First report of Cladosporium cladosporioides causing leaf blight on Sambucus chinensis 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.

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.

Nageia nagi (Thunb.) Kuntze belongs to the family Podocarpaceae with shiny green branches and leaves, which is widely distributed in East Asia and the Southern Hemisphere. The leaves, roots and fruits of N. nagi have been used as herbal medicine to treat rheumatism, arthritis and venereal diseases (Abdillahi et al. 2011). In September 2022, leaf spot symptoms were found on approximately 30% of the leaves of N. nagi trees in a community located at the Economic and Technological Development Zone, Nanchang City, Jiangxi Province, China. Following the initial infection, the leaf lesions extended outwards from the top in a circular pattern, appearing as a dark brick color, and later changed to yellow and became dry, with a darker brown margin surrounding them. Ten symptomatic leaves were randomly selected, and a small piece of leaf tissue (5mm ×5mm) located between the health and infected tissues was cut and surface-desinfected with 70% ethanol for 30 s and 1% sodium hypochlorite (NaClO) for 30 s sequentially. After rinsing three times in sterile distilled water, all the small pieces of leaves were placed on potato dextrose agar (PDA) plates, followed by incubation at 28℃ for 3 days. Ten isolates, cultured on each PDA plate, appeared olive green with a granular surface, and an uneven white edge, and finally turned greenish black. The conidia were hyaline, with ellipsoidal to subglobose shapes and spore sizes of 5.5-8.3 × 7.2-12.0 μm (width × length) (=7.2±0.71 × 9.9±1.3 μm, n=40). These morphological characteristics are consistent with those of Phyllosticta species. To confirm the species, three representative isolates, JFRL 03-768, JFRL 03-769 and JFRL 03-770 were selected for further identification. The internal transcribed spacer (ITS) region, actin (ACT), translation elongation factor 1-alpha (TEF1-a), and glyceradehyde-3-phosphate dehydrogenase (GPD) genes of the three isolates were amplified and sequenced with the primers V9G/ITS4 (Carbone and Kohn 1999), ACT-512F/ACT-783R (Carbone and Kohn 1999), EF-728F/EF-2 (O´Donnell et al. 1998) and Gpd1-LM/Gpd2-LM (Myllys et al. 2002; Guerber et al. 2003), respectively. All sequences had been deposited into GenBank (ITS: OQ195332, OQ195333 and OQ195334; ACT: OQ207621, OQ207622 and OQ207623; TEF1-a: OQ207624, OQ207625 and OQ207626; GPD: OQ207627, OQ207628 and OQ207629). A maximum likelihood phylogenetic tree was constructed using the IQtree V1.5.6 (Ngugen et al. 2015) based on the concatenation of multiple sequences (ITS, ACT, TEF1-a and GPD). In the cluster analysis, the representative isolates (JFRL 03-768, JFRL 03-769 and JFRL 03-770) were positioned within a clade comprising of Phyllosticta styracicola. Subsequently, the pathogenicity of P. styracicola was determined by wound inoculation of healthy 2 year-old N. nagi plants, and this experiment was repeated for three times. Briefly, for each isolates, six disinfected leaves were wounded with a sterile scalpel, and then inoculated with 10-μl drop of the conidial suspension (1 × 106 conidia/ml). Another six disinfected leaves were inoculated with 10-μl drop of sterile water as a control group, and all plants were incubated at 28°C with 80% humidity. After 15 days, a similar spot lesion appeared on the leaves of the experimental group. P. styracicola was successfully re-isolated, and then subjected to morphological identification and molecular sequencing (ITS, ACT, TEF1-a and GPD genes). Whilst, the control leaves showed no symptoms. Previous studies have reported that P. styracicola could result in the development of lesions on Styrax grandiflorus leaves in China (Zhang et al. 2013). To our knowledge, this is the first report that P. styracicola can cause leaf spot on N. 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.

