First Report of Leaf Spot Caused by Phyllosticta styracicola on Nageia nagi in China.

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.

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.

Peanut (Arachis hypogaea) is an important economic and oil crop in China. In September 2022, leaf spots wereobserved on peanut in Luoyang city, Henan province, China (34°49'N, 112°37'E). The disease occurred on about 30% ofthe peanutleaves in only one 0.5-acre field. Symptoms appeared primarily as brown spots, that varied in shape, and appeared round, oval or irregular. In addition, some disease patches exhibited a concentric ring pattern. Small pieces (5×5 mm) of five diseased leaves were surface disinfected in 3% NaClO for 2 minutes, rinsed three times in sterile distilled water, dried on sterilized filter paper, and cultured on potato dextrose agar (PDA) at 25°C for 3 days. Five isolates with uniform characteristics were obtained and subcultured by transferring hyphal tips to fresh PDA. The colonies of the isolates were circular and the margins were clean. The colonies showed white coloration, and after 5-7 days of incubation on PDA plates, concentric rings with dark green sporodochia appeared on the surface of the colonies. The conidiophores branched repeatedly. The conidiophore stipes unbranched, hyaline, 10.0 to 23.2×1.5 to 3.3 μm (n=50). The conidia were rod-shaped or long oval and single-celled, measuring 4.6 to 8.6×1.4 to 3.1 μm (n=100). Based on these characteristics, the five isolates were identified as Paramyrothecium foliicola (Lombard et al 2016). Genomic DNA was extracted from the representative isolates LH-1-1 and LH-1-2. The internal transcribed spacer (ITS), RNA polymerase II second largest subunit (RPB2), calmodulin (CmdA), and translation elongation factor 1-alpha (tef1) loci were amplified and sequenced using the following primer pairs: ITS1/ITS4 (White et al. 1990), RPB2-5F2/RPB2-7cR (O'Donnell et al. 2007), CAL-228F/CAL-2Rd (Carbone & Kohn 1999), and EF1-728F/EF2 (O'Donnell et al. 1998), respectively. BLASTn analysis revealed that the sequences of ITS (OR352397.1 and OR417392.1), RPB2 (OR413573.1 and OR420678.1), CmdA (OR413572.1 and OR420677.1), and tef1 (OR413574.1 and OR420679.1) had 99 to 100% (553/558 bp, 721/721 bp, 597/598 bp, and 384/389 bp) similarity to P. foliicola (MN593634.1, MN398038.1, OM801785.1, MK335967.1). A phylogenetic tree based on the Maximum Likelihood method also confirmed that the two isolates converge on the same branch as P. foliicola. Pathogenicity tests were performed using leaves of 60-day-old peanut plants (cv. Zhonghua 8). Briefly, uninfected healthy leaves (non-wounded) were inoculated with 30-µl drops containing a spore suspension (5×105 conidia/ml) of LH-1-2, and peanut leaves inoculated with sterile distilled water served as controls. All treatments were incubated in an incubator at 25℃ and high relative humidity with a 12:12 hour light-dark cycle. After 5-7 days, inoculated leaves showed symptoms similar to those observed in the field, while no symptoms were observed on control leaves. The pathogenicity tests were repeated three times. The fungus was reisolated from the infected leaves and identified as P. foliicola based on morphological and molecular characteristics, thus fulfilling Koch's postulates. P. foliicola has previously been reported to cause leaf spot of tomato and mung bean, stem canker of cucumber (Huo et al. 2022; Sun et al.2020; Huo et al. 2021). To our knowledge, this is the first report of P. foliicola causing leaf spot on peanut in the world. Identification of this pathogen will be helpful in monitoring peanut diseases and developing disease control strategies.

