Report Of Leaf Spot Research Articles

First Report of Leaf Spots Caused by Pestalotiopsis nanjingensis on Photinia × fraseri 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.

First Report of Leaf Spot Caused by Paramyrothecium foliicola on Peanut in China.

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.

First report of Diaporthe eres causing leaf spot on Rhododendron latoucheae Franch. in China.

Rhododendron latoucheae Franch. is an evergreen shrub with charming fragrance and large and abundant flowers that make it highly attractive as an ornamental species. The species is distribution in southwest China covers several different habitats and environments (Zhang, et al. 2022). From May to July in 2023, symptoms of leaf spot were observed on R. latoucheae over a wide portion of the Baili Azalea Forest Area (27°10' to 27°20'N, 105°04' to 106°04'E), Guizhou Province, China. About 500 plants were surveyed, and the incidence of leaf spot on R. latoucheae leaves was 12%, significantly reducing their ornamental and economic value. The affected leaves had irregular, grey white lesions with a clear blackish brown boundary and faint black conidiomata in a brown center. To isolate the pathogen, 15 symptomatic leaves were collected from 10 plants. A few black dots were picked from the lesions with a sterilized needle, plated on water agar, and incubated at 25°C for 24 h to observe spore germination (Choi et al. 1999). Then the germinated spores were transferred onto PDA for further purification and morphological observation. Three single-spore isolates (GULJ1-L7, GULJ1-L8, and GULJ1-L9) identical in morphology were obtained. The isolate GULJ 1-L7 was used for further study. Colonies on PDA irregular grew white felty aerial mycelium, becoming white felted aerial mycelium in the centre and grey-brown mycelium at the marginal area on the upper surface, while the lower surface presents alternating white, tan and taupe. Colony with conidiomata irregularly distributed over agar surface. In the representative isolate, darkly pigmented pycnidia (flask-shaped) were produced over the colony surface on PDA after about 30 days, and oozed milky or yellowish mucilaginous drops. The fungus produced two types of conidia, α and β. Regular α conidia were 5.15-10.29 × 1.54-3.33 μm (n = 50), hyaline, elongated, biguttulate and non-septate. Beta conidia were 20.34-31.91 × 1.01-1.90 μm (n = 50), aseptate, hyaline, smooth, spindle shaped, slightly curved to bent. The morphological features were consistent with the description of Diaporthe eres (Pereira, et al. 2022). The pathogen was confirmed to be D. eres by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), the partial translation elongation factor 1-alpha (TEF) genes and the calmodulin (CAL) using primers ITS1/ITS4, Bt-2a/Bt-2b, EF1-728F/EF1-986R, and CAL-228F/CAL-737R, respectively (Tao et al. 2020). Sequences from PCR amplification were deposited in GenBank with accession numbers OR740563 (ITS), OR754301 (TUB), OR754298 (TEF), and OR754295 (CAL) respectively. BLAST searches of the sequences revealed 99.07% (533/538 nt), 100% (490/490 nt), 99.69% (317/318 nt) and 98.95% (376/380 nt) homology with those of D. eres AR5193T from GenBank (KJ210529.1, KJ420799.1, KJ210550.1 and KJ434999.1), respectively. Phylogenetic analysis (MEGA 7.0) using the maximum-likelihood method placed the isolate GULJ1-L7 in a well-supported cluster with D. eres CBS 138694T. The pathogen was thus identified as D. eres based on the morphological characterization and molecular analyses (Feng, et al. 2013; Tao, et al. 2020). The pathogenicity of GULJ1-L7 was tested through a pot assay. Due to the difficulty of artificial planting wild R. latoucheae, we conducted a pot essay to detect the pathogenicity of GULJ1-L7 using a closely related Rhododendron delavayi Franch. Ten healthy R. delavayi plants were scratched with a sterilized needle (0.45 mm in diameter) on three leaves per plant. Plants were inoculated by spraying α and β spore mixture suspension (106 spores ml-1) of GULJ1-L7 onto leaves until runoff, and the control leaves were sprayed with sterile water. The plants were maintained at 25°C and 75% relative humidity in a growth chamber. The pathogenicity test was repeated three times. After 14 days, the treated leaves developed brown lesions similar to those in the field, whereas the control had no symptoms. The same fungus was reisolated from the infected leaves and identified based on a morphological characterization and molecular analyses. These results fulfilled Koch's postulates. To our knowledge, this is the first report of leaf spot on R. latoucheae caused by D. eres in China. The fungal pathogen identification will provide valuable information for prevention and management of leaf spot disease associated with R. latoucheae.

First Report of Leaf Spot Caused by Corynespora cassiicola on Jasminum sambac in Pakistan.