Trollius chinensis is widely distributed in east Asian countries that include China, Siberia, and Japan, with antibacterial, antiviral, anti-inflammatory and analgesic activity for medical applications. In August 2021, leaf blight was observed on nearly 80~95% of T. chinensis plants growing in Daxinganling (51.43°N, 126.39°E) from Heilongjiang Province, China. Initial symptoms were gray-black necrosis, wilting progressing from the leaf margin, and eventual defoliation. Six T. chinensis plants with typical symptoms were randomly collected, and three fresh leaf samples were collected from each plant. Diseased leaf pieces that measured 5 mm square were disinfected in 75% ethyl alcohol for 30 s and 7% NaClO for 60 s, rinsed three times in sterile distilled water, and placed on potato dextrose agar (PDA). Twelve fungal isolates, obtained by single-spore isolations, were selected for further. These isolates produced colonies that measured 63 to 73 mm in diameter after 7 days growth on PDA. Colonies were black to brown in color with gray-white aerial hyphae on their surfaces, neat edges, olive green on the back. The isolates produced conidia that were ovate to pear-shaped, brown to black in color, with 1 to 4 transverse septa and 0 to 1 oblique septa, smooth surfaced, parietal cells extending into the beak, and measured 12.5 to 37.5 × 5.0 to 12.5 μm(n=150). Conidiophores were dark, erect or curved, branched, with pronounced spore marks, and measured 35.0 to 50.0 × 4.0 to 5.0 μm(n=150). All twelve fungal isolates were morphologically similar to Alternaria alternata (Simmons 2007). Two representative isolates jlh01 and jlh02 were used for molecular identification. The internal transcribed spacer (ITS) region, RNA polymerase second largest subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), and Alternaria major allergen (Alt a 1) were amplified with the primers ITS4/ITS5, RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou 2013), gpd1/gpd2, EF1-728F/EF1-986R (Nishikawa and Nakashima 2020) and Alt-for/Alt-rev (Woudenberg et al.2015). The resulting sequences were deposited in GenBank (ITS, OM095427, OM108099; RPB2, OM131213, OM131214; GAPDH, OM201165, OM201166; TEF1, OM131211, OM131212; Alta1, OM201167, OM201168). Phylogenetic tree results showed 100% similarity between jlh01, jlh02 and the type strain CBS 118812. Morphological and molecular analysis results confirmed the identity of the fungus as A. alternata. Pathogenicity tests were done by spraying water-spore suspensions containing 106 spores per ml of A. alternata isolates jlh01 and jlh02 on leaves of six healthy T. chinensis plants, separately. Six control plants were sprayed with distilled water and both sets of plants covered with plastic bags and placed in a greenhouse maintained at 25⁰ C. Plastic bags were removed from all plants after 48 h. Black brown lesions and concentric rings developed on spore-inoculated plants after 15 days and control plants remained symptomless. The pathogenicity tests were conducted three times. A. alternata was reisolated and identified based on morphological and molecular traits, thus fulfilling Koch's postulates. To our knowledge, this is the first report of A. alternata causing leaf blight on T. chinensis in China. Based on the plant's medicinal value, this report provides the basis for further research and control of T. chinensis leaf blight.

Wampee (Clausena lansium [Lour.] Skeels) is a tropical fruit. In July 2022, leaf spot symptom was observed in wampee (cv. JIXIN) in a field ((21°25'N, 110°10'E, about 100 ha ), Guangdong Province, China. Disease incidence was around 70% (n = 100 investigated plants from about 2 ha). Leaf spots were round or irregular with a clear yellow halo around a brown, necrotic lesion. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces. The surfaces were disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Thereafter, the samples were rinsed thrice in sterile water, placed on potato dextrose agar (PDA), and incubated at 28 °C in the darkfor 3 days. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty isolates were obtained. Three representative single-spore isolates (CLCT-1, CLCT-2, and CLCT-3) from the twenty isolates were confirmed to be identical based on morphological characteristics and ITS analysis and used for further study. The colonies on PDA were gray white at first, subsequently turning grayish to dark gray, with numerous black microsclerotia and setae. Conidia were hyaline, aseptate, falcate with pointed ends, and 16.5 to 22.3 × 2.5 to 3.2 μm (n = 30). Morphological characteristics of the isolates were consistent with the description of Colletotrichum truncatum (Schwein.) Andrus & W. D. Moore (Sawant et al. 2012). For molecular identification, the colony PCR method (Lu et al., 2012) was used to amplify the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) loci of the isolates using primer pairs ITS1/ITS4, GDF1/GDR1, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). The sequences were submitted to GenBank under accession numbers OP740964 to OP740966 (ITS), OP800837 to OP800839 (GAPDH), and OP800843 to OP800845 (ACT). The sequences of the three isolates were 100% identical (ITS, 547/547 bp; GAPDH, 290/290 bp; and ACT, 266/266 bp) with those of C.truncatum (accession nos. GU227869, GU228261, and GU227967) through BLAST analysis.. In addition, a phylogenetic tree was generated on the basis of the concatenated data from sequences of ITS, GAPDH, and ACT that nested within the clade containing C. truncatum (the type strain CBS 112998) by the maximum likelihood method. From the combination of the morphological and molecular characteristics, the isolates were determined to be C.truncatum. A pathogenicity test was performed in a greenhouse at 24 to 30°C with 80% relative humidity. Wampee plants (cv. JIXIN, n =5, 1-month-old) were inoculated with a spore solution (1 × 105 per mL) until it run-off. Whereas control plants were sprayed with sterile distilled water. Leaf spots were observed on the inoculated plants after 10 days while the control ones remained healthy. The pathogen re-isolated from all the symptomatic leaves was identical to the inoculation isolates in terms of morphology and just ITS analysis, but unsuccessful from the control plants. C.truncatumhas also beenreportedto be thecausalagent of anthracnose disease in multiple crops (Diao et al. 2014;Villafana et al. 2018; Stella de et al. 2021), thus, this is the first to report C.truncatum causing leaf spot on C. lansium in China. This study provides an important reference for the control of the disease due to the high host range ofC.truncatum.