Daphne odora Thunb. an evergreen shrub with scented flowers, is used for ornamental purposes but it also has medicinal benefits (Otsuki, et al. 2020). In August 2021, leaf blotch symptoms were observed on roughly 20% of leaves of D. odora var. marginata plants in Fenghuangzhou Citizen Park, Nanchang city (28°41'48.12″ N, 115°52'40.47″ E), Jiangxi Province, China. Brown lesions first appeared on the edges of leaves, which eventually dried and died (Fig. 1A). For fungal isolation, 12 symptomatic leaves were randomly collected, the edges between diseased area and healthy area were cut into small pieces (4×4 mm), surface-sterilized by dipping in 70% ethanol for 10 s and 1% sodium hypochlorite for 30 s, and then rinsed three times with sterile distilled water. Leaf pieces were then plated on potato dextrose agar (PDA) and incubated at 28 ℃ for 3-4 days. A total of 10 isolates were recovered from the diseased leaves. The pure colonies of all fungal isolates had similar characteristics, and three isolates were randomly selected (JFRL 03-249, JFRL 03-250 and JFRL 03-251) for further study. Colonies of this fungus were gray and uneven, with a granular surface, and irregular white edges, finally turning black on PDA (Fig. 1B, C). Pycnidia were black, globose and 54-222 μm in diameter (Fig. 1D). Conidia were hyaline, single-celled, and nearly elliptical, which ranged from 7 to 13 × 5 to 7 μm (n=40) (Fig. 1E). These morphological characteristics were consistent with those described for the fungus Phyllosticta spp. (Wikee et al. 2013a). To confirm the fungal identity, the internal transcribed spacer (ITS) region, actin (ACT), translation elongation factor 1-alpha (TEF1-a), glyceradehyde-3-phosphate dehydrogenase (GPD) and RNA polymerase II second largest subunit (RPB2) genes were amplified using primers ITS5/ITS4, ACT-512F/ACT-783R, EF-728F/EF2, Gpd1-LM/Gpd2-LM and RPB2-5F2 /fRPB2-7cR, respectively (Wikee et al. 2013b). The sequences of the selected isolates were 100% identical. Hence, sequences of one representative isolate JFRL 03-250 were deposited in GenBank (OP854673, ITS; OP867004, ACT; OP867007, TEF1-a; OP867010, GPD; and OQ559562, RPB2). BLAST search analysis in GenBank showed 100% similarity with those of P. capitalensis (GenBank accession nos. ITS, MH183391; ACT, KY855662; TEF1-a, KM816635; GPD, OM640050 and RPB2, KY855820). From a phylogenetic perspective, a maximum likelihood phylogenetic tree was constructed by using IQtree V1.5.6 based on multiple sequences (ITS, ACT, TEF1-a, GPD and RPB2) (Nguyen et al. 2015), and the cluster analysis resulted the representative isolate JFRL 03-250 within a clade comprising Phyllosticta capitalensis (Fig. 2). Based on morphological and molecular characters, the isolate was identified as P. capitalensis. To confirm pathogenicity and fulfill Koch's postulates, 6 healthy potted plants were inoculated with 1× 106 conidia/ml suspension of isolate JFRL 03-250 by spraying on the leaves, whereas 6 plants were sprayed with sterile distilled water to serve as control. All potted plants were incubated at 28°C, 80% relative humidity and 12-h light/12-h dark alternating conditions in a climate cabinet. After 15 days, similar symptoms were observed in the inoculated leaves as in the field (Fig. 1F), whereas control leaves remained asymptomatic (Fig. 1G) and P. capitalensis was successfully re-isolated from the symptomatic leaves. Previously, P. capitalensis has been reported to cause brown leaf spot disease of various host plants around the world (Wikee et al. 2013b). However, to our knowledge, this is the first report of brown leaf spot caused by P. capitalensis on D. odora in China.

Radermachera hainanensis Merr. plants are native in south-central and southeast of China. Plants produce large flowers, and are widely cultivated in China as ornamentals. In April 2020, R. hainanensis Merr. plants grown in Cixi Lvpin Garden (30°26'54″N, 121°25'48″E), Zhejiang Province, were found to have many black circular necrotic lesions. In the early infection stage, the lesions appeared in lower leaves as small black circular spots which developed later into large spots (11 to 38 mm diameter) with grey centers and chlorotic edges. Ultimately, the spots spread and merged. Moreover, infected leaves showed premature leaf fall. Disease intensity reached approximately 20% of plants in the affected field (0.5 ha). After effective chemical control, this disease did not spread to other healthy plants in the same garden. To identify the causative pathogen associated with the disease, ten symptomatic leaves were collected from ten different plants. Leaf tissues were cut from the lesion margins and sterilized as follows: surface sterilized with 75% ethanol for 30 seconds and washed three times in sterile distilled water. The leaf tissues were then dipped into 10% sodium hypochlorite for 3-4 minutes, then washed three times in distilled water and dried on a sterile filter paper. After drying, the surface-sterilized leaf discs were cut to small pieces (3×3 mm) and transferred to potato dextrose agar (PDA) plates and incubated at 28°C for 2 to 3 days under 12 h photoperiod. A total of 15 isolates were obtained from the affected leaves, and all the isolates displayed the same colony characteristics. Then, three single-spore isolates were randomly selected (F2, F5 and F8) for further study. The fungal colonies were dark green with a granular surface, and irregular white edges, later turning black. Conidia were one-celled, oval, and narrow at the end with a single apical end, measuring from 7.8 to 11.1 × 4.6 to 5.9 μm (av. 9.5 × 5.2 μm, n=50). These morphological characteristics were consistent with the description of Phyllosticta capitalensis (Wikee et al. 2013; Guarnaccia et al. 2017). The identity of three representative isolates were confirmed by a multilocus approach. The DNA of three isolates were extracted and partial sequences of ribosomal internal transcribed spacer (ITS), actin (ACT), and translation elongation factor 1-alpha (TEF1-α) were amplified and sequenced as previously described (White et al. 1990; O'Donnell et al. 1998; Carbone & Kohn et al. 1999). The three selected isolates shared 100% identical sequence of ITS, ACT and TEF1-α. Then representative isolate F8 was selected for further study. BLAST analysis in GenBank showed that the obtained sequence of ITS (MZ317550) had 99% identity to P. elongata isolate eSX25240811. Other two sequences of ACT (MZ326837) and TEF1-α(MZ326839) showed 99% and 98% identity to P. capitalensis isolate YLWB01, respectively. The phylogenetic trees were constructed by Bootstrap method with 1000 replications using Maximum Likelihood model implemented in the MEGA 7. Results showed that the isolate F8 clustered with P. capitalensis with 100% bootstrap support. Pathogenicity of strain F8 was tested by Koch's postulates. A pathogenicity test was performed in a greenhouse with 80% relative humidity at 28°C. 20 healthy plants were sprayed with a 1×106 conidia ml-1 suspension (three leaves from each individual plants) and another 20 healthy plants were sprayed with sterile distilled water (three leaves from each individual plant) as control. Conidia was obtained from PDA plates after 7 days of incubation in the biochemical incubator at 28°C and concentration was counted in hemacytometer. After 15 days, disease symptoms were observed on all inoculated leaves, whereas the control plants remained asymptomatic. After that, P. capitalensis was re-isolated only from the infected leaves and identified by morphological and sequence analyses. Early identification of P. capitalensis as a causal agent for black spot is crucial to employ effective disease management strategies to control disease in the field. P. capitalensis has been reported on many crops in China (Cheng et al. 2019; Tang et al. 2020; Liao et al. 2020). However, to our knowledge, this is the first report of black spot disease caused by P. capitalensis on Radermachera hainanensis Merr. in China.