Jasminum sambac L. is a species of jasmine native to a small region in the eastern Himalayas and is cultivated worldwide as an ornamental plant (USDA-ARS 2016). In Pakistan, it is cultivated for ornamental purposes throughout the country. The flowers of this plant are traditionally used in the preparation of essential oils and for making jasmine tea. The flowers and leaves also have been used in folk medicine to treat breast cancer, epilepsy, ulcers and promote wound healing (Al-Snafi 2018). In December, 2017, almost 10 leaves of 3 plants of J. sambac growing plant nursery of Gehlan, Pattoki, Punjab a province of Pakistan were observed with leaf spot disease. Infected leaves exhibited circular to sub-circular spots with indistinct margins and grey papery centers delimited by dark brown rims. For further microscopic study, the infected leaves were examined under a stereomicroscope. For the isolation and cultural studies of infecting fungus, infected parts of leaves were surface sterilized in 1% NaOCl for about 10 seconds, washed twice in sterilized distilled water, plated on potato dextrose agar (PDA) medium and incubated at 25°C for 4 days. Pure cultures were obtained having colonies of light to dark brown color. Conidia (n=20) were light brown to pale olivaceous brown, smooth, obclavate to cylindrical in shape, 99.5-118.5 μm in length and 12.5-15.0 μm in width, with mostly 3 to 14 pseudosepta. Conidiophores (n=20) were straight to slightly curved, unbranched, and pale to light brown in color. Based on the morphological characteristics of the colonies and conidiophores and conidia, the pathogen was identified as Corynespora cassiicola (Berk and M.A. Curtis) C.T. Wei. (Berkeley & Curtis 1968; Lu et al. 2021; Wei 1950). Genomic DNA was extracted following using modified CTAB method (Gardes and Bruns 1993) and internal transcribed spacer (ITS) region was amplified with ITS1 and ITS4 primers (White et al. 1990). The ITS sequence generated of about 553 bp and deposited in GenBank (accession no. MN954556), was found more than 99% similar to previously deposited sequences of C. cassiicola (GenBank accession nos. MN339671, EU364535, FJ852574, MK139711, EU131374) as verified through BLASTn and phylogenetic tree construction. A pathogenicity test was performed for fulfilling Koch'spostulates. Conidial suspension (105 conidia/ml) of the recovered isolate was sprayed on the 5 healthy leaves of 2-month-old seedling of J. sambac. Mock inoculated plants sprayed sterile distilled water were used as a control. The seedlings were covered with plastic bags to maintain high humidity at 24 to 28°C for a week. Identical disease symptoms to those observed in nursery plants were observed on the leaves of the inoculated plants in 7 days but not mock inoculated plants and results were reconfirmed. The reoccurred fungus was isolated from the diseased spots of the inoculated leaves to complete Koch's postulates and identified microscopically. A representative sample of leaves with lesions was deposited in the LAH herbarium, Department of Botany University of the Punjab, Pakistan (LAH35691). Previously, C. cassicola has been found infecting Jasminum mesnyi in China and Jasminum sp. in Florida (Alfieri et al. 1984; Zhang et al. 2018). The best of our knowledge, this is the first report of leaf spot caused by C. cassiicola on J. sambac in Pakistan. It will establish a foundation for future studies of management strategies for this plant disease caused by C. cassiicola.

First report of Epicoccum nigrum causing leaf spots on Campomanesia guazumifolia (Camb.) Berg in Brazil.

Campomanesia guazumifolia is a native tree that produces fruit that can be consumed fresh or used by industry (Donadio et al., 2002). In February 2022, in the experimental area of the Universidade Tecnológica Federal do Paraná - Brazil, disease was observed in 22 trees, with 50% to 80% severity in crown leaves. Symptoms were small, irregular, or circular-shaped, dark-brown lesions with yellow halos (Figure S1). As the disease progressed, the lesions increased in size, without distinction between mature and young tissues, causing complete leaf wilting. Twenty symptomatic leaves from 11 trees grown in the same orchard line were collected. For fungal isolation, the leaf surfaces were disinfected with 0.5% NaOCl solution for 1 min, rinsed in sterile distilled water, and dried on sterile filter paper. Five fragments of diseased leaf tissue were placed on a potato dextrose agar medium. The morphological characteristics of the colony, such as filamentous mycelium and golden yellow on the upper part, with the presence of circular to ovoid and multicellular conidia (mean 21.00 µm x 24.45 µm, n = 30) of the nine isolates, coincided with the description of the fungus of the genus Epicoccum (Valenzuela-Lopez et al., 2018). Further identification of one of these nine isolates was confirmed by amplifying and sequencing three loci (ITS, β-tubulin, and RPB2) using the ITS1/ITS4, Bt2a/Bt2b, and 5F2/7cR primer pairs, respectively (White et al., 1990, Glass and Donaldson, 1995, O'Donnell et al., 2007). A single representative isolate (Cgen01) was analyzed and submitted to GenBank (OR020968, OR079879, and OR079878). The Bayesian Inference was used to reconstruct the phylogenetic trees (Figure S2), starting from random trees for 5,000,000 generations, using MrBayes v. 3.2.1 (Ronquist et al., 2012). The isolate clustered together with the isolate of Epicoccum nigrum (Chen et al., 2017) with a high posterior probability (0.98). For the pathogenicity tests, four young, healthy branches containing 20 leaves were spray-inoculated with 1.5 mL of conidia suspension of Cgen01 (106 conidia mL-1), covered with perforated transparent plastic bags, and moistened with distilled water in the orchard. The air temperature ranged from 14ºC to 25ºC. Sterile distilled water was used as a control. Three replicates (pathogen and control) on different trees were evaluated. After five days, the fungus was re-isolated from the symptomatic lesion, showing morphological characteristics similar to those of Cgen01. Control branches did not show fungal growth. The inoculation test was conducted twice and similar symptoms were observed. This is the first report of leaf spots caused by E. nigrum on C. guazumifolia in Brazil. E. nigrum, an endophytic fungus described as a mycoparasite, showed phytopathogenic behavior in this study, causing spots and loss of leaves in C. guazumifolia, drastically reducing the production of photoassimilates and affecting the quality of the fruits.

First Report of Leaf Spot Caused by Botrytis cinerea on Strawberry in China.