Leymus chinensis is a dominant plant in the Eurasian steppe and plays a crucial role in ecological protection and restoration due to its functions in sand fixation, soil conservation, and ecosystem stabilization (Liu et al. 2022). In August 2024, leaf spot disease was first observed on L. chinensis in a grassland of Abaga Banner, Xilinhot City, Inner Mongolia, China (43°54′47″N, 115°34′1″E). Investigations covering 0.25 hectares revealed that the disease incidence stood at 15%. Initial symptoms appeared as elliptical spots with brown edges and grayish-white centers. These spots gradually expanded into elongated lesions at later stages, eventually causing leaf wilting and, in severe cases, plant death. Thirty leaves displaying typical leaf spot symptoms were collected for pathogen isolation. Tissue pieces (5 mm×5 mm) from lesion margins were surface-sterilized (70% ethanol, 20 s; 1% NaClO, 2 min), rinsed three times with sterile distilled water, and placed on PDA. The plates were incubated at 25 ℃ in the dark for 5 days. Hyphal tips from actively growing colonies were picked and transferred onto fresh potato dextrose agar (PDA) plates for purification, yielding 10 isolates with consistent morphological characteristics. All isolates formed gray, circular colonies with gray edges, and the reverse sides of the culture plates showed gray pigmentation.Two representative isolates, YH1 and YH2, were selected for detailed characterization. Conidia were clavate to drum-shaped, measuring 10.85-34.96 × 5.86-13.76 μm (n=30), with 3-5 transverse septa, 0-2 longitudinal septa. These morphological characteristics were consistent with descriptions of Alternaria species (Simmons 2007). For molecular identification, genomic DNA of isolates YH1 and YH2 was extracted using a commercial kit (Aidlab Novel Plant Genomic DNA Extraction Kit). The internal transcribed spacer (ITS) region, translation elongation factor (TEF1-α) gene, and Alternaria major allergen (Alt a 1) gene were amplified and sequenced using primer pairs ITS1/ITS4 (White et al. 1990), Ef728M/Tef1R (Stępień et al. 2012), and Alt-for/Alt-rev (Woudenberg et al. 2015), respectively. The obtained sequences were deposited in GenBank under accession numbers: ITS (PX644766, PX644767), TEF1-α (PX653172, PX653174), and Alt a 1 (PX653171, PX653173). BLASTn analysis revealed 100% (ITS), 99.73% (TEF1-α), and 100% (Alt a 1) sequence similarity with Alternaria alternata. A phylogenetic analysis based on the combined dataset of the three genes placed isolates YH1 and YH2 within the A. alternata clade. Pathogenicity tests were conducted by spraying leaves of fifteen healthy L. chinensis plants (three plants per pot, five pots total) with a conidial suspension (1×10⁶ conidia/mL, containing 0.2% Tween 20) until runoff. Fifteen control plants were sprayed with sterile water containing 0.2% Tween 20. All plants were maintained in a greenhouse at 25 ℃, 80% relative humidity, under a 12 h light/dark cycle. The experiment was repeated twice. Ten days post-inoculation, symptoms identical to those observed in the field developed on inoculated leaves, while control plants remained symptomless. The fungus re-isolated from the lesions was confirmed as A. alternata based on morphology and molecular analysis, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of A. alternata infecting L. chinensis in China. This study provides essential information for the diagnosis, understanding of disease epidemiology, and development of control strategies for this newly identified 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.

