First Report of Leaf Spot Caused by Nigrospora coryli on Tobacco in China

The rose apple (Syzygium samarangense (Blume) Merr. & L.M.Perry) plant has been commonly cultivated in Thailand. In May of 2022, leaf spot disease of rose apple was discovered in Chiang Mai Province, Thailand, with approximately 30% disease incidence. The typical symptoms initially showed brown spots (0.1 to 0.5 mm in diameter) with a yellow halo surrounding. These spots then expanded with black edges and the infected leaves appear blighted and desiccated. In humid conditions, pale yellow conidiomata formed on the lesions. Small pieces (5 × 5 mm2) of the margins between lesions and the healthy tissue were surface disinfected with 1% NaClO for 1 min, 70% ethanol for 30 s, and washed three times with sterile distilled water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25 ºC for three days. Three fungal isolates (SDBR-CMU419, SDBR-CMU420, and SDBR-CMU421) were obtained that exhibited similar morphology. Fungal colonies appeared white to gray with cottony mycelia after incubation on PDA at 25 ºC for one week. All fungal isolates produced asexual morph on PDA. Setae were 5590 × 2.53.5 µm, brown with 13-septa, cylindrical base, and tip rounded. Conidiophores were hyaline to pale brown, septate, and branched. Conidiogenous cells were hyaline to pale brown, cylindrical to ampulliform, 2050 µm long (n = 50). Conidia were one-celled, hyaline, smooth-walled, aseptate, straight, cylindrical, end round, guttulate, 1017 × 35 µm (n = 50). Appressoria were mostly formed from mycelia, oval to irregular, brown to dark brown, smooth-walled, 610 × 57 µm (n = 50). Morphologically, all fungal isolates resembled to Colletotrichum (Weir et al. 2012; Jayawardena et al. 2021). The internal transcribed spacer (ITS) region of the ribosomal DNA, actin (act), β-tubulin (tub2), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified using primer pairs ITS5/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), T1/T22 (O'Donnell and Cigelnik 1997), CL1C/CL2C (Weir et al. 2012), and GDF1/GDR1 (Templeton et al. 1992), respectively. The ITS (ON740892 to ON740894), act (ON759242 to ON759244), tub2 (ON759245 to ON759247), CAL (ON759248 to ON759250), and GAPDH (ON759251 to ON759253) sequences were deposited in GenBank. Multi-gene (combined data set of ITS, GAPDH, CAL, act, and tub2) maximum phylogenetic analyses indicated that all fungal isolates clustered with C. siamense ICMP 18578 (type strain) with strong statistical (99% ML) support. For pathogenicity test, asymptomatic leaves, stems and fruits detached from healthy plants were surface disinfected using 0.1% NaClO for 3 min, washed three times with sterile distilled water, and air-dried. A uniform wound (3 pores, 1 mm in width) was made at the equator of each leaf, stem and fruit using aseptic needles. Mycelial plugs (5 mm in diameter) and conidia suspensions (1 × 106 conidia/ml) of each fungal isolate grown on PDA at 25 ºC for one week were used to inoculate both wounded and unwounded samples by the detached method (Huda‑Shakirah et al. 2022; Suwannarach et al. 2022). Plugs of PDA and sterile distilled water were used as controls. Ten replications were performed for each treatment and the experiment was repeated twice. All inoculated samples were incubated in a moist chamber at 25 ºC with 90% relative humidity. The disease severity index was used to evaluate the specimens (Acar et al. 2008; Ngegba et al. 2017). After one week, both wounded and unwounded leaves that inoculated with mycelial plugs and conidia suspensions showed brown leaf spots and a weak infection. Mycelial plugs inoculated on both wounded and unwounded fruits revealed a moderate infection, but inoculation of conidia suspensions showed a weak infection. No symptoms of disease were observed on the inoculated stems. Control leaves, stems and fruits remained asymptomatic. The pathogen C. siamense was re-isolated from spot and rot lesions on PDA in order to fulfill Koch's postulates. Phoulivong et al. (2012) reported that C. siamense is a causal agent of fruit rot in rose apples cultivated in Lao and Thailand. To our knowledge, this is the first report of C. siamense causing leaf spots on rose apple plants in Thailand. Importantly, these findings will provide crucial information for epidemiologic studies and in the development of appropriate management strategies for this newly emerging disease.

Rhododendron simsii (indoor azalea) is widely cultivated for its high ornamental value (Xu et al. 2021). In April to May 2023, a leaf spot disease occurred in a field study at the Baili Azalea Forest Area (27°12'N, 105°48'E), Guizhou Province, China. About 500 plants were investigated, and the results showed that the incidence of leaf spot was 20 ~ 30%. To study this disease, 10 plants showing severe symptoms were collected. Initially, the symptoms were round or irregularly shaped brown spots (1 to 10 mm). With time, the spots enlarged and merged. Symptomatic leaves were washed with sterile distilled water, and 5 × 5 mm pieces of the infected tissues were removed. After surface sterilization (30 s with 75% ethanol, 2 min with 3% NaOCl, then washed three times with sterilized distilled water), the leaf pieces were dried and placed on potato dextrose agar (PDA) and incubated at 25℃ for 5 days. Fungal colonies developed from leaf tissues, and the germinated spores were transferred onto PDA for further purification and morphological observation. Three isolates (GUBJ23, GUBJ24, and GUBJ12) with similar morphology were obtained from five affected leaves. The representative strain GUBJ23 was selected for further study. On PDA the mycelium was initially white but with sporulation turned gray and then black. Black, single-celled conidia, spherical to sub-spherical, from 11.80 to 21.39 × 13.38 to 21.83 μm (n = 50) in diameter were borne singly on hyaline vesicles at the tips of conidiophores. These morphological characteristics were similar to those of Nigrospora sphaerica (Wang et al. 2017). To confirm the identification, primer pairs for the internal transcribed spacer (ITS) region (ITS5/ITS4), β-tubulin (TUB2) (Bt-2a/Bt-2b), and the translation elongation factor 1-alpha (TEF1-α) (EF1-728F/EF1-986R), were used for PCR amplification of DNA from strain GUBJ23 (Carbone and Kohn 1999; Glass et al. 1995; White et al. 1990). The resulting sequences were deposited in GenBank with accession numbers OR818025 (ITS), OR835150 (TUB2), and OR835147 (TEF1-α). BLAST searches of the sequences revealed 99.80% identity (503/504 bp) of the ITS sequence, 100.00% identity (395/395 bp) of the TUB2 sequence, and 100.00% identity of the TEF1-α sequence (241/241 bp) with N. sphaerica LC7294 (accessions KX985932, KY019602, and KY019397, respectively.) Based on a combined dataset of ITS, TEF1-α, and TUB2 sequences, a phylogenetic tree was constructed using the maximum likelihood method and confirmed that isolates GUBJ23, GUBJ24, and GUBJ12 were N. sphaerica (Wang et al. 2017). Leaves of three healthy R. simsii plants were spray-inoculated with a spore suspension (105 conidia/mL), and an additional three plants were sprayed with sterile water. These plants were incubated at 25℃ in 75% relative humidity. After 5 to 7 days of inoculation, 0.5 to 1.8 mm spots appeared on the leaves. At 10 to 14 days after inoculation, grayish brown, semicircular or irregular lesions appeared on the leaves, usually with a diameter of 0.8 to 3 mm. The symptoms were like symptoms seen on naturally infected leaves, while the control leaves remained asymptomatic. The pathogen was re-isolated from diseased leaves and identified by morphological characterization and molecular analyses (ITS, TUB and TEF1-α), and the reisolated pathogen was identical to N. sphaerica. Thus completing Koch's postulates. According to previous research, N. sphaerica is a widely distributed phytopathogenic fungus that has a wide host range (Wang et al. 2017). This study is the first to identify N. sphaerica as the cause of leaf spot disease in R. simsii. Given the popularity of R. simsii as a pot plant and landscape shrub in Asia and othr regions, the occurrence of leaf spot disease seriously affects its ornamental and economic value. Therefore, it is crucial to establish and implement effective disease management practices to reduce impact of the disease.