Deutzia crenata Sieb. et Zucc, native to Japan, with white flowers in early summer, is a high quality ornamental shrub widely planted in China.In October 2021, a new leaf spot disease was observed on approximately 70% of the 320 D. crenatatreesgrowing in NanjingBotanical Garden, Jiangsu Province, China. The disease started as irregular small gray spots on the leaf of D. crenata that coalesced into larger lesions. Infected leaves turned yellow (Figure S1A) and leaves with multiple spots withered. To isolate the pathogen, leaf sections (3 to 4 mm) were excised from the lesion margin, surface sterilized in 75% alcohol for 30 s and then in 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃in the darkness. Pure cultures were obtained by monosporic isolation. The colony of a representative isolate (L-1), growing on PDA was circular, white, and cottony, and the surface undulate and pale luteous (Figure S1B). The reverse was similar in color (Figure S1C). The conidial masses were black and appeared over PDA plates after 12 days (Figure S1D). Conidia [18.3 to 28.4×5.4 to 8.5 µm (mean 24.5×6.7 µm)] (n=35) were fusiform to ellipsoid and four-septate (one basal and one apical cell hyaline, and three brown median cells), with two to three apical appendages (Figure S1E). These characteristics were consistent with the description of Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). Three regions of the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1α), and β-tubulin (TUB) genes (GenBank Accession No. OM663738, No. OM687134 and No.OM687133, respectively) were amplified and sequenced with the primers pairs ITS1/ITS4 (Innis et al. 1990), EF1-526F/EF1-1567R (Maharachchikumbura et al. 2014) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The obtained sequences were 95.4-99.8% similar to those from Neopestalotiopsis sp. accessions in GenBank. A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate L-1 clustered in the N. ellipsospora clade with 98% bootstrap support (Figure S2). To test pathogenicity, three detached healthy leaves and three one-year-old D. crenata seedlings were inoculated with 20 μL conidia suspension (1×106 spores/mL) on the left sides of leaves. The right side of each leaf was inoculation with 20 µL of sterile water as the experimental control. All plants were covered with clear polyethylene bags and incubated in a greenhouse (Institute of Botany, Jiangsu Province and Chinese Academy of Sciences) at 25℃, 80% relative humidity, and a 12-h light/dark cycle. The experiment was repeated three times. After 5 days of inoculation, leaf spots typical of those observed in the orchards were observed on the left sides of all inoculated leaves and the right sides did not have any leaf spot symptoms (Figure S1F-G). The same fungus was isolated from the diseased spots of the inoculated leaves to complete Koch,s postulates (Figure S1H). N. ellipsosporais known to cause leaf spots on Camellia sinensisand sweet potato, infects fruits of Ardisia crenata in China (Maharachchikumbura et al. 2014; Maharachchikumbura et al. 2016; Wang et al. 2019), and causes stem spots on Acanthopanax divaricatus in Korea (Yun et al. 2015). This is the first report ofN. ellipsosporacausing leaf spot onD. crenata in the world. The occurrence of this disease needs to be monitored, because it can reduce the ornamental value of D. crenata. This finding provides the foundation to further investigate the biology and epidemiology of this disease so that effective strategies can be developed to manage this disease.

Pecan (Carya illinoensis) is an important tree for commercial nut production in North America and widely cultivated in China. In September 2019, leaf spot symptoms were observed on the leaves of C. illinoensis in an ecologic orchard in Chuzhou, Anhui, China (32°10′20″N, 118°20′12″E), with a disease incidence of 90%. The initial symptoms appeared as small circular to irregular dark brown or black spots on the leaves. The lesions enlarged and coalesced into large necrotic areas, which later resulted in leaf abscission and stunting. Disease symptoms were not observed on the fruits. To isolate the pathogen, leaf fragments (3 to 4 mm) from symptomatic leaves were surface sterilized with 75% ethanol for 30 s and 0.1% HgCl₂ solution for 30 s, rinsed three times in sterile distilled water, placed on potato dextrose agar (PDA) plates, and incubated at 25°C in the darkness. Pure cultures were obtained by monosporic isolation. The colony of a representative isolate, CZ-15, growing on PDA was olivaceous and circular, with abundant aerial mycelium, and light brown on the reverse with white borders on PDA. The conidia were ellipsoidal, subellipsoidal, to ovoid with a short conical beak at the tip, light brown to dark brown with one to six transverse and zero to three longitudinal septa and were in the range of 17.15 to 28.41 × 7.54 to 18.54 µm (n = 50). Based on observed cultural and morphological features, this fungus was tentatively identified as Alternaria alternata (Simmons 2007). Genomic DNA was extracted from single conidial cultures of representative isolate CZ-15, and the internal transcribed spacer (ITS), 18S ribosomal RNA (SSU), 28S ribosomal RNA (LSU), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), and anonymous region (OPA10-2) genes were amplified with the primers described by Woudenberg et al. (2015). The obtained sequences showed 98 to 99% similarity with those from A. alternata accessions in GenBank. The sequences from this isolate were deposited in GenBank under the following accession numbers: ITS, MN636274; SSU, MN636283; LSU, MN636275; GAPDH, MN650588; TEF1, MN650589; and OPA10-2, MN650590. A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate CZ-15 clustered in the A. alternata clade with 99% bootstrap support. To test pathogenicity, 10 detached healthy leaves and 10 1-year-old C. illinoensis plants were inoculated by excising 5-mm mycelial plugs from a 7-day-old colony grown on PDA and placing them on the adaxial surfaces of leaves. As a control treatment, 10 additional detached leaves and potted seedlings were inoculated with 5-mm PDA plugs without mycelia. All plants were covered with clear polyethylene bags and incubated in a growth chamber at 23 ± 5°C, 80% relative humidity, and a 12-h light/dark cycle. The experiment was repeated three times. Seven days after inoculation, the symptoms were similar to those on the original infected plants, whereas the control leaves remained symptomless. A. alternata was reisolated from the lesions and morphologically identified, confirming Koch’s postulates. To our knowledge, this is the first report of A. alternata associated with leaf spot disease on C. illinoensis. This study provides a foundation to further investigate the biology, epidemiology, and management of this disease.