In the winter of 2022, circular or irregular leaf spots were observed on strawberry (Fragaria × ananassa) planted in commercial fields (cultivar 'xuetu', 'mengzhifu') in Yinzhou, Ningbo, Zhejiang, China (N29°48'48″, E121°39'47″), with disease incidence ranging from 10 to 15% in a field approximately 0.67 ha in size. The estimated crop loss associated with this disease was ~10%. Symptoms included circular or irregular lesions with brown halos and wheel marks, which eventually developed into leaf blight and petiole decay, but spore masses were seldom found on the leaf surface. In severe cases, leaves withered and abscissed. To isolate the causal agent, ten diseased leaves from ten different plants were collected, surface-sterilized with 75% ethanol for 50 s, rinsed twice with sterile distilled water, cut into small pieces (0.5 cm × 0.5 cm), and plated on potato dextrose agar (PDA), then incubated at 25°C in darkness for 5 days. Isolates , which displayed one kind of colony morphology were consistently obtained from each of the ten samples, and 58 single-conidium isolates with the same colony morphology were obtained. The isolation frequency was 58 of 60 samples. The colonies that grew on PDA produced white mycelia, which sporulated after 1 week, producing typical Botrytis-like gray spores. Three isolates (NBCM-1, NBCM-2, NBCM-3) were selected for identification and pathogenicity assays. Conidia were round to ellipsoid, 9.2 to 14.3 μm long (n=50), and 6.4 to 9.2 μm wide (n=50). Sclerotia were not observed on PDA. Based on these characteristics, the pathogen was tentatively identified as Botrytis cinerea (Zhang 2001). PCR was conducted for each of the three isolates to amplify the G3PDH, HSP60, RPB2, NEP1, and NEP2 genes, which are typically used for molecular identification of Botrytis species (Staats et al. 2005; Liu et al. 2016). The resulting amplicons were sequenced, and the sequences were processed using BLAST in the National Center for Biotechnology Information. Sequences of the three isolates were deposited in GenBank (accession nos. OR052082 to OR052086, OR493405 to OR493414). BLASTn analyses showed that isolates were 99 to 100% identical to B.cinerea reported causing leaf spot on strawberry in California; accession numbers MK919496 (G3PDH, 883/883 bp), MK919494 (HSP60, 992/992 bp), and MK919495 (RPB2, 1081/1081 bp). The resulting concatenated data set of G3PDH-HSP60-RPB2-NEP1-NEP2 was used to conduct a multilocus phylogenetic analysis (MLSA) using the maximum likelihood method. The MLSA tree indicated that the three isolates belonged to Botrytis cinerea. To test for pathogenicity, three 1-month-old strawberry (cultivar 'xuetu') plants were inoculated with each isolate (NBCM-1, NBCM-2, NBCM-3). A noninoculated control (sterile water only) was also included. The strawberry plants were inoculated by spraying with conidia suspension (1.0 × 105/ml) until run-off. Inoculations with sterile water served as controls. All plants were kept at 28/25°C (day/night), under a 12:12-h light/dark photoperiod. All plants were covered with transparent plastic bags to maintain humidity for the first 48 h, after which the bags were removed. After 4 to 7 days, leaf spot symptoms similar to those observed in the field were observed in all inoculated plants, while the controls remained healthy. The experiment was repeated three times. The pathogen was reisolated from the inoculated leaves and again identified as B. cinerea, with the same methodology used for the initial identification. Leaf spot caused by B. cinerea on strawberry was recently reportedin California (Mansouripour and Holmes 2020) and Florida (Marin and Peres 2022). To our knowledge, this is the first report of B. cinerea causing leaf spot on strawberry in China. The pathogen is also the causal agent of Botrytis fruit rot on strawberry. Given the high variability of this pathogen (Marin and Peres 2022), further studies on its occurrence, spread, management, and control are required. The identification of this pathogen provides a basis for further research on its management and control.

First Report of Leaf Spot Caused by Boeremia exigua on Panax ginseng in Liaoning, China

First Report of Leaf Spot Caused by <i>Boeremia exigua</i> on <i>Panax ginseng</i> in Liaoning, China

Leaf Spot Caused by Didymella glomerata on Chaihu (Bupleurum falcatum L.) in China.

Bupleurum falcatum is a Apiaceae family herbal medicinal plant, which has the functions of soothing liver, relieving depression, relieving fever, dispelling stagnation, and regulating menstruation. B. falcatum roots have been used in Chinese herbal formbulary for at least 2000 years (Ahmadimoghaddam et al. 2021). In June 2021, infected leaves of B. falcatum that had dark brown, circular, elliptical or irregular shaped lesions or severely withered were obtained in Yichang (30.75 ° N，111.24 ° E), Hubei, China. Disease incidence was approximately 40% in the 20 hm2 B. falcatum plantation base. Fifteen small pieces (3 mm) were cut from the junction between disease and health of surface sterilized (with 75% alcohol) leaves and then plated on potato dextrose agar (PDA). After 3 days incubation, eight isolates with the same colony morphology were sub-cultured and purified by hyphal tip isolation. Isolate CHYB1 cultured on potato dextrose agar (PDA) was selected for identification. The colony was initially white and later producing gray and brown. Pycnidia were dark, spherical or flat spherical, and 78.3 to 137.4 µm in diameter. Conidia were oval mostly, smooth, aseptate, and 18 the size was 3.7 to 5.1 × 1.6 to 2.5 µm. Following DNA extraction, PCR was performed using the TSINGKE 2×T5 Direct PCR Mix kit. Target areas of amplification were the internal transcribed spacer (ITS) and beta-tubulin gene (TUB2) using ITS1/4 (White et al. 1990) Btu-F-F01/Btu-F-R01 primers (Wang et al. 2014), respectively. BLAST analysis of the ITS sequence (MZ818334.1) had 99% similarity to a 498 bp portion of D. glomerata sequence in GenBank (KR709012.1) and TUB2 sequence (OL439060) had 100% similarity to a 323 bp portion of D. glomerata sequence in GenBank (LT592974.1). All isolates (CHYB1-8) were taken for a pathogenicity test in laboratory on surface-disinfested leaves of B. falcatum. Mycelial plugs (5 mm) were excised from the margin of colony cultured for 5 days, and placed on surface-disinfested leaves of potted B. falcatum which involved creating small wounds. The potted plants were placed in a closed bucket to keep 80% relative humidity. Controls were inoculated with non-colonized PDA plugs (5 mm). All treatments had three replicates. On the inoculated B. falcatum, the leaves of B. falcatum appeared brown spot and been covered with off-white hyphae 7 DPI. By comparision, the control leaves had no symptoms. The pathogen was reisolated from the inoculated leaves and exhibited same morphological characteristics and ITS sequence as those of D. glomerata. D. glomerata was reported to cause round leaf spot on Sophora tonkinensis Gagnep and black spot disease of Actinidia chinensis in China (Pan et al. 2018; Song et al. 2020). To our knowledge, this is the first report of leaf spot caused by D. glomerata on B. falcatum in China.