Polygonatum sibiricum Redouté, a perennial herb in the genus of Polygonatum, is extensively used as a medicinal plant in China. In May 2023, serious leaf spot disease of P. sibiricum with incidence of thirty percent in a 0.4-ha field was observed in the Maojian District of Shiyan City (110°43'51"E 32°32'6"N), Hubei Province, China. Symptoms first appeared as yellowish brown spots throughout the middle portion as well as around the margin of leaves. Lesions often had a brown border and could be surrounded by a yellow halo. Symptoms ranged from a few lesions scattered across leaves to lesions densely covering large sections of leaves. Six samples of symptomatic leaves were cut into 5-mm pieces, surface disinfected with 70% ethanol for 30 s and 1% NaClO for 5 min, washed three times with sterile distilled water, dried on sterilized filter paper and then cultured on potato dextrose agar (PDA) for 3 to 5 days at 26°C. The hyphal tips were cut from the fungal mycelium and transferred to new PDA for further purification. Twelve colonies (HJZ 1-12) with similar morphology were obtained from the purified samples after 7 days. Colonies on PDA were initially grayish white, and then turned Olive gray with concentric zonation. The conidiophores were brown, erect, and geniculate branched with an average of 4.88 μm (n=20) in width. Conidia were oblong or ovate, obtuse round on both sides, straight, 2 to 3 transverse septations and three or four cells with 13.75 to 26.61×6.17 to 9.83 μm (n=50) in size. Morphological characteristics of the representative isolate HJZ-2 and HJZ-3 were consistent with the description of Curvularia sp (Tan et al. 2018). The internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified using primers ITS1/ITS4 and Gpd1-LM/Gpd2-LM (White et al. 1990; Berbee et al. 1999). Sequences were deposited in GenBank with accession numbers PQ269826/PQ732211 for ITS and PQ280046/PQ738619 for GAPDH. BLAST analysis in GenBank showed that the sequences ITS and GAPDH had >99 to 100% nucleotide identities (ITS: 99.18%, 610/608 bp, MG250427.1; 100%, 577/608 bp, MG250427.1; GAPDH: 100%, 509/509 bp MT515338.1; 100% 524/530 bp, MT515298.1) with C. buchloes. Also, the phylogenetic tree based on genes of ITS and GAPDH by the neighbor joining method showed that isolate HJZ-2 and HJZ-3 clustered together with C. buchloes. Based on morphological and molecular characteristics, isolate HJZ-2 and HJZ-3 were determined to be C. buchloes. For the pathogenicity test, fifteen leaves of two-year-old ten healthy P. sibiricum plants were inoculated with a spore suspension (1×106 spores/ml). Sterilized distilled water was used to inoculate the other ten plants as control. All inoculated plants were put in a greenhouse with 80% relative humidity at 25°C. After 5 days, the leaves of inoculated pathogen revealed similar symptoms with those observed in the field and the control group showed no symptoms. The pathogens were reisolation from the infected leaves and identified as C. buchloes by morphological characteristics and sequencing data of ITS and GAPDH genes. The experiments were repeated three times. Previously, C. buchloes was reported causing leaf spots on alfalfa in Pakistan (Haq et al. 2021). This is the first report of C. buchloes causing leaf spots on P. sibiricum in China. This study will provide an important reference for the control of the disease. The epidemiology of this disease should be investigated in further research.

Tobacco (Nicotiana tabacum L.), a broad-leaved annual in the Solanaceae family, is commercially cultivated for its valuable leaves (Chen et al. 2020). In a 0.5-hectare field of tobacco (cv. Yunyan 87) planted in May 2023 in Guizhou Province, China, leaf spot disease was observed with an incidence of 29–37%, based on an assessment of 500 randomly selected plants. Initial symptoms appeared as small, circular or oval white spots (1-2 mm) with distinct brown margins on leaves. As the disease advanced, spots enlarged to 5–8 mm, became sunken, and turned into necrotic lesions with central tissue often collapsing to form irregular holes. A total of seven symptomatic leaves, each collected from different tobacco plants (cv. Yunyan 87), were used for pathogen isolation following standard protocols of the tissue transplanting method. Bits (5 × 5 mm) of leaf tissue cut from the lesion-healthy tissue junctions were surface-sterilized with 75% ethanol for 30 s and 1% sodium hypochlorite for 1 min, rinsed three times with sterile distilled water, and then transferred onto potato dextrose agar (PDA) medium. Following 7 days of incubation at 25°C in the dark, nine isolates with uniform morphology were recovered. On PDA, the colonies were initially white but became dense and developed velvety aerial hyphae within the 7-day period. Subsequently, one isolate, designated wz, was chosen for further study for its representative morphology among all recovered isolates as well as its stable and consistent growth on PDA, ensuring experimental reproducibility (Fig. S1). Genomic DNA of the isolate wz was extracted for molecular identification. The internal transcribed spacer (ITS) region, RNA polymerase II second largest subunit (RPB2), large subunit (LSU), beta-tubulin (TUB2) genes were amplified using primers ITS1/ITS4 (White 1990), fRPB2-5F/fRPB2-7cR (Liu et al. 1999), LROR/LR5 (Rehner and Samuels 1994), and BT2Fd/BT4Rd (Li et al. 2017), respectively. The resulting sequences were deposited in GenBank (accession nos. ITS: PX724618; RBP2: PX753868; LSU: PX745185; TUB2: PX761597). BLAST analysis confirmed that the sequences from isolate wz shared high nucleotide identity with those of Xylaria sp. ZS-2021c isolate 138. The ITS (99.14%), RBP2 (100%), and TUB2 (99.33%) sequences all showed near-complete to complete identity with their respective homologs in isolate 138 (GenBank accessions: MZ648853, PP320372, and MZ695792). Phylogenetic reconstruction based on the concatenated ITS-RBP2-TUB2 dataset further supported the placement of isolate wz within the Xylaria sp. ZS-2021c clad (Zhu et al. 2024) (Fig. S2, Table S1). The pathogenicity of isolate wz was assessed using six healthy tobacco seedlings (cv. Yunyan 87) of five- to six-leaf stage. The leaves were inoculated using a 5-mm mycelial plug. Control plants were treated similarly but with PDA-only plugs. Each treatment was replicated three times. Plants were grown in a high-humidity environment maintained with sterile moistened cotton and incubated in a greenhouse set at 25°C and 80% relative humidity. Disease development was monitored daily. After seven days, all inoculated leaves exhibited leaf spots consistent with field observations, characterized by irregular to circular white spots often surrounded by brown margin. As symptoms advanced, the lesions progressed to necrosis, with central tissue disintegration ultimately resulting in the formation of holes. Control plants remained completely asymptomatic throughout the study. The pathogen was re-isolated from the margins of lesions and confirmed to be morphologically and genetically identical to the original inoculated strain, thereby fulfilling Koch’s postulates. In addition, no asexual or sexual spores were observed despite multiple induction methods, including ultraviolet radiation, host inoculation, temperature variation, and mycelial washing, consistent with some sterile Xylaria isolates (Liu et al. 2008). Xylaria arbuscula has previously been reported as a pathogen causing leaf spot on flue-cured tobacco in China (Xie et al. 2021). To our best of knowledge, this is the first report of Xylaria sp. ZS-2021c causing leaf spot on tobacco in China. This work establishes a critical foundation for subsequent epidemiological investigations, studies of pathogen diversity, and the development of targeted management strategies to safeguard tobacco production.