Sarcandra glabra is an important Chinese medicinal plant, which was widely cultivated under forest in south China. Guangxi province is the main producing areas of this herb. In June 2019, a serious leaf disease was found causing severe defoliation in the S. glabra plantation under bamboo forest in Rongan country, Guangxi province (109°13'N''E). About 70% of the plants in the plantation (300 ha) showed the similar symptoms. Initially, circular lesions appeared on young leaves as black spots (about 1 to 2 mm). Then, the spots gradually enlarged usually with an obvious yellowish margin (6 to 8 mm). Finally, the lesions coalesced and formed irregular, black, and large necrotic areas, resulting in the leaf abscission. For pathogen isolation, small pieces of tissue (5×5 mm) taken from 25 diseased leaves were sterilized with 75% ethanol for 30 s, subsequently, soaked in 0.1% HgCl2 for 2 min, rinsed three times in sterile distilled water, dried, and then placed aseptically onto the potato dextrose agar (PDA) plates, and incubated at 28 °C (12 h/12 h light/dark). Three days later, the isolates were placed on a new PDA plate for subsequent purification and sporulation. 20 pure fungal isolates were obtained from single spores. Of which, 15 isolates showed similar morphological characteristics.The colonies on PDA were round, dense, gray edge and dark gray in center area. Conidia in culture were appeared light brown, cylindrical in shape, with 0 to 8 septa, and 55 to 165 μm × 5.2 to 13.5 μm in size (mean = 106.2 μm × 8.6 μm, n = 30). These morphological characteristics resemble those of Corynespora sp. (Berk. & M.A. Curtis) C.T. Wei (Ellis et al. 1971). A single-spore isolate (ZD5) was selected from the 15 fungal isolates for a subsequent molecular identification. The genes of internal transcribed spacer (ITS) of ribosomal DNA, β-tublin, and actin were amplified with the primer pairs ITS-1/ITS-4 (White et al. 1990), β-tubulin 2-Bt2a/Bt2b (Glass and Donaldson 1995), ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. And the ITS, β-tublin, and actin sequences were deposited in the GenBank database with the accession numbers MW362446, MW367029, and MW533122. Blast analysis and neighbor-joining analysis based on ITS, β-tublin, and actin sequences using MEGA 6 revealed that the isolate was placed in the same clade as C. cassicola with 100% bootstrap support. Pathogenicity test was performed on the two-year-old potted S. glabra. Six-mm-diameter mycelial plugs were attached to the healthy leaves of S. glabra for co-culture, while the control group was attached with PDA. All plants were covered with plastic bags for 2 days in order to maintain high humidity and cultured in a greenhouse at 28 °C with a 12-h/12-h light/dark cycle. The symptoms appeared 2 days after co-culture were identical to those observed in the field. The same fungus was re-isolated from the lesions, and further morphological characterization and molecular assays, as described above.The control leaves remained symptomless during the pathogenicity tests. According to the previous literatures, C. cassicola is a plant pathogenic fungus with a broad host range, which can damage diverse tropical plants including Salvia miltiorrhiza (Lu et al. 2019), Solanum americanum (Wagner and Louise 2019), Vitex rotundifolia (Yeh and Kirschner 2017), Cucumis sativus, Lycopersicon esculentum (Hsu et al. 2002), Carica papaya (Tsai et al. 2015),and so on. To our knowledge, this is the first report of C. cassicola causing leaf spot on S. glabra in China.