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.

Kadsura coccinea (Lem.) A. C. Smith is an evergreen liana widely cultivated in China for its economic importance in traditional medicine. Many phytochemical studies on the stems and roots of K. coccinea have shown a variety of biological activities, such as anti-hepatitis, anti-HIV, and anti-tumor (Yang et al. 2020). In July 2021, symptoms of leaf spot were observed in a plantation of K. coccinea in Longan (23°03´N, 107°54´E), Guangxi province, China. The incidence of this disease was 36%, and severity varies from approximately 20 to 40% of leaf surface coverage. Symptoms began as small brown spots that expanded into irregular to nearly flower-shaped lesions. To isolate the pathogen, leaves with spots were collected, sterilized with 75% ethanol for 15 s followed by 2% sodium hypochlorite for 120 s, rinsed three times in sterilized distilled water, cut into 5 × 5 mm pieces, and placed onto potato dextrose agar (PDA) plates. The plates were kept in an incubator at 26°C in the dark for at least 2 days. A total of 27 fungal colonies of similar morphology out of 30 pieces of infected tissues were isolated. Four representative isolates (HBB1 to HBB4) were selected to study for further characterization. Fungal colonies were initially grayish-white and then turned greenish-gray on PDA. The black pycnidium and immature conidia appeared over PDA plates after 18 days. The immature conidia were colorless and transparent, elliptical, and had a single-cell structure. After 5 days, the immature conidia gradually become black and develop into mature conidia. The mature conidia were dark brown and two-celled with longitudinal striations, 20.41-29.93 × 12.42-17.19 μm (average 26.07×14.51 μm; n = 100). For DNA-based identification, the internal transcribed spacer (ITS) region, translation elongation factor 1 alpha (EF1-α), and β-tubulin (TUB) genes of the isolates were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Sequences were submitted to GenBank (Accession nos. MW045412 to MW045415 for ITS, MW065559 to MW065562 for EF1-α, and MW065555 to MW065558 for TUB). A phylogenetic analysis was conducted using the Maximum Likelihood method on concatenated sequences of the three genes, which showed that the four Chinese isolates from K. coccinea were clustered with reference isolates of Lasiodiplodia theobromae including the ex-neotype CBS 164.96. Pathogenicity tests were performed on young, fully expanded leaves of 2-year seedlings. A 10 μL conidial suspension (1×106 conidia/mL) was inoculated on each wound on the left-half leaf and a 10 μL sterile water was inoculated on each wound on the right-half leaf (control). Each treatment was repeated three times. Inoculated leaves were wrapped in plastic bags for 5 days and plants were maintained in a growth chamber at 27°C, 85% relative humidity. Brown leaf spots appeared 5 to 6 days after inoculation, whereas the control leaves treated with sterile water showed no symptoms. All re-isolations from spots produced colonies with the same morphological characters as L. theobromae, completing Koch's postulates. To our knowledge, this is the first report of L. theobromae causing leaf spot on K. coccinea in China and worldwide. Severe leaf disease caused by L. theobromae threatens K. coccinea production. The disease threatens K. coccinea growth, and effective control measures should be identified to reduce losses.