Bletilla striata (named "Bai Ji" in Chinese) is a plant from the Orchidaceae family that has been employed in traditional Chinese medicine for thousands of years in China. Polysaccharides extracted from B. striata have been shown to have an effect on Alzheimer's disease (Lin et al. 2021). Since 2021, leaf spots have been observed in the B. striata plantation in Chongqing, China. Out of 200 plants, the disease incidence was estimated at 56%, and the disease index was estimated at 32%. The symptoms were necrotic lesions with brown edges and yellow halos; severe infection caused the infected leaves to become blighted, dry and fall off. To identify the causal agent, eighteen leaves with typical symptoms were collected from the B. striata plantation (30.60°N, 108.64°E). The margins of infected tissue areas were cut into small pieces (5×5 mm), surface sterilized with 70% ethanol for 1 min, and rinsed twice with sterile distilled water. The tissue was then surface sterilized in 3% sodium hypochlorite for 2 min, followed by three rinses with sterile water. The tissue was then placed onto potato dextrose agar (PDA) plates and incubated at 25°C for 3 days, pure cultures of fungal isolates were obtained by single-spore isolation, stored on PDA slants and maintained at 4°C. Colonies of the fungal isolates showed three color types, ranging from grayish white to green above with olive green on the reverse, but conidial characteristics were more similar and indicated this was a single fungus. Conidiophores were single, lateral from hyphae or terminal; straight or curved; smooth-walled with 1 to 8 septa; pale brown; usually with only one pigmented terminal conidiogenous site, sometimes with one additional lateral conidiogenous locus; sometimes slightly swollen at the apex; and 15 to 170 μm long, 2.5 to 4.5 μm wide. Conidia were in short or moderately long chains of 2-8 conidia normally, sometimes with more; rarely branched; normally 14.07 to 50 × 5.24 to 10 μm in size; ellipsoid, fusiform, long ellipsoid, obclavate or ovoid with 1 to 11 transverse septa and 2 to 4 longitudinal septa; beakless or with subcylindric or cylindric secondary conidiophores, analogous to the beak 4.25 to 58.6 μm long, 3.2 to 4.8 μm wide. The fungal isolates were tentatively identified as Alternaria sp. The representative isolate BJ8 was selected for the pathogenicity test. The leaves of six healthy plants of B. striata (two years old) grown in pots were washed with sterile water. Ten mL of conidial suspension (1×106 conidia mL-1) contained in 0.05% Tween 80 buffer was brushed onto upper and lower surfaces of all the leaves on three plants, while other plants were brushed with 10 mL 0.05% Tween 80 buffer to serve as controls. Plants were placed in a greenhouse at 25°C and 95±1% relative humidity after inoculation and observed for symptoms. The symptoms initially developed as irregular brown necrotic lesions on the inoculated leaves after 7 days, with a yellow halo around the lesions, consistent with the symptoms in the field. Leaves on the control plants did not produce any symptoms. For molecular identification, the genomic DNAs of representative isolates BJ5, BJ6, and BJ8 were extracted. The internal transcribed spacer (ITS) region and RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-alpha (TEF1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were used for polymerase chain reaction (PCR), using primers ITS5/ITS4, GPD1/GPD2, EF-1F/EF-1B and RPB27cR/RPB25F2, respectively (White et al. 1990; Berbee and Pirseyedi et al. 1999; Carbone and Kohn 1999; Liu et al. 1999). The neighbor-joining tree revealed that these isolates are clustered together with the reference strain of A. burnsii. The sequences were deposited in NCBI GenBank BJ5 [ITS: OP897263; GAPDH: OQ544937; TEF1: OQ544941; RPB2: OQ544939], BJ6 [ITS: OP897262; GAPDH: OQ544938; TEF1: OQ544942; RPB2: OQ544940], and BJ8 [ITS: OK285209; GAPDH: OK340046; TEF1: OK340047; RPB2: OQ544936]. All three isolates showed 100% similarity with A. burnsii CBS 107.38 [ITS: KP124420; GAPDH: JQ646305; TEF1: KP125198; RPB2: JQ646457] ex-type sequence, thus the pathogen causing the leaf spot on B. striata was identified as A. burnsii. A. burnsii is an important pathogenic fungus causing blight of cumin (Shekhawat et al. 2013). Furthermore, Al-Nadabi et al. (2018) found that A. burnsii can cause leaf spots on wheat and date palms, and Sunapao et al. (2022) reported that A. burnsii can infect coconuts (Cocos nucifera), causing dirty panicle disease. This is the first report of A. burnsii causing leaf spot on B. striata in China. The new discovery shows that since A. burnsii can readily adapt to a variety of climatic conditions, controlling the fungus is crucial for the healthy growth of B. striata in the future. This study will provide a basis for further elucidating the pathogenic mechanism and development of effective control measures for this disease.