First report of Pseudopithomyces chartarum causing leaf spot on Tetrapanax papyrifer in Guizhou, China.

Tetrapanax papyrifer (Hook.) K. Koch, widely utilized in clinical practices in traditional Chinese medicine, is a medicinal plant whose dried stem pich exhibits activities such as lactation induction, diuresis, and anti-inflammatory effects. The species is native to the southwest of China, such as Guizhou and Yunnan provinces. It thrives in sunlight and warmth and is planted in fertile valleys in the region (Zhang et al. 2023). In July 2021, a leaf spot-like disease was observed on approximately 15% of T. papyrifer (Big T. papyrifer) in a field in Shibing County (127.2°E, 25.2°N), Guizhou Province, China. The symptomatic leaves displayed irregular, watery dark brown lesions with black conidiomata in gray centers and surrounded by yellow halos. To identify the causal agent leading to the disease, 15 symptomatic leaves from five trees in one field were collected. These leaves underwent surface sterilization, which included 30s in 75% ethanol, 2 min in 3% NaOCl, and three times of washing with sterilized distilled water. Thereafter, small pieces of the symptomatic leaf tissues (0.2 × 0.2 cm) were plated on PDA and incubated at 25°C for seven days (Fang 2007). Three isolates were obtained based on the improved single spore isolation method proposed by Gong et al. (2010), and named as GUTC 321, GUTC 523 and GUTC 873. The fungal colonies on PDA were villiform, creamy-white, whorled, and sparse aerial mycelium on the surface with black, gregarious conidiomata. The conidia were ellipsoid, mid brown to dark brown, mainly with 3-4 transverse septa, usually divided by longitudinal septum, often constricted at the septa, 21.8 (12.6-34.5) × 13.9 (8.8-19.8) µm (n=50). The morphological features were consistent with the descriptions of Pseudopithomyces chartarum (Ariyawansa et al. 2015). All three isolates exhibited identical morphological properties. The potential pathogen was confirmed as P. chartarum by amplification and sequencing of the internal transcribed spacer regions (ITS), large subunit ribosomal (LSU) and translation elongation factor 1 alpha (TEF1) genes with primers ITS4/ITS5, LROR/LR7 and EF-983F/EF-2218R, respectively (Ariyawansa et al. 2015; Jayasiriet al. 2019). BLASTn analyses of the sequences showed 100% identity among the three isolates and a high homology (ITS, 99.8%: 598/599; LSU, 100%: 853/853; and TEF1, 100%: 871/871) with those of P. chartarum sequences in GenBank (MT123059, OK655822, and MK360080, respectively). The sequences of the genes from isolate GUTC321 were deposited in GenBank under accession numbers OP269599 (ITS), OP237015 (LSU), and OR069689 (TEF1). Phylogenetic analyses of the concatenated ITS-LSU-TEF1 sequence (2,685 bp) of GUTC 321 using PhyloSuite 1.2.2 with PartitionFinder model revealed that the isolate clustered closely with P. chartarum isolate CBS 329.86T (Cecilia 1986). The pathogenicity of GUTC 321 was tested thereafter on ten healthy T. papyrifer plants grown in pots in growth chamber. The plants were inoculated by spraying with spore suspension (106 spores mL-1) of GUTC 321 or sterile water (control) onto leaves that had been slightly injured with sterilized SiO2 (0.1-0.25 mm) until runoff. The plants were maintained at 25°C in the growth chamber, and monitored for symptom development. Local lesions began to appear on all GUTC 321-inoculated leaves, but not on those of the control plants, 48 hours after inoculation. Seven days after the inoculation, lesions similar to those observed on field plants occurred on GUTC321-inoculated plants but not on the control plants, the lesions observed only in inoculated leaves. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses (ITS, LSU and TEF1) from the infected leaves thus fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot on T. papyrifer caused by P. chartarum in China. Considering the significance of T. papyrifer in Chinese medicine, approximate management measures need to be developed and applied to control the disease in the crop.

Identification and fungicide sensitivity of Alternaria tenuissima causing leaf spot of Tetrapanax papyrifer newly reported in China

AbstractTetrapanax papyrifer, the rice‐paper plant, is an evergreen shrub and is traditionally used as a herbal medicine in China. In October 2021, a serious leaf spot disease was extensively observed on T. papyrifer in Guizhou, China. Given the limited information available about the aetiology of this disease, isolations were performed, the morphological characteristics and multi‐locus phylogenetic analysis of the fungus associated with the disease were described, and the pathogenicity tests and Koch's postulates were completed. To our knowledge, this is the first report of leaf spot on T. papyrifer caused by Alternaria tenuissima in China. Among 11 fungicides tested, 10% difenoconazole WDG had the best inhibition effect in vitro with EC50 values at 0.1 μg mL−1. This study provides new information for the identification and chemical control of Alternaria leaf spot of T. papyrifer.

First report of leaf spot on sweet potato (Ipomoea batatas) caused by Alternaria alternata in Bulgaria

For the first time in Bulgaria, leaf spotting on sweet potatoes was investigated, and the causative agent, Alternaria alternata, was reported. The collected morphological data aimed to determine the identity of the fungus. According to Koch, the primary isolates from the damaged parts are identical to those produced from pathogenicity tests. In a laboratory we tested sixty-two leaves which had similar symptoms, with slight to medium-sized lesions with concentric circles and a yellowish halo. In general, the attack from Alt. alternate affected 7% of all plants and 32% of the examined leaves. The pathogen tests proved that the pathogen could also cause damage to tubers during storage. Knowing the etiology of diseases is essential for taking preventive measures and thus preserving the quality and quantity of agricultural production. Keywords: Alternaria alternata, sweet potato, leaf spots, pathogen, pathogenicity

First Report of Leaf Spot Caused by Alternaria alternata on Cyclocodon lancifolius in China.

