12 h/12 h light/dark on PDA, the colony diameter reached 78.2 to 82.7 mm. The colonies were light orange, turned pale pink with light orange beneath. The conidia were single-celled, aseptate, cylindrical, smooth-walled, straight, hyaline with both ends bluntly rounded, measuring 12.3 to 16.8 × 4.3 to 5.6 μm (n = 100; average=14.5 × 5.1μm). These morphological characteristics were consistent with the description of C. karstii (Zhao et al. 2021). For molecular identification, the genomic DNA of the representative isolate JM202010 was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The internal transcribed spacer (ITS) [ITS1/ITS4 (White et al., 1990)], calmodulin (CAL) [CL1C/CL2C (Weir et al., 2012)], actin (ACT) [ACT512F/ACT-783R (Carbone & Kohn, 1999)], chitin synthase (CHS-1) [CHS-79F/CHS-345R (Carbone & Kohn, 1999)], β-tubulin (TUB2) [BT2A/BT2B (O’Donnell et al., 1997)], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [GDF/GDR (Templeton et al. 1992)] were amplified. Sequences were deposited in GenBank (ITS: OL454787, CAL: OL518966, ACT: OL518967, CHS-1: OL518968, TUB2: OL518969, and GAPDH: OL518970). BLAST results indicated that the ITS, CAL, ACT, CHS-1, TUB2 and GAPDH sequences showed >99% identity with Colletotrichum karstii sequences at NCBI (GenBank MW494453.1, MW495036.1, MG387951.1, MW495038.1, MW495042.1, and MG602034.1). The conidial suspension (1 × 106 conidia/ml) was sprayed on the leaves of 4-year-old J. mimosifolia plants (10 plants) and inoculated for pathogenicity test. Fifteen leaves of each plant (10 pots in total) were inoculated with spore suspensions on both sides of the leaves. An equal number of control leaves was sprayed with sterilized distilled water as a control. Finally, all pots were kept in a greenhouse at 26°C under a 16 h/8 h photoperiod and 60 to 68% relative humidity. The inoculated plants showed symptoms similar to those of the original diseased plants, but the controls remained asymptomatic. Colletotrichum karstii was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated thrice, which showed similar results, confirming Koch’s postulates. To our knowledge, this is the first report of brown leaf spot on J. mimosifolia caused by C. karstii in China. C. karstii was previously reported as the causal agent of anthracnose on Fatsia japonica (Xu et al. 2020) and Nandina domestica (Li et al. 2017) in China. This finding provides an important basis for further research on the control of this disease.

The Pharbitis purpurea (L.) Voisgt, a member of the Convolvulaceae, is a graceful plant with an air purifying function and ornamental values. It is often cultivated in parks and roadsides. In April 2021, leaf spots (with approximately 67.9% disease incidence) were observed on P. purpurea grown in Xichang city (27°49′N; 102°16′E). More than 1000 square meters of planting area were investigated. Initially, yellowish-brown spots were of different sizes with a yellow irregular border, and slightly sunken necrotic lesions. Gradually, the necrotic lesions expanded and developed into brown spots that often coalesced and expanded to cover the entire leaves. Finally, the leaves wilted, died and fell off. For fungal isolation, infected tissues from ten samples were cut into small pieces of (2.5 × 2.5 mm) sterilized with 3% NaOCl for 30 s and 75% ethanol for 60 s, rinsed three times with sterilized water, blot-dried and cultured on potato dextrose agar (PDA) at 25°C in dark for 8 days. After culturing for 8 days, the colony diameter reached 75.2 to 79.7 mm. The pure colonies were grayish-white with pale yellowish borders and grayish black and pale yellowish borders on the reverse side. The conidia were hyaline, single-celled, cylindrical, smooth-walled, subcylindrical with obtuse to slightly rounded ends, measuring 11.6 to 17.9 × 3.7 to 5.8 μm (n = 100; average=14.7 × 4.9μm). These morphological characteristics were consistent with the description of Colletotricum siamense (Zhang et al. 2021). For molecular identification, the genomic DNA of the representative isolate LBH202104 was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). Partial of internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified using the primers ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, and GDF/GDR, respectively (Weir et al. 2012). BLAST results of obtained sequences (ITS: OM948680, ACT: OM959361, CAL: OM959366, and GAPDH: OM959364), showed >99% identity with C. siamense sequences (MN305712, MZ461478, MK141754, and MK361203) in GenBank. Based on morphology and phylogenetic analysis, the representative isolate was identified as Colletotrichum siamense (Fig. S1&S2). For pathogenicity test, the conidial suspension (1 × 106 conidia/ml) was sprayed on the leaves of 4-year-old eight potted P. purpurea plants. Fifteen leaves of each plant were inoculated. For negative controls, 8 plants were sprayed with sterilized distilled water. Finally, all pots were kept in a greenhouse at 26°C under a 16 h/8 h photoperiod and 68 to 75% relative humidity. The inoculated plants showed symptoms similar to those of the original diseased plants, while controls remained asymptomatic. C. siamense cultures were re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated thrice, which showed similar results, confirming Koch’s postulates. To our knowledge, this is the first report of leaf spot caused by C. siamense on P. purpurea worldwide. The identification of this pathogen provides a foundation for the management of Leaf spot in P. purpurea.