Melon (Cucumis melo L.) is a member of the Cucurbitaceae family, an important economical and horticultural crop, which is widely grown in China. In May 2020, fruit rot disease with water-soaked lesions and pink molds on cantaloupe melons was observed in several greenhouses with 50% disease incidence in Ningbo, Zhejiang Province in China. In order to know the causal agent, diseased fruits were cut into pieces, surface sterilized for 1 min with 1% sodium hypochlorite (NaClO), 2 min with 75% ethyl alcohol, rinsed in sterile distilled water three times (Zhou et al. 2018), and then placed on potato dextrose agar (PDA) medium amended with streptomycin sulfate (100 μg/ml) plates at 25°C for 4 days. The growing hyphae were transferred to new PDA plates using the hyphal tip method, putative Fusarium colonies were purified by single-sporing. Twenty-five fungal isolates were obtained and formed red colonies with white aerial mycelia at 25°C for 7 days, which were identified as Fusarium isolates based on the morphological characteristics and microscopic examination. The average radial mycelial growth rate of Fusarium isolate Fa-25 was 11.44 mm/day at 25°C in the dark on PDA. Macroconidia were stout with curved apical and basal cells, usually with 4 to 6 septa, and 29.5 to 44.2 × 3.7 to 5.2 μm on Spezieller Nährstoffarmer agar (SNA) medium at 25°C for 10 days (Leslie and Summerell 2006). To identify the species, the internal transcribed spacer (ITS) region and translational elongation factor 1-alpha (TEF1-α) gene of the isolates were amplified and cloned. ITS and TEF1-α was amplified using primers ITS1/ITS4 and EF1/EF2 (O'Donnell et al. 1998), respectively. Sequences of ITS (545 bp, GenBank Accession No. MT811812) and TEF1-α (707 bp, GenBank Acc. No. MT856659) for isolate Fa-25 were 100% and 99.72% identical to those of F. asiaticum strains MSBL-4 (ITS, GenBank Acc. MT322117.1) and Daya350-3 (TEF1-α, GenBank Acc. KT380124.1) in GenBank, respectively. A phylogenetic tree was established based on the TEF1-α sequences of Fa-25 and other Fusarium spp., and Fa-25 was clustered with F. asiaticum. Thus, both morphological and molecular characterizations supported the isolate as F. asiaticum. To confirm the pathogenicity, mycelium agar plugs (6 mm in diameter) removed from the colony margin of a 2-day-old culture of strain Fa-25 were used to inoculate melon fruits. Before inoculation, healthy melon fruits were selected, soaked in 2% NaClO solution for 2 min, and washed in sterile water. After wounding the melon fruits with a sterile needle, the fruits were inoculated by placing mycelium agar plugs on the wounds, and mock inoculation with mycelium-free PDA plugs was used as control. Five fruits were used in each treatment. The inoculated and mock-inoculated fruits were incubated at 25°C with high relative humidity. Symptoms were observed on all inoculated melon fruits 10 days post inoculation, which were similar to those naturally infected fruits, whereas the mock-inoculated fruits remained symptomless. The fungus re-isolated from the diseased fruits resembled colony morphology of the original isolate. The experiment was conducted three times and produced the same results. To our knowledge, this is the first report of fruit rot of melon caused by F. asiaticum in China.

Nai plum (Prunus salicina var. cordata cv. Younai) is one of the most popular fruit crop in South China. In July 2023, a fruit rot of nai plum with about 5 % disease incidence was observed in a fruit market of Changsha city, Hunan Province, China. Initially, small, brown lesions appeared randomly on the fruit surface, with disease progression, the lesions gradually expanded and developed into soft rot. To isolate possible fungi from rotten fruits, small pieces (2 × 2 mm) from the periphery of 10 infected fruits were surface-sterilized using 70% ethanol for 10 s, rinsed three times in sterile distilled water, air dried, and then placed onto potato dextrose agar (PDA) plates and incubated at 28℃ for three days. Emerging colonies were subcultured by hyphal tiptransfer on fresh PDA. A total of ten isolates with similar morphology were obtained. Fungal colonies were initially white, gradually turning gray and eventually becoming black, and aerial hyphae were dense and fluffy. Conidia were hyaline, single celled, ellipsoidal to fusiform, and range from 12.7 to 20.0 μm long (avg. 16.9 ± 2.39 μm) × 5.3 to 7.3 μm wide (avg. 6.3 ± 0.82 μm). These morphological characteristics of these isolates matched those of Neofusicoccum parvum (Phillips et al. 2013). To future confirmation of the identify, the internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF1-a), and beta-tubulin TUB2) genes of two representative isolates (JXNP1 and JXNP2) were amplified and sequenced using primer sets ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999; Phillips et al. 2013), and BT2A/BT2B (Glass and Donaldson 1995), respectively. The sequences of both isolates were deposited in GenBank for the ITS (accession nos. OR899331 and OR899332), TEF1-a gene (accession nos. OR909890 and OR909891) and TUB2 gene (accession nos. OR909892 and OR909893). BLAST analysis showed 99-100% identity