Farfugium japonicum (L.) Kitam (with the common name leopard plant) is known as a garden and medical herb, and belongs to the family Asteraceae. In May 2019, a leaf spot disease was observed on the upper leaf surface of F. japonicum in Changsha city, Hunan province, China. More than 98% of the F. japonicum plants were infected in a garden of Donghu district (28°13' N; 112°56' E). Leaf symptoms included small (1 to 10 mm in diameter), brown spots that were circular, tan to gray in the center and distinct brownish-yellow margins. Severely affected leaves were blighted and plants were dying. For isolation, symptomatic leaf tissue was surface sterilized, rinsed in sterile distilled water, and plated on potato dextrose agar (PDA) amended with a 50 μg/ml streptomycin sulfate followed by incubation at 25°C in darkness. By a single-spore isolation technique, pure fungal cultures were obtained and displayed gray-brown and gray-white aerial mycelia after five days of incubation. One representative isolate (HnAa-1) was selected for further studies. Conidia of HnAa-1 were olive brown, obpyriform, either branched or unbranched with a short beak, 1 to 5 transverse septa, and 0 to 3 longitudinal or oblique septa. The conidia were 10 to 35 μm long and 2 to 12 μm wide. HnAa-1 was identified as an Alternaria sp. on the basis on morphological characterization by Simmons (1). Further identification to species level was made by molecular analyses. DNA of HnAa-1 was extracted from the regions internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and partial Alt a 1 major allergen (ALT) gene. Amplification and sequencing was carried out with the method described by Woudenberg et al.(2) . BLASTn searches showed that the ITS, GAPDH and ALT sequences had the highest similarity with A. alternata strains, with 100% (548/548) identities for ITS (GQ169728), 100% (567/567) identities for GAPDH (MK903028) and 99.36% (466/469) identities for ALT (MN184998). Moreover, the ITS, GAPDH and ALT sequences had more than 99% identities with the epitype CBS 916.96 of A. alternata (ITS: AF347031; GAPDH: AY278808; ALT: AY563301). The ITS, GAPDH and ALT sequences of HnAa-1 were submitted to GenBank (Accession No. MT767170, No. MW115639 and No. MW316727). Pathogenicity tests were conducted by spraying a 10 ml conidial suspension (1.0 ×105 conidia /mL) on surfaces of leaves of three healthy plants (8-week-old). Leaves of three healthy plants were sprayed with sterile distilled water as a control treatment. All inoculated plants were maintained in growth chamber at 25°C with a 12-h photoperiod. The pathogenicity test was repeated twice. After five days inoculation, typical brown spots and necrotic lesions similar to those observed in the field, had developed on all inoculated plants but not on water-treated control plants. Alternaria alternata was re-isolated from the symptomatic tissue of inoculated plants but not from the control plants, and re-identified with morphological and molecular methods, which fulfilled Koch's postulates. This host-pathogen association has been reported in Korea (3), but it is the first report of A. alternata causing leaf spots on F. japonicum in China. Since A. alternata is a ubiquitous and very important plant pathogen causing leaf spot diseases in over 100 species plant, the occurrence of this disease is a serious threat to F.japonicum and might lead to economic losses. Therefore, appropriate prevention strategies to F.japonicum should be adopted.

Passion fruit (Passiflora edulis Sims), which is native to South America, is an important fruit crop in tropical and subtropical countries. Passion fruit growing areas have increased rapidly in southern China. In 2018 to 2019, circular spots on passion fruit were observed in Shangsi, Guangxi, China (21°15'N, 107°98'E). The disease occurred from June to April of the following year. The disease incidence was generally between 10% to 30%, but could reach up to 50% in purple passion fruit 'Tainong No.1'. The initial lesions on the fruits were small, with a brown center and a greasy margin, and then became sunken and lighter brown with a diameter of about 1 cm in later stages. The spots on the leaves were often surrounded by a yellow halo and turned into larger lesions after coalescence.. Five typical symptomatic fruit and leaves were collected from Shangsi county for the presumed pathogen isolation. Section of the samples were surface sterilized to isolate the fungus on potato dextrose agar (PDA) at 28°C. Five fungal isolates with similar morphology on PDA were obtained by single spore isolation. Colonies at the age of 7 days accompany with flourishing aerial hyphae, showed surface color varying from white to grey. Conidia were ovate or elliptic, light brown to brown, with 2 to 5 diaphragms, 0 to 4 longitudinal-oblique diaphragms, and mostly 8.2 to 36.7 μm × 5.4 to 15.8 μm. The morphology of the fungus resembled Alternaria alternata (Fr.) Keissl (Simmons, 2007). Each of the five isolates (SF-001, SF-002, SF-003, SF-004 and SF-005) was molecularly identified using genomic regions of 18S nrDNA (SSU), 28S nrDNA (LSU), RNA polymerase second largest subunit (RPB2), internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-alpha (TEF1) (Jayawardena et al., 2019). Nucleotide sequences of SSU (MZ275254, ON055696, ON055697, ON055698 and ON055699), LSU (MZ275253, ON062947, ON062948, ON062949, ON062950), RPB2 (MZ275251, ON055377, ON055378, ON055379 and ON055380), ITS (MW866522, MW866523, ON053451, ON053452 and ON053453), GAPDH (MZ286628, ON055381, ON055382, ON055383 and ON055384) and TEF1 (MZ275255, ON055373, ON055374, ON055375 and ON055376) were deposited in GenBank database. The LSU, GAPDH and TEF1 sequences showed 100% identity with A. alternata in NCBI (KX609773, MK683852 and MK637432, respectively). The SSU, RPB2 and ITS sequences showed 99% identity to A. alternata (U05194, MK605898 and MN856409, respectively). In pathogenicity test (Zhang et al., 2020), 3-month-old grafted 'Tainong No.1' seedlings and mature fruit were used. Five-mm-diameter mycelial plugs taken from 7-day-old PDA colonies of each of 5 isolates were placed on the leaves and fruit that were wounded with a sterilized needle to form 3 pinpricks. Sterile PDA plugs were used as control. Three plants and three fruits were used in each treatment, and the test was repeated twice. The inoculated plants and fruit were kept in plastic bags and grown in a chamber at 28℃. Typical lesions were observed on inoculated plants and fruit after 3 days, but the controls remained healthy. A. alternata was consistently reisolated from these typical lesions. Previously, leaf spot on passion fruit caused by A. alternata has only been recorded in New Zealand (Rheinländer, 2010). To our knowledge, this is the first report of A. alternata (Fr.) Keissl. causing leaf spot on passion fruit in China. The identification of the pathogen may help to take effective management strategies of controlling this disease.