Cyclocodon lancifolius Bunge in the family Campanulaceae, and commonly known as Hong Guo Ginseng, is found in the Indonesia, Philippines, Vietnam, Japan, and China. The leaves and roots of C. lancifolius are widely used as tonics by ethnic minorities in Guizhou and Hunan Provinces in China. In addition, the fruit is edible, and it is a new resource for both medicine and food. In June 2022, symptoms of leaf spot (Fig 1 A and B.) were observed on C. lancifolius plants in the medicinal plant greenhouse of Guizhou University of Traditional Chinese Medicine (106°61'E, 26°39'N), Guizhou Province. The incidence of leaf spot on C. lancifolius was approximately 40 to 70% of all leaves in canopy. Early symptoms on leaves were small circular or irregular brown spots. As the disease progressed the lesions gradually expanded, and multiple lesions coalesced to form large irregular brown spots. Eventually the seedlings died and leaves of mature plants wilted. In order to isolate the pathogen, ten leaf pieces (5×5 mm) were cut from the junction of the diseased and the healthy tissues, surface sterilized with 75% ethanol for 30 s, 0.1% mercuric chloride (HgCl2) solution for 60 s, rinsed in sterile water three times, finally dried and placed on potato dextrose agar (PDA) and cultured in the dark at 27°C for 4 days. Five purified fungal isolates were obtained by single spore isolation. The colonies were olivaceous to dark olive with white margins and abundant aerial mycelia. On potato carrot agar (PCA) medium, these fungi produced septate conidiophores. Conidia were obclavate or ellipsoid, brown, with one to four transverse septa and one to two longitudinal septa. Spores measured 7.64 to 14.20 × 3.32 to 6.38 µm (n=50). These morphological characteristics are consistent with Alternaria alternata (S. P. Wiltshire. 1933). To further confirm the identification, four genomic DNA regions including the ribosomal DNA internal transcribed spacer (ITS), translation elongation factor 1-a gene (TEF), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), and Alternaria major allergen gene (Alt a1) were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), TEF1-728F/TEF1-986R (Carbone and Kohn 1999), gpd1/gpd2 (Berbee et al. 1999), RPB2-5F/RPB2-7cR (Liu et al. 1999), and Alt-for/Alt-rev (Hong et al. 2005), respectively. Sequences were deposited in GenBank with accession Nos. ITS: OQ128111, OQ690707, and OQ690708; TEF: OQ200380, OQ700996, and OQ700998; GAPDH: OQ200378, OQ700993, and OQ700995; RPB2:OQ200379, OQ701002, and OQ701004; Alt: OQ675614, OQ700999, and OQ701001. In a BLAST search, the sequences were 99-100% identical with corresponding sequences of A. alternata. A maximum likelihood phylogenetic tree was constructed with the combined sequence data sets of ITS, TEF, GAPDH, RPB2, and Alt a1 using MEGA 11. The isolate DHY0, DHY1, and DHY3 clustered with A. alternata (J. H. C. Woudenberg et al. 2015) (Fig. 2). To fulfill Koch's postulates, leaves on three healthy 3-month-old potted C. lancifolius seedlings were wounded with sterile needles and inoculated with 5 mm diameter mycelium, which was covered moist by sterile cotton for 24 h. Sterile water was used as the control. After inoculation, the plants were incubated at 27°C, 85% relative humidity, and a 12 h photoperiod. The experiment was repeated three times. Fifteen days after inoculation, all the leaves showed leaf spot symptoms that were similar to those observed in the greenhouse, while control leaves were asymptomatic (Fig. 1). A. alternata was successfully re-isolated from the symptomatic leaves and identified by morphology and the molecular methods described above. This pathogen has been reported to cause a leaf disease in a wide range of vegetables (Zhang et al. 2021), flowers (Zhang et al. 2022), and medicinal plants (Xing et al. 2020). To the best of our knowledge, this is the first report of A. alternata causing leaf spot on C. lancifolius in China. The accurate identification of this pathogen will provide a basis for the prevention and control of C. lancifolius leaf spot disease in the future.

First report of leaf spot caused by Canariomyces microsporus in hulless barley in China

AbstractFrom September 2021 to September 2022, leaf spots appeared in hulless barley seedlings in the experimental field of Qinghai University (36°62′ N, 101°77′ E) with an incidence of 35%–60%. In this study, four diseased samples were collected from different experimental sites at Qinghai University for analysis. Moreover, four strains with the same colony and microscopic morphology were isolated. A representative QK‐1 strain was used for DNA extraction and sequencing via PCR amplification using a multi‐gene phylogenetic analysis (ITS, tub2 and EF‐1α). BLAST search revealed that ITS, tub2 and EF‐1α sequence shared 99.59% (accession MH860852), 98.97% (MK926899) and 95.37% (MN078437) nucleotide sequence similarity with isolate CBS276.74 of C. microsporus. The fungus that caused leaf spot of the hulless barley was then identified as Canariomyces microspores. Similar symptoms were observed on hulless barley seedlings after control inoculation with the QK‐1 strain, consistent with the Koch's hypothesis. This is the first study reporting on C. microsporus in Hulless barley in China, suggesting that barley is a new host of C. microsporus.

Leaf Spot caused by Alternaria alternata on Macleaya cordata 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.

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

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.

First report of leaf spot caused by Epicoccum latusicollum on Hemerocallis citrina in China.