with the ex-type strain of N. parvum (CMW9081) for ITS, TEF1-a and TUB2. A maximum likelihood phylogenetic tree was constructed using IQtree web server based on combined ITS, TEF1-a and TUB2 data set. The phylogenetic tree revealed that two isolates clustered with N. parvum in a clade with 90% bootstrap support. Based on morphological and molecular data analysis, the isolates were identified as N. parvum. To confirm the pathogenicity, five healthy nai plum fruits were wounded by using a sterile needle after surface sterilization with 75% ethanol, then a 5-mm-diameter mycelial disc of isolate JXNP1 was taped to the wound, the control fruits were taped with sterile agar plugs. All fruits were incubated at 25 ℃ with 80% humidity. After five days, typical naturally occurring fruit rot symptoms appeared on the fruits which inoculated with N. parvum, whereas control fruits remained asymptomatic. To fulfill Koch's postulates, the pathogen was re-isolated from the inoculated fruits and comfirmed as N. parvum by morphological and molecular analysis. Previous studies reported that N. parvum caused fruit rot on various common fruits in China, including loquat, kiwifruit and citrus (Lei et al. 2013; Zhai et al. 2019; Zhou et al. 2013). To our knowledge, this is the first report of N. parvum causing postharvest fruit rot on nai plum in China. This finding provides critical insights for the management of the high-risk disease on plum in China.

Hami melon (Cucumis melo var. saccharinus) is designated as a geographical indication product in China due to its pleasant aroma, crisp texture, sweetness, and coloration (Zhu et al., 2021). In June 2024, approximately 50% (556 out of 1110) post-harvest Hami melons (cv. Xizhoumi 25), cultivated and stored in Turpan, Xinjiang, China (42.88°N, 90.22°E), exhibited initial symptoms of dark brown discoloration after 20 days of storage at 15°C, resulting in fruit rotting accompanied by black-gray fungal growth. To identify the pathogen, segments (5 mm2) from the margins of rotted tissue were excised from five randomly selected symptomatic Hami melons, subjected to surface sterilization twice with 75% ethanol, rinsed three times with sterilized water, and then placed onto potato dextrose agar (PDA) medium. Five morphologically identical single-spore isolates (XJ-1 to XJ-5) were obtained following Leslie and Summerell (2006). On PDA at 25°C in darkness, their average radial mycelial growth rate during the linear growth phase (days 1–3) was 23.67 ± 0.5 mm/day (n = 75; 5 isolates × 5 replicates × 3 time points). Colonies covered the plates by day four and turned dark gray with abundant aerial mycelium by day seven. Conidia from all five isolates (n = 50; 10 conidia per isolate) were dark brown, thick-walled, and exhibited ellipsoid or ovoid shapes with one septum, measuring 19 to 24 × 9 to 13 µm. These morphological features of the five isolates were consistent with Diplodia species (Phillips et al., 2013). For species identification, the internal transcribed spacer (ITS) region, beta-tubulin (TUB), and translational elongation factor 1-alpha (TEF1-α) genes were PCR amplified and DNA sequenced using primer pairs ITS1/ITS4, BT2a/BT2b, and EF1-728F/EF1-968R, respectively (Úrbez-Torres et al., 2008). Multiple sequence alignments with ClustalW revealed that the obtained ITS, TUB, TEF1-α sequences of all five isolates were 100% identical. BLASTn search in GenBank indicated the consensus sequences of the representative isolate XJ-1 (ITS: PQ316093.1; TUB: PQ309646.1; TEF1-α: PV356091.1) shared 98.18 to 100% similarity with reference sequences of Diplodia seriata De Not. (KX464107.1, MT587382.1 for ITS; KX464833.1, MT592548.1 for TUB; KX464597.1, MT592090.1 for TEF1-α). A phylogenetic analysis utilizing the concatenated nucleotide sequences (ITS, TUB, and TEF1-α) grouped all five isolates (XJ-1 to XJ-5) within the D. seriata species complex clade, with a bootstrap support value of 97%. To verify pathogenicity, 20 healthy Hami melon fruits (cv. Xizhoumi 25) were surface-sterilized with 75% ethanol and 2% sodium hypochlorite (NaClO), and then rinsed with sterile distilled water (Zhou et al., 2019). Ten fruits were subsequently wounded with a sterile hole punch and inoculated with mycelium plugs (6 mm in diameter) from a 3-day-old XJ-1 culture. An equal number of fruits inoculated with sterile mycelium-free PDA plugs served as controls. All the inoculated fruits were maintained in an artificial climate chamber at 20°C, with 90% humidity and a 12-h light/12-h dark cycle. After 10 days, all the inoculated fruits exhibited dark brown and water-soaked lesions consistent with those observed on naturally infected fruits, whereas controls remained healthy. The re-isolated fungus from symptomatic fruits matched the original D. seriata isolates in morphology and in ITS, TUB, and TEF1-α sequences, thereby satisfying Koch’s postulates. The pathogenicity test was repeated three times with the same results. To our knowledge, this is the first report of Diplodia seriata causing postharvest fruit rot on Hami melons in China, which will aid in developing future management strategies for this disease in China.