In June 2023, yellowish-brown leaf spots with elevated black small spots were observed on tobacco in Zheng'an County, Zunyi City, Guizhou Province, China (107°4′-107°41′E, 28°9′-28°51′N, average altitude 1200 meters). About 20% of tobacco plants in a 20-acre field showed significant growth suppression and yield reduction. The chemical balance of tobacco leaves was disrupted, leading to deteriorated leaf appearance quality, which affected the flavor characteristics and combustion performance of cigarettes, further reducing purchase prices and farmer income. The symptomatic tobacco leaves were collected to isolate the causal agent. Symptomatic tissues were surface-sterilized with 75% ethanol for 45 seconds, rinsed 3 to 4 times with sterile distilled water, and dried with sterile cotton, then transferred to the potato dextrose agar (PDA) plates, which were incubated at 26°C in the dark for 5 to 7 days (Fan et al. 2021). Three cultures were obtained with an isolation frequency of 16% by hyphal transfer method. The fungus produced white colonies on PDA and appeared reddish-brown on the underside of the plates after one week. The conidia with four septa resembled those of Pestalotiopsis trachycarpicola and were spindle-shaped, ranging from 3.16 to 6.39 × 15.22 to 24.01 μm (n = 50). The apical and basal cells were colorless and triangular, while the middle three septal cells were light brown to brown. One filamentous averaging 4.39 to 9.54 μm in length (n = 50) appendage at the apex, and 2 to 3 averaging 9.20 to 15.92 μm (n = 50) appendages at the base. Three loci—internal transcribed spacer (ITS), partial β-tubulin (TUB), and partial translation elongation factor 1-α (EF-1α)—were amplified and sequenced using specific primers for isolated Genomic DNA (Zheng et al. 2023). These sequences have been submitted to GenBank under accession numbers ITS: PP024198, PP648161, PP654438; TUB: PP054320, PP662648, PP662649; EF-1α: PP054321, PP662650, PP662651. The BLAST analysis revealed a sequence homology range of 98.01 to 100% of the strain SVG00116F (accessions: ITS ON238108, TUB OP895026, EF-1α OP895025; Araujo et al. 2023). A phylogenetic tree from the three loci ITS, TUB, and EF-1α was constructed using Maximum Likelihood (ML), and neighborhood connections were made with the sequences of the twelve types of isolates in GenBank, which showed that the three strains clustered in the same clade as Pestalotiopsis trachycarpicola, confirming morphological identification. Pathogenicity tests were conducted twice on healthy tobacco plants with 5-7 leaves. Wound inoculation involved applying 1–3 fungal mycelial plugs (5 mm diameter) per site across three flue-cured cultivars (Yunyan87, K326, Guiyan20). Fungal-free sterile PDA plugs served as controls. The plants were kept in a greenhouse at 28°C and 90% relative humidity. After 10 days, all tobacco except for blank PDA-inoculated tobacco leaves inoculated with all isolates showed necrotic spots, and the spores observed in the lesion were morphologically consistent with P. trachycarpicola. Based on morphological and molecular characteristics, the same pathogen P. trachycarpicola was re-isolated from the inoculated leaves, fulfilling Koch’s postulates. This is the first report of leaf spot caused by P. trachycarpicola in China. The disease spreads widely and causes a decrease in tobacco leaf yield; this first report provides a basis to prevent its future spread and curb further losses.

Tobacco (Nicotiana tabacum L.) is one of the most important cash crops in China. In June 2019, tobacco (cv. Yunyan 87) samples with gray spots surrounded by yellowish ring were collected in Zhengan (107.43° N, 28.55° E), Guizhou province, China. Pieces of leaf tissue (3 mm × 3 mm) that were cut at the junction of diseased and healthy portion were surface sterilized and plated on potato dextrose agar (PDA). After incubation at 25°C in the dark for 7 days, an isolate (T22) was chosen and used for pathogen identification. The colonies had aerial hyphae, initially white and then turned grey, and produced a soluble red pigmen on PDA. The colonies were floccose aerial mycelia, dark grey, with pale brown hyphae, and produced conidia on oatmeal agar. Conidia were ovoid or ampulliform, black, smooth. Based on morphological characteristics, isolate T22 was identified as Nigrospora aurantiaca (Wang et al. 2017). For molecular identification, the large subunit (LSU) and internal transcribed spacer (ITS) of ribosomal RNA, β-tubulin (TUB) and translation elongation factor 1-alpha (TEF1) genes of T22 were amplified by PCR with the primer sets LROR/LR5, ITS1/ITS4, Bt2a/Bt2b and EF1-728F/EF2 (Suwannarach et al.2019), then PCR products were sequenced. Their GenBank accession numbers were MT341787, MT328649, MT348395 and MT348394, respectively. Phylogenetic tree of combined LSU, ITS, TUB, and TEF sequences showed that isolate T22 was assigned to N. aurantiaca strain (CGMCC 3.18130 and LC 7034) with 100% bootstrap support. Based on morphological characteristics and multi-gene molecular analysis, isolate T22 was identified as N. aurantiaca. To fulfill Koch's postulates, PDA plugs grown with N. aurantiaca were placed on the leaves of four tobacco plants (cv. Yunyan 87) at the 10-leaf stage. Leaves inoculated with PDA only plugs served as the controls. Treated plants were maintained in a greenhouse with temperatures ranging from 18 to 28 °C. Five days after inoculation, typical symptoms were observed on inoculated leaves but not on the controls. N. aurantiaca was re-isolated from the diseased leaves but not from the controls. To our best of knowledge, this is the first report of N. aurantiaca causing leaf spot on tobacco in China. N. aurantiaca has been reported to cause leaf spot on Castanea mollissima in China (Luo et al. 2020). Due to potential serious damage caused by the disease in this region, proper disease management practices should be developed and implemented.

Red dragon fruit (Selenicereus undatus (Haw.) Britton & Rose) is an emerging high-value crop in Ecuador, particularly in Manabí Province, where its cultivation provides a profitable alternative to traditional crops. Between April 2018 and March 2023, symptomatic fruits were observed in Rocafuerte and Santa Ana cantons, with an incidence ranging from 7 to 11%. Initial symptoms appeared as small, slightly sunken brown spots that enlarged and turned olive to black, developing a superficial powdery mycelium. Lesions expanded rapidly, causing extensive epidermal decay and abundant sporulation. Five symptomatic fruits were collected from each canton. Fungal isolates were obtained from 9 out of 10 surface-sterilized (70% ethanol, 1% NaOCl) tissue fragments from fruit lesions, which were plated on potato dextrose agar (PDA) and incubated at 28°C for 4 days. Colonies were circular, moderately growing, with abundant cottony gray to dark brown aerial mycelium. Conidia were straight, pale brown, 18–30 × 8–12 µm, with 2–6 transverse septa, characteristic of the genus Curvularia (Manamgoda et al. 2012). Morphological identification was confirmed by sequencing the internal transcribed spacer (ITS), of isolates FP180444 and FP230447, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor 1-alpha (TEF1α) genes of isolate FP180444, using ITS1/ITS4, gpd1/gpd2 and TEF1-983F/TEF1-2218R primers respectively (Ahmadpour et al., 2020). BLAST analysis showed 100% (ITS), 99.7% (TEF1α), and 99.8% (GAPDH) identity with C. cactivora CBS 580.74 (GenBank accession nos. MN688803, MN688857, and LT715853). Sequences were uploaded to GenBank with accession nos. PX485998, PX485999 , PX551765, and PX551766. A multilocus phylogeny based on concatenated ITS, GAPDH, and TEF1α sequences was generated using RAxML v8.2.12 with the GTR+GAMMA model and 100 bootstrap replicates, with Bipolaris sorokiniana CBS 110.14 as the outgroup. The isolates grouped with C. cactivora (Petr.) M.B. Ellis reference strains with a bootstrap support of 100, confirming its identity. To further confirm the identity and explore genomic features, whole-genome sequencing of isolate FP180444 was performed using Illumina NovaSeq. The C.cactivora FP180444 genome is available at NCBI with accession number SAMN52968965. Pathogenicity was confirmed by inoculating five-month-old healthy plants and fruits with 0.5 ml of a 1 × 10⁵ conidia/ml suspension from monoconidial cultures from isolates FP180444 and FP230447, by syringe infiltration. Controls were treated with sterile distilled water. After 15 days at 28°C and 77% relative humidity, inoculated fruits and cladodes developed typical brown to black lesions, while controls remained symptomless. Three plants and three fruits were used per isolate, and the experiment was conducted twice. The fungus reisolated from infected tissues was confirmed as C. cactivora by microscopic observation and ITS sequencing, fulfilling Koch’s postulates. To our knowledge, this is the first report of Curvularia cactivora causing fruit rot of red dragon fruit in Ecuador, and the first genome assembly of a C. cactivora isolate. Given the increasing commercial importance of dragon fruit in the country, the presence of this pathogen may pose a risk to yield, fruit quality, and market access, while the genome resource will facilitate future studies on pathogenicity, host adaptation, and disease management.