Hemerocallis citrina is a popular vegetable crop. Its eatable flower buds contain abundant nutrients, especially lecithin (Guo et al., 2022). In March 2021, leaf spot disease was observed on 90% cultivated H. citrina seedlings in Dazhou city (31°17'56″ N, 107°31'59″ E), Sichuan, China. Totally, 15 diseased seedlings were sampled (three samples per 666 m2). The symptomatic leaves were cut into pieces (5 × 3 mm), superficially disinfected with 70% ethanol for 20 s and 1% Sodium hypochlorite (NaClO) for 40 s, and washed with sterile distilled water six times. The disinfected tissues were incubated on PDA amended with streptomycin sulfate (50 mg/L) in dark at 25 ℃. Two days later, hyphal tips from the edges of growing colonies were transferred to fresh PDA plates. Finally, 40 purified isolates were obtained. Using primer pairs ITS1/ITS4 (Glass & Donaldson, 1995), amplified rDNA internal transcribed spacer (ITS) regions indicated that these isolates belonged to different genera, mainly including Epicoccum, Fusarium and Colletotrichum. Six isolates of Epicoccum genus similar in morphology, named HHC46, HHC47, HHC491, HHC492, HHC51 and HHC58, were selected for identification. Cultured on oatmeal agar for 7 days, colonies were initially white and villose. Fourteen days later, mycelia started to secrete scarlet pigment. The NaOH spot test showed color changed from green to red, identical to that in Epicoccum species (Boerema et al., 2004). Meanwhile, colonies produced abundant conidia. Conidia were ellipsoidal, aseptate, and 4.1 to 6.5 × 1.3 to 2.9 µm (n = 30). Chlamydospores were also observed, globose to subglobose. The morphological features were similar to those of Epicoccum latusicollum (Xu et al., 2022). The DNA sequences of Beta-tubulin (TUB2) and DNA-directed RNA polymerase II second largest subunit (RPB2) of six isolates were amplified and sequenced, using primer pairs Bt2a/Bt2b (Glass & Donaldson, 1995), and RPB2-5f2/RPB2-7cr (O'Donnell et al., 2012), respectively. BLASTN searches indicated our ITS (OP107240 - OP107245), TUB2 (OP131865 - OP131870) and RPB2 (OP131871 - OP131876) sequences except one TUB2 (OP131867), showed 100% identity to the corresponding sequences of E. latusicollum CGMCC:3.18346 (KY742101, KY742343 and KY742174, respectively). There was a nucleotide divergence between OP131867 and reference sequence. Based on concatenated ITS, TUB2 and RPB2 sequences, the constructed phylogenetic tree of Epicoccum species, confirmed that our isolates were E. latusicollum. To test pathogenicity, 2-year-old healthy seedlings of cultivar "chuanhuanghua No.1" were sprayed with conidial suspension of HHC51 (105 conidia/mL), with controls treated with sterile distilled water. Each treatment (biological replicates = 3) was incubated in a greenhouse (at 25°C under 90% relative humidity, 16/8 h light/dark cycle). The experiment was repeated twice. After 18 days, leaf spot symptom in inoculated seedlings appeared. Whereas, non-inoculated controls showed no symptom. The pathogens were re-isolated from diseased leaves and identified as E. latusicollum, based on morphology and molecular methods described above. E. sorghinum was previously reported as causal agent of leaf spot in H. citrina (Ma et al., 2021). To our knowledge, this is the first report of E. latusicollum causing leaf spot in H. citrina worldwide. Our study will assist with monitoring disease distribution in H. citrina and host diversity of E. latusicollum (Chen et al., 2017).

First Report of Leaf Spot on Soybean Caused by Epicoccum sorghinum in Heilongjiang Province, China.

Soybean (Glycine max (Linn.) Merr.) is one of the important oil crops in China. In September 2022, a new soybean leaf spot disease was found in Zhaoyuan County, Suihua City, Heilongjiang Province, China. Symptoms of the initial formation of irregular brown lesions on the leaves, dark brown inside, the periphery is yellow, vein chlorotic yellow, severe leaf spots connected into pieces, late fall off, not the same as previously reported soybean leaf spot (Fig. 1A). The leaf samples of infected plants were collected, and the leaf tissue (5 × 5 mm) was cut from the edge of the lesion, and then surface sterilized with 3% sodium hypochlorite for 5 min, rinsed with sterile distilled water for 3 times, and inoculated on potato dextrose agar (PDA) at 28°C. Isolates growing around the tissues from samples were subcultured on PDA, and 3 isolates were obtained using the single-spore isolation method. The fungal hyphae were white or grayish white in early stage, and the hyphae with light green concentric ring appeared on the front of the colony after 3 days, appeared orange, pink or white convex, irregular shape, reddish brown on the front of the colony for 10 days and black spherical pycnidium can be produced in the hyphae layer for 15 days (Fig.1D, E). Conidia were oval, hyaline, unicellular, aseptate, and 2.3 to 3.7 × 4.1 to 6.8 μm (n=30, Fig. 1F). Chlamydospores were subglobose, light brown, unicellular or multicellular, and 7.2 to 14.7 × 12.2 to 43.9 μm (n=30, Fig. 1H, I). Pycnidia mostly spheroid, brown, and 47.1 to 114.4 × 72.6 to 167.4 μm (n=30, Fig. 1G). A cetyl trimethyl ammonium bromide method was used to extract DNA from 7-day-old. Internal transcribed spacer (ITS), RNA polymerase II (RPB2) and β-tubulin (TUB) gene were amplified using ITS1/ITS4 (White et al. 1990), RPB2-5F/RPB2-7cR (Liu et al. 1999) and BT2a/Bt2b (O'Donnell et al. 1997) primers respectively. The sequences obtained by polymerase chain reaction (PCR) were sequenced and the results showed that the DNA sequences of the 3 isolates were identical. Therefore, the sequence of isolate DNES22-01, DNES22-02 and DNES22-03 was submitted to GenBank. According to BLAST search, the ITS (OP884646), RPB2 (OP910000) and TUB (OP909999) sequences showed 99.81% similarity to Epicoccum sorghinum strain LC12103 (MN215621.1), 99.07% to strain P-XW-9A (MW446946.1), and 98.85% with the strain UMS (OM048108.1), respectively. Phylogenetic analysis by maximum likelihood method (MEGA7.0) generated based on the ITS, RPB2 and TUB sequences indicated that the isolates formed a supported clade to the related E. sorghinum type sequences. Isolates was found to be most closely related to E. sorghinum and far from other species. Based on morphological and phylogenetic characteristics, isolates DNES22-01, DNES22-02 and DNES22-03 was identified as E. sorghinum (Bao et al. 2019; Chen et al. 2021; Zhang et al. 2022). At the 4-leaf-stage, 10 soybean plants were inoculated by spraying with a conidial suspension (1 × 106 spores·ml-1). Sterile water served as a control. The test was repeated 3 times. All samples were incubated in a growth chamber at 27°C. Symptoms typical developed on the leaves after 7 days, but control samples remained healthy (Fig.1B, C). The fungus was reisolated from symptomatic tissues and identified as E. sorghinum by morphology characteristics and molecular characterization. To our knowledge, this is the first report of E. sorghinum causing leaf spot on soybean in Heilongjiang, China. The results can provide the basis for future studies on the occurrence, prevention, and management of this disease.