Macleaya cordata is a perennial herb that belongs to the Papaveraceae and is typically prescribed as a traditional antibacterial medicine in China (Kosina et al. 2010). The extract from M. cordata has been widely used in the manufacturing of natural growth promoters as an alternative to antibiotic growth promoters in the livestock industry (Liu et al. 2017), and the products are marketed in 70 countries such as Germany, China, etc (Ikezawa et al. 2009). During the summer of 2019, symptoms of leaf spot were observed on M. cordata (cv. HNXN-001) in two commercial fields (approximately 1, 300 m2 and 2, 100 m2) of Xinning county, Shaoyang City, Hunan Province, China, where approximately 2 to 3% of the plants were affected. The initial symptoms were irregular black and brown spots on the leaves. The lesions expanded and coalesced, eventually leading to leaf blight. Six symptomatic basal leaf sections from six plants from two fields were surface disinfested in 0.5% NaClO for 1 min, then 75% ethanol for 20 s, rinsed in sterile water three times, air dried, and placed onto potato dextrose agar (PDA), one dish for samples from a single leaf. Plates were incubated at 26°C in darkness. Nine strains with similar morphological characters were isolated, and one representative isolate ( BLH-YB-08) was used for morphological and molecular characterization. The colonies on PDA were grayish-green with white round margins. Conidia were typically obclavate to obpyriform, brown to dark brown, and 12.0 to 35.0 × 6.0 to 15.0 μm, and with 1 to 5 transverse septa and 0 to 2 longitudinal septa (n=50). Isolates were identified as Alternaria sp. on the basis of mycelial characteristics, color, and conidial morphology. To confirm identity of the pathogen, DNA was extracted from isolate BLH-YB-08 with the DNAsecure Plant Kit (TIANGEN, Biotech, China). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), actin (ACT), 28S nrDNA (LSU), 18S nuclear ribosomal DNA (SSU), histone 3 (HIS3), internal transcribed spacer (ITS) region of ribosomal DNA, and translation elongation factor 1-α (TEF) genes ( Berbee et al. 1999; Carbone and Kohn. 1999; Glass and Donaldson. 1995; White et al. 1990.) were amplified and sequenced. Sequences were deposited into the GenBank database. They were 100% sequence identity of GAPDH (OQ224996) with A. alternata strain AA2-8 (MH65578; 578/578bp), 100% sequence identity of RPB2 (OQ190460) with A. alternata strain SAX-WN-30-2 ( MK605877; 933/933bp), 100% sequence identity of ACT (OQ923292) with A. alternata strain FCBP0352 (OL830257; 939/939 bp), 100% sequence identity of LSU (OQ891167) with A. alternata XL14 (MG839509 ; 908/908 bp), 100% sequence identity of SSU (OQ139544) with A. alternata strain BJ19.4.1(OM736063; 1,067/1,067 bp), 100% sequence identity of HIS3 (MT454856) with A. alternata YJ-CYC-HC2 (OQ116440 ; 442/442 bp), 100% sequence identity of ITS (MT212225) with A. alternata CS-1-3 (OQ947366; 543/543bp), and 100% sequence identity of TEF (OQ190461) with A. alternata strain YZU 221185 (OQ512730; 252/252 bp). To test pathogenicity, the isolate BLH-YB-08 was cultured on PDA for 7 days to prepare conidial suspensions and the spore concentration adjusted to a final concentration of 1×106 spores/ml. The leaves of five potted 45-day-old M. cordata (cv. HNXN-001) plants were sprayed with conidial suspensions, and five control potted plants were wiped with 75% alcohol and washed five times with sterile distilled water. They were then sprayed with sterile distilled water. Plants were placed in a greenhouse at 25 to 30°C with 90% relative humidity. Pathogenicity tests were conducted twice. Fifteen days after inoculation, lesions were found on inoculated leaves, and the symptoms were the same as those in the field, whereas the controls were healthy. A fungus was consistently isolated from the inoculated leaves and identified as A. alternata by DNA sequencing of the GAPDH, ITS, and HIS3 genes, fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot on M. cordata caused by A. alternata in China. Understanding its etiology may help to control this fungal pathogen, thus reducing economic losses. Funding: Hunan Provincial Natural Science Foundation General Project (2023JJ30341) Hunan Provincial Natural Science Foundation Youth Fund (2023JJ40367) Seed Industry Innovation Project of Hunan Provincial Science and Technology Department Special project for the construction of Chinese herbal medicine industry technology system in Hunan Province "Xiangjiuwei" Industrial Cluster Project of the Ministry of Agriculture and Rural Affairs.