Amaranthus hybridus (=A. patulus), often called green amaranth, is an annual herbaceous plant of the Amaranthaceae. This plant is considered a harmful weed in the agricultural context of North America and has expanded its distribution to Asia and Europe. In Korea, it has become a problematic invasive issue, leading to economic losses due to reduced crop yields and rising weed management costs (Park et al., 2014), although its seeds and young leaves are edible and frequently consumed. In October 2020, we observed leaf spot symptoms on A. hybridus plants that were growing within perilla farms (Perilla frutescens var. japonica) located in Damyang (35°14'07"N, 126°59'40"E), Korea, with a disease incidence of 20 to 30% of the inspected plants. The initial signs appeared as grey to brown dots on the leaves, which gradually expanded into irregular, brown patches with a diameter of 2-3 cm. A single spore was isolated from the diseased leaf under a dissecting microscope, placed onto a 2% water agar plate, and incubated in darkness at 25°C for three days. Pure cultures were obtained by transferring single hyphal tips onto potato dextrose agar (PDA) plates. Five single-spore isolates were the same in the cultural and morphological examination, and a representative isolate (P309) was preserved at the Korean Agricultural Culture Collection (KACC49813), Korea. Colonies appeared light gray to white with regular margins and reached 4 to 5 cm in diameter after a week. After two weeks, black patches of spores were often visible in the aerial mycelia. Conidiophores were brown to pale brown, often branched, thick-walled, and measured 6.8 × 2.7 µm (n = 30). Conidia were single-celled, dark brown, globose to ellipsoid, and measured 6.8 × 5.0 µm (n = 50), with a ratio of length/width of 1.1 to 1.6 (n = 50). These morphological characteristics matched those of Arthrinium arundinis (Crous et al., 2013). For molecular identification, genomic DNA was extracted from conidia and mycelia on two-week-old PDA culture of the KACC49813. PCR was performed for the internal transcribed spacer (ITS) (primers ITS1/ITS4, White et al. 1990), the large subunit (LSU) rDNA (primers LROR/LR5, Vilgalys et al. 1990), the beta-tubulin gene (TUB) (primers T1/Bt-2b, O'Donnell and Cigelnik 1997), and the translation elongation factor 1-alpha (TEF) (primers EF1-728F/EF-2, Crous et al. 2013). A BLASTn search of the resulting sequences of ITS (560 bp; OL744431), LSU (881 bp; OL744432), TUB (790 bp; PP084934), and TEF (445 bp; PP084935) revealed 100 % similarity (e-value=0.0, coverage=100%) to previously reported sequences of Arthrinium arundinis (e.g. MF627422 for ITS, KF144930 for LSU, KF144973 for TUB, and KY705146 for TEF), confirming the identity of the Korean isolate. Pathogenicity assays were performed twice by spraying 1 ml of a conidial suspension (1.1 × 104 conidia per mL) onto the leaf surface of sixteen healthy A. hybridus plants. Sixteen control plants were sprayed with sterile water. All plants were kept in a growth chamber at 80% relative humidity and 23 °C with a 12-h light/dark cycle. Three weeks after the inoculation, initial symptoms mirroring the aforementioned ones appeared, while the control plants remained symptomless. Fungal isolates were successfully re-isolated from the inoculated leaves, and their identity as A. arundinis was confirmed by DNA sequencing, thus fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot caused by A. arundinis on Amaranthus hybridus, not only in Korea but globally. Arthrinium arundinis has also been reported as a plant pathogen on some agricultural crops (Ji et al. 2020; Liao et al. 2022; Farr and Rossman 2023), suggesting its polyphagous behavior. Then, this pathogen could represent a potential risk to the cultivation of edible amaranth in Korea and other crops where Amaranthus species are spread as weeds. For this reason, continuous monitoring is necessary to assess the impact of this fungus on Amaranthus and other crops.

As a globally cultivated economic crop, tobacco (Nicotiana tabacum) is known for its addictive properties, which arise from the mildly irritating and psychoactive compounds it contains (Hu et al. 2010). Tobacco leaves are susceptible to a range of fungal and bacterial diseases during production and curing, including target spots, brown spots, wildfire, and powdery mildew (Guo et al. 2024). During a survey conducted in June 2025 in Zhengan (107.43° N, 28.55° E), Guizhou Province, China, tobacco (cv. Yunyan 87) plants were found affected by a leaf spot disease, with an incidence rate ranging from 41% to 47%. Initially, symptomatic leaves developed irregular, yellowish-brown spots that gradually expanded and turned necrotic, eventually acquiring a whitish appearance. To investigate the disease, six severely symptomatic plants were selected for pathogen isolation using the tissue transplanting method. From each plant, pieces (5 × 5 mm) of leaf tissue taken from the border between diseased and healthy tissue were surface-sterilized with 75% ethanol for 30 s, followed by 1% sodium hypochlorite for 1 min, and then rinsed three times with sterile distilled water before being placed on potato dextrose agar (PDA) medium. After incubating at 25°C in the dark for 7 days, a total of nine fungal isolates with similar morphology were obtained. One representative isolate, designated YB13, was selected for further identification (Fig. S1). The fungal colonies on PDA exhibited abundant aerial mycelia and were white in color, and covered the whole plates (90 mm in diameter) in seven days. After 10 days of incubation at 28°C, the fungus produced black, ovoid, smooth, and aseptate conidia with 12-15 μm in diameter. For molecular identification, genomic DNA was extracted from isolate YB13. The internal transcribed spacer (ITS) region, along with the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUB2), and translation elongation factor 1-alpha (TEF1-α) genes were amplified using primers ITS1/ITS4 (White 1990), gpd1/gpd2 (Berbee et al. 1999), BT2Fd/BT4Rd (Li et al. 2017), and EF1-728F/EF1-986R (Carbone and Kohn 2019) respectively. The resulting sequences have been deposited in GenBank under the following accession numbers: ITS: PX736263; GAPDH: PX556631; TUB2: PX556632; and TEF1-α: PX711191. BLAST analysis of the sequences from isolate YB13 revealed high identity with those of Nigrospora coryli isolate W18. Specifically, the ITS sequence shared 99.14% identity with isolate W18 (GenBank: PP218065), the TUB2 sequence shared 99.71% identity with isolate W18 (GenBank: PP320372), and the TEF1-α sequence shared 100.00% identity with isolate W18 (GenBank: PP461302). A multilocus phylogenetic analysis based on a concatenated dataset of ITS, TEF1-α, and TUB2 genes further confirmed that isolate YB13 clusters within the N. coryli clade (Fig. S2). Pathogenicity of the isolate YB13 was confirmed on five healthy tobacco plants (cv. Yunyan 87) at seedling stage (four to five leaves). To wound the leaves, a 4 mm² area on each was lightly scratched with a sterile needle, after which a 5-mm diameter mycelial plug was placed on the wound. Control leaves were inoculated with PDA-only plugs. Following inoculation, leaves were maintained under high humidity by enclosing the treated plants in transparent plastic bags containing sterile water-soaked cotton at the base to maintain approximately 80% relative humidity. Plants were incubated in a greenhouse at 25°C. All experiments were performed in triplicate. The leaf disease development was observed and recorded daily. After 7 days, all inoculated leaves developed leaf spots consistent with symptoms observed in the field. Lesions appeared as irregular to circular spots, 5–12 mm in diameter, with a yellowish-brown color and often a chlorotic halo. As symptoms progressed, the lesions turned necrotic, developing dry, whitish centers surrounded by a darker margin and a yellow halo. In contrast, control plants remained completely asymptomatic. The pathogen was re-isolated from lesion margins and confirmed to be identical to the original inoculated strain based on colony morphology and DNA sequencing, thereby fulfilling Koch’s postulates. N. coryli has previously been reported as an endophyte within the stem of Corylus heterophylla at Mycorrhizal Seedling Cultivation Center in Guizhou, China (Wang et al. 2024). To our best of knowledge, this is the first report of N. coryli causing leaf spot on tobacco in China. These findings underscore the importance of continued pathogen surveillance and provide a basis for epidemiological studies and the development of management strategies for this emerging disease.