Photinia × fraseri Dress, belonging to the Rosaceae family, is widely cultivated as an ornamental plant in China. In July 2022, the leaf spot symptoms were observed on over thirty P. × fraseri plants in an approximately 2-hectare park in Xinjian District, Nanchang City, Jiangxi Province, China (28°43′02″ N, 115°44′01″ E), with a disease incidences of roughly 10% . At first, small, grayish-white lesions appeared on the leaf edges, later expanding into 2 to 10 mm circular or irregular spots. These spots turned grayish-white to brown, with dark brown margins. Eventually, some lesions’ centers dried and died. For fungal isolation, ten symptomatic leaves were randomly collected. The edges between the diseased and healthy tissues were cut into small pieces (4 × 4 mm). These pieces were then surface-sterilized by dipping in 70% ethanol for 30 s and 1% NaClO for 30 s. Subsequently, they were rinsed three times with sterile distilled water. Leaf pieces were then transferred to potato dextrose agar (PDA) medium and incubated at 25 °C for 3–4 days. Eight isolates with similar colony morphology were collected from diseased leaves. Colonies of this fungus on PDA were nearly round, white, and had sparse aerial mycelium on the surface with black, gregarious conidiomata. The conidia were nearly cylindrical, smooth, hyaline, and 4-septate, measuring 16.7 to 24.3 × 4.2 to 6.6 µm (mean 20.9 × 5.3 µm, n=50). The three middle cells were smooth, doliiform, and brown, with concolorous septa that were darker than the rest of the cell. They measured 11.8 to 17.0 µm long (mean 14.1 µm, n=50). The basal and apical cells were triangular and transparent. The basal cells had a mean length of 4.7 µm and were equipped with a basal appendage, while the apical cells had two appendages with a mean length of 17.7 µm(n=50). The characteristics of these isolates match those of Pestalotiopsis species (Maharachchikumbura et al. 2014). To identify them accurately, three representative isolates, namely JFRL 03-161, JFRL 03-162, and JFRL 03-226, were selected for further analysis. The internal transcriptional spacer (ITS) region, β-tubulin (TUB2) and translation elongation factor 1-alpha (TEF1-α) gene were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), BT2a/BT2b (Glass and Donaldson 1995), and EF1-526F/EF1-1567R (Maharachchikumbura et al. 2012), respectively. All sequences (ITS: OR342044-OR342046, TUB2: OR343299-OR343301, and TEF1-α: OR343302-OR343304) were deposited in GenBank. A BLASTn homology search revealed 99-100% identity to Pestalotiopsis nanjingensis CSUFTCC16 (ex-type). The sequences included ITS (OK493602, 486/486 nucleotides), TUB2 (OK562377, 438/439 nucleotides), and TEF1-α (OK507972, 478/478 nucleotides). The maximum likelihood analyses were performed for the combined ITS, TUB2 and TEF1-α data sets using IQtree web server (Trifinopoulos et al. 2016). The resulting phylogenetic tree demonstrated a strong association: the three isolates clustered tightly with P. nanjingensis forming a clade with robust 99% bootstrap support. This clustering, consistent with both morphological and molecular characteristics, confirmed the identity of the fungus as P. nanjingensis. To evaluate its pathogenicity, we obtained 3-year-old P. × fraseri ‘Red Robin’ plants, which were purchased then potted in a controlled climate chamber. We surface sterilized six healthy leaves of P. × fraseri with 70% ethanol and created wounds using a sterile needle. Subsequently, we inoculated a 50 μL conidial suspension (1 × 106 conidia/mL) of the isolate JFRL 03-161 on these wounded leaves. In parallel, another six leaves from P. × fraseri were inoculated with sterile distilled water, serving as the control group. All potted plants were incubated under conditions of 26 °C and 80% humidity. After seven days, all leaves inoculated with isolate JFRL 03-161 displayed symptoms similar to those observed in the field, whereas the control leaves remained unaffected. To fulfill Koch’s postulates, we re-isolated P. nanjingensis plants from the symptomatic leaves and identified it based on morphological and molecular characteristics. It has been reported that two species of Pestalotiopsis, namely P. microspora and P. trachicarpicola can caused damage to the leaves of P. × fraseri in China (Xu et al. 2022; Zhu et al. 2021). However, to our best knowledge, this is the first report on leaf spot caused by P. nanjingensis on P. × fraseri in China. Therefore, it is necessary to pay more attention to the leaf spot disease of P. × fraseri caused by Pestalotiopsis species and develop appropriate control strategies.