Morel mushroom (Morchella spp.) is a valuable mushroom, which has extremely high nutritional and economic value. In the early March of 2022, a serious rot disease was observed on approximately 30% fruiting bodies at an M. esculenta farm of Suzhou City, Anhui province, China. A white mold-like hyphae was initially present on the pileus, which then gradually spread to the whole fruiting body, eventually resulting in softening of the fruiting body and death. This disease developed rapidly at relatively high temperature (>20°C) and humidity (>85%), resulting in approximately 80% loss of yield. Twenty infected tissues were cut into small pieces (5 × 5 mm) and placed on potato dextrose agar (PDA) and incubated at 25°C for 5 days. Fifteen morphologically similar isolates were obtained and purified using the single spore isolation technique. Colonies of these isolates were yellowish-white, and tomentose with thick aerial hyphae after 7 days at 25°C on PDA plates. Conidiophores were dimorphic: primary conidiophores were Verticillium-like, secondary conidiophores were penicillate. Primary conidiophore stipe length ranged from 68.6 to 180.5 µm, and the base width was 3.2-5.6 µm. Phialides were solitary, straight, generally slightly tapering towards the tip, each producing a small, hyaline drop of conidia. Secondary conidiophores stipe length ranged from 68.4 to 120.5 µm, the base measured 3.3-6.1µm. Phialides were straight to slightly curved, slightly flask-shaped, with widest point below the middle, slightly tapering in the upper part, without visible collarette. Conidia were colorless, smooth, slightly curved, and distally broadly rounded with an average size of 6.3 to 8.2 × 2.4 to 3.7 μm (n=30). These isolates were initially identified as Clonostachys rosea based on morphological features (Schroers et al. 1999). To confirm the identity of C. rosea, primers ITS1/ITS4 (White et al. 1990) and EF1-728F/ EF1-986R (Carbone and Kohn 1999), were used to amplify the internal transcribed spacer (ITS), and translation elongation factor 1-alpha (EF-1α) genes of the representative isolate 5-3-2. These sequences were deposited in GenBank (GenBank accession nos. ON614093 and ON630916) and had 100% and 99.45% nucleotide identity with Clonostachys rosea E5R(17) and Clonostachys rosea KGSJ26 (GenBank accession nos. MK752437 and MT462122), respectively. Single conidium were isolated and multiplied on PDA for pathogenicity testing. To fulfill Koch's postulates, pathogenicity tests were performed using the fruiting bodies. Spores of C. rosea isolates 5-3-2 were collected and diluted with sterile distilled water at a concentration of 1 × 106 conidia/ml. Five healthy fruiting bodies were inoculated with 1 ml of the spore suspension, which were maintained in an artificial climate chamber at 22°C and 85% humidity. Sterile water inoculated on other 10 healthy fruiting bodies served as control. Mycelia grew rapidly and overgrew more than half of the fruiting bodies within 3 days. The fruiting bodies died five days after inoculation. Clonostachys rosea isolates were re-isolated from symptomatic fruiting bodies and identified by the methods described above. The control group showed no symptoms. The experiment was conducted twice. To our knowledge, this is the first identification of Clonostachys rosea as the causal agent of the Morchella sextelata rot.