Rubber is an economically important crop cultivated extensively in Malaysia for latex production. In March 2024, necrotic spots were observed on mature leaves of rubber plants (clone RRIM2004) during surveys in two rubber plantations, in Sungai Buloh, Selangor state and Kota Tinggi, Johor state, with a disease incidence of 80%. Initial symptoms on leaves appeared as light-yellow, circular, semi-circular to irregular lesions (2–5 mm in diameter) on the adaxial leaf surface, which gradually changed to brown and grey-white spots (Figure 1). Diseased leaves became blighted and the plants defoliated as the disease progressed. To identify the pathogen, fragments (5 × 5 mm) were excised from the margin of the diseased leaf tissues, surface-sterilised with 1% sodium hypochlorite solution for three minutes, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA) and incubated at 28°C with a 12 h photoperiod for 7 days. Ten single-spore isolates were obtained from sampled leaves, all isolates exhibited a Pseudopestalotiopsis-like morphology and two representative isolates (PA1 and PA2) were selected for further study. Colonies on PDA were whitish with dense aerial mycelia, forming black gregarious conidiomata and the reverse side was whitish to pale yellow (Figure 2). Conidia were fusoid to ellipsoid, straight to slightly curved, 4-septate, ranging from 21 to 30 ±6.5–9 µm (n = 30) and septa darker than the rest of the cell (Figure 3). The basal cells were conic with a truncate base, hyaline and thin-walled, 2.5–5.0 µm long. Three median cells were doliiform, 13.5 to 19.5 µm long, hyaline, subcylindrical, thin-walled, with 2–3 tubular apical appendages arising from the apical crest, unbranched, filiform, 17–25 µm long. The basal appendages were singular, tubular, unbranched, centric, 3.5–7.0 µm long. On the basis of morphology, both representative isolates were identified as Pseudopestalotiopsis (Maharachchikumbura et al. 2014). The internal transcribed spacer (ITS) region of rDNA and translation elongation factor 1-alpha (TEF1-α) gene of isolates PA1 and PA2 were amplified using the ITS5/ITS4 and EF1-728F/EF1-986R primer set, respectively (White et al. 1990; Carbone and Kohn 1999). BLASTn analysis of the resulting ITS and TEF1-α sequences indicated 99% identity to ex-holotype Pseudopestalotiopsis ampullacea strain LC6618. The ITS (GenBank Accession Nos. PP779714 and PP779715) and TEF1-α (PP785033 and PP785034) sequences were deposited in the GenBank databases. Phylogenetic analysis using the maximum likelihood analysis based on the concatenated ITS-TEF1-α indicated that the Ps. ampullacea PA1 and PA2 isolates form a strongly supported clade (82 bootstrap value) to the ex-holotype culture of Ps. ampullacea LC6618 and both isolates were most closely related to other Pseudopestalotiopsis species (Figure 4) (Kumar et al. 2024). Five healthy leaves from 6-month-old rubber plants (clone RRIM2004) were inoculated with either isolate PA1 or PA2 according to Liu et al. (2025). Control leaves were mock-inoculated using sterile water. Seven days post-inoculation, necrotic lesions developed on inoculated leaves, closely resembling symptoms observed on naturally infected rubber leaves in the field, whereas the control leaves remained asymptomatic (Figure 5). Pseudopestalotiopsis ampullacea was re-isolated from all symptomatic tissues, verified by molecular identification, confirming Koch's postulates. This is the first report of Ps. ampullacea causing leaf spot symptoms on Hevea brasiliensis in Malaysia. The pathogen is primarily known to infect palm species, particularly oil palm (Ismail et al. 2017). The occurrence of this disease needs to be monitored because it poses a significant threat with the potential to reduce latex production by 28%–46% (Kusdiana and Saputra 2022), adversely affecting overall yield and profitability. Therefore, preventive strategies need to be developed to reduce the incidence of the disease in the field. We thank the Diagnostic Unit, Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, for their support in the plant disease diagnosis. Universiti Putra Malaysia supported this work through the Putra Grant Initiative (GPI), vote project number 9758500.