Bitter gourd (Momordica charantia L.), a member of the Cucurbitaceae family, is an economically important vegetable crop cultivated in India, valued for its nutritional and medicinal properties (Mallikarjuna et al. 2023). In May 2024, leaf spot disease symptoms were observed on bitter gourd in Dakshin Charilam (23.630 N, 91.320E), Sepahijala, Tripura, India with disease incidence of leaf spot ranged from 6% to 11% (n = 100 plants) in a 1-ha field. Symptoms included small, water-soaked, angular, gray or straw-colored spots with a yellow halo. As the disease progressed, spots became dry and dropped leaving irregularly shaped holes in the leaves. Ten symptomatic diseased leaves were randomly collected from 10 individual plants and cut into small pieces (5.0 mm2) at the junction between diseased and healthy tissue using a sterilized scalpel. The pieces were disinfected with 1% sodium hypochlorite (NaOCl) solution for 2 min and subsequently samples were washed thoroughly with sterilized distilled water three times. Disinfected tissue was transferred to Petriplates containing potato dextrose agar (PDA) and incubated at 25±2°C. Out of ten symptomatic leaves, eight isolates with similar morphological characterizations were isolated and purified. Two isolates (Bitter G-3 and Bitter G-3′) were selected for further morphological and molecular identification. After 7 days at 25±2ºC, colonies on PDA were fast growing, dark greyish black, with hyaline to olivaceous grey, septate, smooth-walled, 3–6.5 (x̄ = 4) μm wide, sporulating hyaline form. Conidia were aseptate, ovoid to ellipsoid (6.2 ± 1.01 × 14.5 ± 0.96 μm; n = 20). Genomic DNA was extracted from 7 days old freshly harvested mycelia using a fungus genomic DNA extraction kit (Biomiga, U.S.A.) and the partial internal transcribed spacer (ITS) region and translation elongation factor 1-alpha (tef1-α) gene were amplified, sequenced with primer pairs ITS4 / ITS5 (White et al. 1990) and EF1- 983F/EF1-2218R (Rehner and Buckley 2005), respectively, and submitted to GenBank (GenBank accession nos. PV248196, PV962840 for ITS and PV274565, PV976015 for tef1). BLASTn searches with the obtained sequences in GenBank revealed 99.79 % identity [466/467 bp; 100% Query Cover; and 0 gaps] with L. theobromae CBS 164.96 (Type; Sequence ID: NR_111174.1) based on ITS gene region and 100 % identity [314/314 bp; 100% Query Cover; and 0 gaps] with L. theobromae isolate CUZ044 (Sequence ID: PV441873.1) based on tef1 gene. A phylogenetic tree was constructed by MEGA11.0 using the Neighbor-Joining method (Saitou and Nei, 1987). Based on morphology and molecular analysis, the fungus was identified as L. theobromae. For pathogenicity test, twelve healthy leaves of three healthy bitter gourd plants were inoculated with a 5-mm-diameter mycelial disc and an additional three healthy plants inoculated with sterile PDA discs as control (He et al. 2021). After 5 days of inoculation, L. theobromae inoculated leaves of bitter gourd exhibited leaf spot symptoms similar to those observed in the field, while the control plants had no symptoms. The pathogen was reisolated (isolate Tri BG-3) from the symptomatic leaves in three successive trials, and identified as L. theobromae, thus fulfilling Koch’s postulates. DNA sequence for ITS and tef1-α gene were submitted to GenBank (GenBank accession nos. PV962841 for ITS and PV976016 for tef1). To the best of our knowledge, this is the first report of L. theobromae causing leaf spot in bitter gourd in India and it is crucial to monitor the occurrence of this pathogen in other bitter gourd growing areas as a potential threat to bitter gourd production.

Acorus calamus var. angustatus Besser, a perennial herb of the Araceae family, was first reported in the ShenNong'sHerbalClassic and is widely distributed in southern China (Li 1979). It is important in traditional Chinese medicine for treating heart, stomach, and brain ailments (Lam et al. 2016). In March 2024, leaf spots were found on its leaves in a traditional Chinese medicine planting base in Yuexi County (30°91'56″ N, 116°19'24″ E), Anhui Province, with an incidence of about 35%. The symptoms began as small, light brown lesions that expanded, resulting in necrotic lesions ranging from 1 to 8 mm in diameter with brown halos. 3 × 3 mm sections, including both symptomatic and asymptomatic tissues, were cut from six infected plants. They were disinfected in 75% ethanol for 30 s, washed three times with sterile distilled water, transferred to Petri dishes containing potato dextrose agar (PDA) and incubated at 25 °C in the dark. Purified fungal isolates were obtained by the single-spore isolation method. The colonies on PDA initially appeared white, gradually became olive-green with 1 to 3 mm white margins and abundant aerial hyphae, while the reverse was greyish green to black. The conidia were light brown, ellipsoidal, obclavate, and 10.0 to 49.5 µm × 4.5 to 16.4 µm (mean 23.9 × 10.2 µm, n=50) in size, with 0 to 6 transverse septa and 0 to 6 longitudinal or oblique septa (n=50). Conidiophores were thick, dark brown, single-celled, with multiple conidial scars, measuring 12.8 to 146.8 × 2.9 to 6.1 (mean 50.6 × 4.2) µm (n=50). Based on above observations, the pathogen were identified as Alternaria spp. (Simmons 2007). Three representative isolates, SCP-1, SCP-2, and SCP-3 were selected for molecular identification. The Internal transcribed spacer (ITS), Alternaria major allergen (Alt a 1), and translation elongation factor 1-alpha (TEF1) genes were amplified with the primers ITS1/ITS4 (White et al. 1990), Alt-for/Alt-rev (Woudenberg et al. 2015) and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. The sequences of the three isolates were consistent, and the sequences of isolate SCP-1 were submitted to NCBI GenBank (ITS, PP723104; Alt a1, PP708704; TEF1, PP708703). The ITS region of isolate SCP-1 was 100% similar to A. alternata TCS3002 (MN394880, Wang et al. 2023), the Alt a 1 gene was 100% similar to A. alternata CBS 620.83 (KP123868), and the TEF1 gene was 100% similar to A. alternata CBS 916.96 ex-type (KC584634). A phylogenetic tree based on sequences of ITS, Alt a 1 and TEF1 genes was constructed using the neighbor-joining method in MEGA 7 software, confirming the fungus as A. alternata. Three healthy plants of A. calamus var. angustatus were spayed with conidial suspension (1 × 107 conidia/ml) of isolate SCP-1. Three additional plants sprayed only with sterile distilled water were controls. All plants were covered with plastic bags to maintain a relative humidity of 90% for 48 h and incubated at 25 °C under a 12-h light photo-period. Twelve days post-inoculation, brown necrotic lesions developed on the inoculated leaves, enlarged, and the symptoms were similar to the original ones. The control plants remained healthy. The fungus was re-isolated from the infected plants and confirmed by morphological traits and molecular methods, fulfilling Koch's postulates. To our knowledge, there are no other reports of this fungus on A. calamus var. angustatus in Anhui, China. This report will help identify the disease based on field symptoms and provide a basis for disease management strategies of A. calamus var. angustatus.