First report of leaf spot caused by Botrytis cinerea on Bletilla striata in Anhui province, China.

Bletilla striata (Thunb.) is a perennial herb plant of the orchidaceous family and is used as an ornamental plant in Europe and the United States. Furthermore, it is important as traditional Chinese medicine (TCM) in East Asian countries, such as China, Japan, Korea, Mongolia, and Myanmar (Gou et al. 2022). In April 2023, a severe disease similar to gray mold occurred in a B. striata plantation in Anqing, Anhui province, China (N30°27'15″, E116°18'32″), causing disease on about 20% of the plants in the field. Early symptoms were characterized by brown spots or stripes on the leaves of B. striata, and as the disease progressed, large brown irregular spots appeared. Eventually disease spots coalesced, covering the entire leaf surface and causing leaf death. A gray mildew layer was observed on the senescent leaves. To investigate the causal agent, 10 plants with typical symptoms were collected from the field. Leaf pieces (5 × 5 mm) from the border of infected areas were soaked in 75% ethanol for 10 seconds, and then transferred into 0.1% mercury bichloride for three min, rinsed three times with sterile water, and transferred to PDA at 25 °C for three days. Pure cultures were obtained by single spore isolation, and the resulting colonies were morphologically similar, indicating a single pathogen, of which the representative BSFC-7 was selected for further study. BSFC-7 colonies were initially white to gray-brown, and cottony aerial hyphae grew over the entire petri dish after five days of incubation. Grayish, branched conidiophores and their terminal unicellular conidia were observed under a microscope after additional two days at 25 °C. Conidia were colorless or gray, elliptical or oval, and 7.06-12.54 × 8.33-13.55 μm (n=30). Sclerotia appeared in BSFC-7 culture up to about two weeks and were black, hard, and round or irregularly shaped (0.81-4.32 × 0.97-5.68 mm, n=20). The morphological characteristics fit the description of Botrytis cinerea (Li et al. 2016). To further identify the species, genomic DNA of BSFC-7 was extracted. PCR analysis was performed with species-specific primer pairs C729+/C729- and two nuclear genes G3PDH and RPB2 with their corresponding primer pairs G3PDH-F/G3PDH-R and RPB2-F/RPB2-R (Rigotti et al. 2002; Aktaruzzaman et al. 2018). The sequences for all three PCR products of C729, G3PDH, and RPB2 (GenBank accession nos. OR287069, OR255923, and OR255924 respectively) exhibited 99 to 100% similarity with other B. cinerea isolates. In the pathogenicity test, detached leaves of B. striata were inoculated with the BSFC-7 isolate. The leaves were soaked in sodium hypochlorite (1%) for two min, washed with sterile distilled water, and then inoculated with 10 µl of conidial suspension (106 conidia/ml). Sterile water was used as control and samples were incubated at 25 °C. After three days, all leaves inoculated with conidia showed dark brown water-soaked lesions similar to those observed in the field, while the control leaves remained healthy. The pathogen was re-isolated from the affected leaves, fulfilling Koch's postulates. B. cinerea is a common pathogen on a wide range of host plant species worldwide and has been reported to infect B. striata in Yunnan province, China (Romanazzi and Feliziani 2014; Zhang et al. 2020). To our knowledge, this is the first report of B. cinerea causing leaf spots on B. striata in Anhui province, China. This study will provide a basis for controlling the prevalence and economic losses of gray mold on B. striata.

First Report of Leaf Spot Caused by Clonostachys rosea on Tea (Camellia sinensis) in China.