Tetrastigma hemsleyanum Diels & Gilg (Vitaceae) is a traditional Chinese medicine species widely distributed in the valley regions of Hubei, China. T. hemsleyanum has the effects of antitumor, anti-inflammatory, antiviral and detoxification (Wang et al. 2024). In October 2023, dark brown spots were observed that up to 50% on the leaves of T. hemsleyanum plants cultivated in a 30 m2 nursery in Shiyan (32.62ºN, 110.78ºE), Hubei, China. Initially, small, dark-brown lesions appeared in the middle of leaves. As the symptoms progressed, necrotic lesions expanded or fused into large, dark brown spots with irregular shape. Five leaf pieces (5 mm×5 mm) from the disease margin were sampled, and were disinfected in 70% ethanol for 20 s, in 1.5% NaClO for 2 min, washed with sterilized water for three times, and were placed on potato dextrose agar (PDA) plates, respectively. The plates were stored at 25 ℃ in the dark. After 7 days, five colonies with similar morphological characteristics were obtained on PDA and two representative isolates RJC1 and RJC3 were used for further study. The RJC1 and RJC3 on PDA initially had white mycelium, which turned light brown after 3 days, then gradually turned black brown after 7 days. Conidia were brown, drumstick-like or clavate, 1-5 transverse and 0-3 longitudinal septa, with darker in septa, usually 12.19-34.07×6.17-15.57 μm in size (n=50). The isolates were identified as Alternaria sp. (Simmons 2007) based on the morphological characteristics. For accurate classification, genomic DNA of two representative isolates was extracted from mycelium after 7 days on PDA using a DNA extraction kit (TSINGKE Biotech Co., Ltd.). Internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1) genes were amplified and sequenced using primer pairs ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999). A BLASTn search revealed that the ITS regions (accessions PQ844677, PQ844678) showed 99.81% and 100% homology with Alternaria alternata Aa1 (OK036714) and PPRI: 26401 (MT505877). The TEF1 regions (accessions PQ876358, PQ999190) showed 99.59% and 99.64% homology with Alternaria alternata Zb-SD1 (OQ585964) and SF-004 (ON055375). A phylogenetic tree was constructed using the neighbor-joining method in MEGA 7. The isolates RJC1 and RJC3 belonged to the same clade with A. alternata, supported by a value of 100%. Based on the morphological characteristics and molecular analysis, RJC1 and RJC3 were identified as A. alternata. For pathogenicity test, five healthy T. hemsleyanum plants were inoculated with a conidial suspension (1.0×105 conidia/mL) and grown in a greenhouse at 25 ℃ with 12 h light/12 h dark. The other five plants were inoculated with sterile water as control. The inoculation was performed three times. Dark brown lesions similar to those in the samples were appeared on leaves 7 days after inoculation, but no symptoms were observed on the control leaves. The same fungus was reisolated from the diseased leaves basing on morphological characterization and sequence analyses, but not from the healthy controls, which was satisfied Koch's postulates. A. alternata has been reported to cause leaf spot disease on citrus (Taycir et al. 2023), Kalanchoe (Sanahuja et al. 2018), and Conocarpus erectus (Fahim et al. 2021). To our knowledge, this is the first report of A. alternata on T. hemsleyanum in China, which will provide theoretical basis for disease diagnosis, occurrence patterns and effective control measures.

Orthosiphon stamineus (Java tea) is a perennial herbaceous plant in the family Lamiaceae and is cultivated extensively in Southeast Asia for its medicinal value (Arifullah et al. 2014). During October 2018, leaf blight symptoms were observed on leaves of ~210 plants O. stamineus grown in experimental plots of a research farm at Faculty of Engineering, Serdang, Selangor, Malaysia (3°00'30.4"N 101°43'19.9"E) with 80% disease incidence. Initial symptoms were brown-to-black lesions on the leaves that enlarged and coalesced until the leaf withered and abscissed. Diseased tissues (4 × 4 mm) of six infected leaves were excised, surface disinfected with 0.5% NaOCl for 1 min, rinsed twice with sterile distilled water, placed onto potato dextrose agar (PDA) plates, and incubated at 25°C with a 12-h photoperiod under fluorescent light for 7 days. A total of six single-spore isolates were obtained from sampled leaves. All isolates exhibited similar morphology and two representative isolates, MK and MK1, were characterized further. Colonies on PDA were initially white then turned dark gray with age and had a pale green underside. Hyphae were branched, septate and hyaline to pale brown. The conidia were one-celled, black, smooth-walled, spherical to subspherical in shape measuring 11.0 μm × 16.5 μm in diameter (n=30) and which are borne on hyaline vesicles at the tip of each conidiophore or formed directly from the mycelia. Based on morphological characteristics, the fungal isolates were identified as Nigrospora osmanthi (Wang et al. 2017). Total genomic DNA of the isolates was extracted from fresh mycelium using DNeasy Plant Mini kit (Qiagen, USA) and the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1) and Beta-tubulin (TUB2) gene regions were amplified using ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999) and Bt-2a/Bt-2b primer set (Glass and Donaldson 1995), respectively. BLASTn analysis showed that the ITS, TEF1 and TUB sequences of the isolates shared 99%-100% identity with Nigrospora osmanthi ex-type strain CGMCC 3.18126 (GenBank accession nos. KX986010, KY019421, KY019461). The sequences of two representative isolates (MK and MK1) were deposited in GenBank (ITS: accession nos. MT645782, MW363019; TEF1: MW366861, MW366862; TUB: MW366863, MW366864). Phylogenetic analysis by the maximum likelihood method based on the concatenated ITS-TEF1-TUB sequences showed that the isolates in this study were clustered in a strongly supported group 98% maximum likelihood with type strain N. osmanthi (Kumar et al. 2016). The pathogenicity of all isolates was confirmed by inoculation on ten healthy leaves of five potted 4-week-old O. stamineus plants using a conidial suspension (1 x 106 spores/ml) produced on 7-days-old PDA cultures. An equal number of plants were sprayed with sterile distilled water only to serve as a control and the treated plants were kept in a growth chamber for 2 weeks at 28 ± 1°C and 95% relative humidity. The experiment was repeated twice. The inoculated leaves developed brown lesions which enlarged into blight symptoms similar to those observed on naturally infected leaves after 5 days of inoculation, while control plants remained healthy. Nigrospora osmanthi was successfully re-isolated from the infected leaves, but not from leaves of non-inoculated control plants, thus satisfying Koch's postulates. . N. osmanthi has been recently reported to cause leaf blight on Ficus pandurata (Liu et al. 2019) and Stenotaphrum secundatum in China (Mei et al. 2019). This disease can cause a significant threat to the cultivation of O. stamineus which has been extensively grown for the production of herbal Java tea. Accurate identification of this pathogen could assist in developing an effective disease management strategy to control this disease.