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae) is traditionally used for hemostasis and detumescence in China. In April 2019, a leaf spot disease on B. striata was observed in plant nurseries in Guilin, Guangxi Province, China, with an estimated incidence of ~30%. Initial symptoms include the appearance of circular or irregular brown spots on leaf surfaces, which progressively expand into large, dark brown, necrotic areas. As lesions coalesce, large areas of the leaf die, ultimately resulting in abscission. To isolate the pathogen, representative samples exhibiting symptoms were collected, leaf tissues (5 × 5 mm) were cut from the junction of diseased and healthy tissue, surface-disinfected in 1% sodium hypochlorite solution for 2 min, rinsed three times in sterile water, plated on potato dextrose agar (PDA) medium, and incubated at 28°C (12-h light-dark cycle) for 3 days. Hyphal tips from recently germinated spores were transferred to PDA to obtain pure cultures. Nine fungal isolates with similar morphological characteristics were obtained. Colonies on PDA were villose, had a dense growth of aerial mycelia and appeared pinkish white from above and greyish orange at the center and pinkish-white at the margin on the underside. Macroconidia were smooth, and hyaline, with a dorsiventral curvature, hooked to tapering apical cells, and 3- to 5-septate. Three-septate macroconidia were 21.2 to 32.1 × 2.4 to 3.9 μm (mean ± SD: 26.9 ± 2.5 × 3.2 ± 0.4 μm, n = 30); 4-septate macroconidia were 29.5 to 38.9 × 3.0 to 4.3 μm (mean ± SD: 33.5 ± 2.6 × 3.6 ± 0.3 μm, n = 40); and 5-septate macroconidia were 39.3 to 55.6 × 4.0 to 5.4 μm (mean ± SD: 48.0 ± 3.9 × 4.5 ± 0.3 μm, n = 50). These morphological characteristics were consistent with F. ipomoeae, a member of the Fusarium incarnatum-equiseti species complex (FIESC) (Wang et al. 2019). To confirm the fungal isolate's identification, the genomic DNA of the single-spore isolate BJ-22.3 was extracted using the CTAB method (Guo et al. 2000). The internal transcribed space (ITS) region of rDNA, translation elongation factor-1 alpha (TEF-1α), and partial RNA polymerase second largest subunit (RPB2) were amplified using primer pairs [ITS1/ITS4 (White et al. 1990), EF-1/EF-2 (O'Donnell et al. 1998), and 5f2/11ar (Liu, Whelen et al. 1999, Reeb, Lutzoni et al. 2004), respectively]. The ITS (MT939248), TEF-1α (MT946880), and RPB2 (MT946881) sequences of the BJ-22.3 isolate were deposited in GenBank. BLASTN analysis of these sequences showed over 99% nucleotide sequence identity with members of the FIESC: the ITS sequence showed 99.6% identity (544/546 bp) to F. lacertarum strain NRRL 20423 (GQ505682); the TEF-1α sequence showed 99.4% similarity (673/677 bp) to F. ipomoeae strain NRRL 43637 (GQ505664); and the RPB2 sequence showed 99.6% identity (1883/1901 bp) to F. equiseti strain GZUA.1657 (MG839492). Phylogenetic analysis using concatenated sequences of ITS, TEF-1α, and RPB2 showed that BJ-22.3 clustered monophyletically with strains of F. ipomoeae. Therefore, based on morphological and molecular characteristics, the isolate BJ-22.3 was identified as F. ipomoeae. To verify the F. ipomoeae isolate's pathogenicity, nine 1.5-year-old B. striata plants were inoculated with three 5 × 5 mm mycelial discs of strain BJ-22.3 from 4-day-old PDA cultures. Additionally, three control plants were inoculated with sterile PDA discs. The experiments were replicated three times. All plants were enclosed in transparent plastic bags and incubated in a greenhouse at 26°C for 14 days. Four days post-inoculation, leaf spot symptoms appeared on the inoculated leaves, while no symptoms were observed in control plants. Finally, F. ipomoeae was consistently re-isolated from leaf lesions from the infected plants. To our knowledge, this is the first report of F. ipomoeae causing leaf spot disease on B. striata in China. The spread of this disease might pose a serious threat to the production of B. striata. Growers should implement disease management to minimize the risks posed by this pathogen.

Flue-cured tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves in China. Around 70% of tobacco production in China occurs in southwest China. In summer of 2019, leaf spot symptoms were observed on ten to twenty percent of tobacco plants in a 2 ha commercial field of Bijie (27.32° N, 105.29° E), Guizhou province, China. The leaf spots were white with dark-brown in edges, irregularly round and oval, and diseased tissue dropped out leaving the leaves ragged in appearance (Fig. 1A, 1B). One diseased leaf from each of five plants was sampled. From five leaves, a total of 15 small (5 mm × 5 mm) pieces of leaf tissue were cut from the edge of the lesions after surface sterilization and placed on potato dextrose agar (PDA) medium. Five fungal colonies that were similar in appearance were isolated and one was purified, BEZ22, was selected arbitrarily for identification. Mycelia of the pathogen was initally white and dense, and then black carbonized mycelia appeared from the center of the colony 7 days' after incubation. Mycelia was white, sparse and radiated when incubated on OA (oatmeal agar) (Fig. 1E, 1F, 1G, 1H). Genomic DNA of the isolate was extracted. The internal transcribed spacers (ITS) with primers ITS1/ITS4 (White et al. 1990), actin (ACT) gene with primers ACT-512F/ACT-738R (Hsieh et al. 2005), beta-tubulin (TUB2) with primers T1/T22 (O'Donnell & Cigelnik 1997) and RNA polymerase II second largest subunit gene (RPB2) with primers fRPB2-5F/ fRPB2-7cR (Liu et al. 1999) were amplified and sequenced, respectively. The generated sequences were deposited in GenBank with accession numbers MT804353 (ITS), MT809582 (ACT), MT799790 (TUB2) and MT799789 (RPB2). Using BLASTN searches, the sequences of each gene above were aligned with the voucher specimum, Xylaria arbuscula 89041211. The number of nucleotides that were similar for ITS (GU300090) was 550/551 (99%); for ACT (GQ421286), 266/266 bp (100%); for TUB2 (GQ478226), 1501/1501 bp (100%); and for RPB2 (GQ844805), 1135/1135 bp (100%), respectively (Fig. 2). A phylogenetic tree was constructed based on these four sequences with a final alignment of 3456 characters (ITS 551, ACT 266, TUB2 1501 and RPB2 1138). Thus, based on morphological and phylogenetic analyses, the isolate BEZ22 was identified as Xylaria arbuscula. To verify pathogenicity, six tobacco plants at seedling stage (5-6 leaves) without visible disease were inoculated using mycelial plugs (5 mm in diameter). Leaves inoculated with PDA only plugs served as controls. After inoculation, all tobacco plants were maintained in a greenhouse with 85% relative humidity at 25 oC under a 12/12 h light/dark cycle. Five days after inoculation, typical early symptoms were observed on the inoculated leaves, and not on the control leaves. Koch's postulates were fulfilled by re-isolation of the pathogen from diseased leaves. Xylaria arbuscula has also been reported as a pathogen of Macadamia in Hawaii (Wenhsiung et al. 2009) and sugarcane in Indonesia (Maryono et al. 2020). However, to our best knowledge, this is the first report of X. arbuscula causing leaf spot on tobacco in China. This leaf spot has the potential to cause serious damage to tobacco in this region that could result in reduced production, consequently disease management of this pathogen should be considered.