Tea (Camellia sinensis), which originated in southwest of China 60 - 70 million years ago, is widely consumed as a beverage for its potential enhancing effect on human health with rich polyphenol content (Pan et al. 2022).From October to December in 2021, a disease with symptoms similar to leaf spot affected the quality and yield of teaPuer (102°73 'E, 25°07' N), Yunnan province, China. Based on the survey, leaf spot symptoms were observed on approximately 60% of tea plants in a 5,700 m2 field. The symptoms initially appeared as shrinking, yellowing, and later became circular or irregular brown spots. To isolate the pathogen, 10 symptomatic leaves were collected from 10 trees, and portions of the diseased tissue (0.5×0.5 cm) were cut at the junction of infected and healthy tissues. After surface sterilization (0.5 min with 75% ethanol and 2 min with 3% NaOCl, washed three times with sterilized distilled water), the disinfected pieces were dried and plated onto potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. Four single-spore isolates, FH-1, FH-5, FH-6 and FH-7, were obtained, these isolates were identical in morphology and in the sequences of internal transcribed spacer region [ITS] and translation elongation factor 1-alpha [TEF] genes. Therefore, the representative isolate FH-5 was used for further study. Fungal colonies were white or light yellow on PDA after 7 days incubated at 28ºC. Conidia were hyaline, round or oval, aseptate, occur singly or in clusters on hyphae or conidia stalks, and measured as 2.94 ± 1.79 × 1.82 ± 0.2 μm (n = 50). Primary conidiophores is Verticillium-like (Fig1.K,L), which generally formed first, 1-3-level verticillate, mostly with divergent branches and phialides, and measured as 16.67 ± 4.39 µm (n = 50). Secondary conidiophores is penicillate (Fig1.I,J), which generally appearing after one week, sometimes even more often branched, and with a length of 16.02 ± 3.83 µm (n = 50). The morphological features were consistent with the descriptions of Clonostachys rosea Schroers H.J. (Schroers et al. 1999). The pathogen was confirmed to be C. rosea by amplification and sequencing of the internal transcribed spacer region (ITS) and translation elongation factor 1-alpha (TEF) genes using primers ITS1/ITS4 and EF1-728F/EF1-986R, respectively (Fu Rongtao 2019). The sequences of PCR products were deposited in GenBank with accession numbers ON332533 (ITS) and OP080234 (TEF). BLAST searches of the obtained sequences revealed 99.22% (510/514 nucleotides) and 98.37% (241/245 nucleotides) homology with those of C. rosea HQ-9-1 form GenBank (MZ433177 and MZ451399, respectively). Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate FH-5 in a well-supported cluster with C. rosea. The pathogenicity of FH-5 was tested through a pot assay. Ten healthy tea plants were scratched with a sterilized needle on the leaves. Plants were inoculated by spraying a spore suspension (105 spores·mL-1) of FH-5 onto leaves until runoff, and the control leaves sprayed with sterile water. Inoculated plants were put in an artificial climate box at 25℃, 70% relative humidity. The pathogenicity test was replicated three times. Symptoms developed on all inoculated leaves but not on the control leaves. Lesions around the wound edge became pale yellow, and brown spots were first observed at 72 h after inoculation, and typical lesions similar to those observed on field plants appeared after two weeks. The same fungus was reisolated and identifiedbased on themorphological characterization and molecular analyses (ITSand TEF)from the infected leaves but not from the noninoculated leaves. In addition, C. rosea has also been reported to cause diseases to broad bean (N. Afshari et al. 2017 ), garlic (Diaz et al. 2022), beet (Haque M.E et al. 2020) and other plants. To our knowledge, this is the first report of leaf spot on tea caused by C. rosea in China. This study provides valuable information for the identification and control of the leaf spot on tea.

First report of leaf spot caused by Curvularia muehlenbeckiae on Fraxinus rhynchophylla Hance in China.

Fraxinus rhynchophylla Hance, is a deciduous trees cultivated on a commercial scale focused on medicinal and wood production. In September 2021, leaf spot was observed on F. rhynchophylla in Heilongjiang Province (127.34°E, 45.19°N), China. These symptoms were observed on 100% F. rhynchophylla plants and the incidence of diseased leaves per plant reached 70% in fields measuring 90 ha. Disease symptoms were small yellow flecks initially, and then turned to gray necrotic spot. Ten diseased leaves were collected randomly from 5 plants and surface disinfested. Tissue samples (2 × 2 mm) were cut at the disease-health junction of the leaves, surface sterilized in 75% ethanol for 30 s, submerged in a 7% NaOCl solution for 3 mins, and rinsed three times with sterile water. Leaf segments were placed onto potato dextrose agar (PDA) and incubated at 26℃ for 5 days. After isolation and purification of monospore, the colonies of the all isolates were inky black, with aerial fluffy mycelium, and concentric whorls on PDA. The conidiophore is septate, single-branched, brown, smooth and 35 - 313 × 2 - 5 μm in size (n = 50), while the conidia are brown, bow-shaped, mostly four cells, with three septa and 12 - 385 × 5 - 20 μm in size (n = 150). The morphological characters matched those of Curvularia muehlenbeckiae (Madrid et al. 2014). DNA was extracted from isolates HQLa and HQLb and used for PCR amplification of RNA polymerase II gene (RPB2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene sequences using the primer fRPB2-SF/fRPB2-7Cr (Schoch et al. 2009), gpd1/gpd2 (Berbee et al. 1999), respectively. The RPB2 (OM984674, OM984675) and GAPDH (OM984672, OM984673) were deposited in GenBank. The phylogenetic tree was constructed by combining other published sequences of RPB2 and GAPDH genes using the maximum likelihood method, and the results showed that the obtained isolates clustered into the same clear branch as C. muehlenbeckiae CBS 144.63 (HG779180, HG779108), with 100% bootstrap support. Combining morphological characteristics and phylogenetic analysis of the fungus, the obtained isolates were identified as C. Muehlenbeckiae. To fulfill the Koch's postulates, pathogenicity tests were carried out on newly grown leaves of F. rhynchophylla. Conidia of the selected isolates grown on PDA plates were flooded with sterile distilled water. Spore suspension was adjusted to 105 spores/mL with the hemocytometer. Three leaves of each plant were disinfected with 1% NaOCl for 2 min, washed with sterilized distilled water three times, and dried with sterile paper towels. Three plants were randomly selected for inoculation under field conditions and each leaf was sprayed with 2 mL of the spore suspension for a total of nine leaves, then the plants were bagged and moistened for 48 h. However, control leaves were sprayed with distilled water. Symptoms were observed nine days after inoculation. No symptoms were observed on control leaves. The same fungus was successfully re-isolated from the lesions. The experiment was replicated three times with the same results and C. muehlenbeckiae identification was confirmed by morphological observations and RPB2 and GAPDH sequencing, indicating that the fungus is the causal pathogen of leaf spot disease on F. rhynchophylla. This is the first report of C. muehlenbeckiae determined as fungal pathogens on F. rhynchophylla plant in China. The results of the study laid the foundation for the future occurrence and epidemiological pattern of the disease and scientific control.