Magnolia grandiflora linn, with large and fragrant flowers, is widely planted in the south of Yangtze River valley in China. It is an excellent street tree and a beautiful ornamental tree for landscaping.In October 2021, a new leaf spot disease was observed on M. grandiflora seedlings and maturetreesgrowing in NanjingBotanical Garden, Jiangsu Province, China. According to statistics, about 300 M. grandifloratrees were planted here, and approximately 60% of M. grandiflora trees suffered from the disease. In the beginning, small black spots appeared on the leaf of M. grandiflora, and then the disease spots were connected into coalesced, and eventually lead to a large area of leaf dead (Figure S1A). To isolate the pathogen, ten diseased leaves were collected from ten plants distributed in different five areas of the botanical garden. The leaf sections (3 to 4 mm) were excised from the margins between healthy and diseased tissues, surface sterilized in 75% alcohol for 30 s and then in 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃in the darkness. Pure cultures were obtained by monosporic isolation. Twenty-three isolates were obtained (the isolate rate of 72%), and identified as Lasiodiplodia sp.. A representative isolate, G-H-1 was used for the further study. The colony of G-H-1, growing on PDA was cotton-like. The primary mycelia was gray and white in the early stage of culture. It gradually turned black gray in the later stage, and the reverse was similar in color (Figure S1B). The pycnidia (fruiting body) was black and appeared over PDA plates after 15 days (Figure S1C). The hyphae of G-H-1 were dark brown, and the conidia were monospora, oval or elliptic, with a size of (9.6 ~ 13.3) µm× (5.7 ~ 8.0) µm (mean 11.7×6.6 µm, n=35) (Figure S1D). In the pycnidia, the conidiophores were inside and produced conidia (Figure S1E). In the early stage, the conidia of G-H-1 were colorless transparent, then gradually turned dark brown with a septum in the center (Figure S1F). These characteristics were consistent with the description of Lasiodiplodia sp. (Alves et al. 2008). The regions of ITS, translation elongation factor 1-alpha (TEF1α) and β-tubulin (TUB) genes (GenBank Accession No.OM698339, No.OM942757, and No.OM942756, respectively) were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Alves et al. 2008) and Bt2a/Bt2b (Glass and Donaldson 1995). The obtained sequences showed 99.05-99.81% similarity with those from L. theobromae accessions in GenBank. A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate G-H-1 clustered in the L. theobromae clade with 96% bootstrap support (Figure S2). To test pathogenicity, three one-year-old M. grandiflora seedlings that previously had been wounded with a sterile needle were inoculated with 20 μL conidia suspension (1×106 spores/mL) on the left sides of leaves. Inoculation with 20 µL sterile water was treated as the control, which were inoculated on the right sides of leaves. All plants were covered with clear polyethylene bags to keep moisture. And inoculated detached leaves were incubated in a greenhouse (Institute of Botany, Jiangsu Province and Chinese Academy of Sciences) at 25℃, 80% relative humidity, and a 12-h light/dark cycle. The experiment was repeated three times. After 5 days of inoculation, typical black spots were found on the left sides of all inoculated leaves and the right sides did not have any leaf spot symptoms (Figure S1G-H). After 25 days of inoculation, perforation occurred at the black spots on the leaves of the inoculated plants, resulting in incomplete leaf (Figure S1I), which is identical disease symptoms to those observed in garden. The same fungus, identified by morphological characteristics and sequencing using ITS, TEF1α and TUB genes, was isolated from the diseased spots of the inoculated leaves to complete Koch,s postulates. The pathogen has a very wide host range. For example, it has been reported to cause dieback and sooty canker on Ficus trees (Abo Rehab et al. 2014), infected trunk of sultana seedless (Tang et al. 2021) and castor bean(Akgul et al. 2015),root ofCitrus (Al-Sadi et al. 2014), date palm,and Mango (Al-Sadi et al. 2013) and Cassia fistula (Deng et al. 2015). But, according to nt.ars-grin.gov, there are no other reports of L. theobromae on M. grandiflora in the world. So, this would be the first one. This study provides an important reference for the biology, epidemiology.