Glyceraldehyde-3-phosphate Dehydrogenase Sequences Research Articles

Structure, function and stability analysis on potential deleterious mutation ensemble in glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for early detection of LUAD

AimsLung adenocarcinoma (LUAD) is the most prominent histological subtype among the lung cancer which is a leading cause in the cancer mortality rate. High mutational and glycolytic rates are the major reported alterations in the lung cancer. Here in our study we are elucidating the structural and functional role of key glycolytic enzyme Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and associated SNPs in LUAD progression. Materials and methodsOur gene expression analysis reveals high expression of GAPDH in the LUAD. In silico tools and analysis were used for the identification and characterization of the deleterious SNPs. Molecular Docking and dynamics simulations (MDS) studies characterized the structural consequences of prioritized deleterious mutations. Key findingsThe sequence based analysis to identify SNPs in GAPDH resulted in 28 deleterious SNPs and 6 SNPs among them showed deleterious and damaging effect. The structural based analysis resulted in 2 stabilizing SNPs of rs ids rs11549328 (D39Y) and rs200102749 (S51Y) in the conserved domain. The IDR and PTM analysis of the GAPDH sequence resulted an IDR region from 191 to 194 positions with an IDR score of 0.511, 0.520, 0.517 and 0.503 with the PTM modifications. SignificanceThe identified deleterious SNPs (D39Y and S51Y) fall in the functional and conserved domain of GAPDH. In addition, the existence of PTMs within the IDR region of the GAPDH may contribute to its enhanced glycolytic activity in LUAD. The results of our study provide potential background deleterious mutants the pathological aspect of GAPDH in LUAD progression.

First Report of Anthracnose Caused by Colletotrichum spaethianum on Zephyranthes candida in China.

Zephyranthes candida, an bulbous perennial plant, are planted in almost every park. In October 2023, anthracnose symptoms were observed on Z. candida leaves in Jiangxi Agricultural University (28.75° N, 115.83°E), Nanchang, Jiangxi Province, China, and the incidence of disease were up to 35% (140 of 400 plants). The lesions extended fromtheleafapextothebase, appearing as a dark brown color, and later changed to yellow and became dry. To isolate the pathogen, 20 symptomatic leaves were collected and cut into small pieces (4×4 mm, one pieces per leave), surface-sterilized with 70% ethanol for 10 s and 1% NaClO for 30 s, rinsed thrice with sterile water, placed onto potato dextrose agar (PDA) plates and incubated at 25℃ for 5 days. Fifteen isolates (15 out of 20) with similar morphological characteristics were obtained. The colonies on PDA presented effuse mycelium, initially white and later pale gray. Conidia were hyaline, curved or slightly curved, aseptate, with a truncate base and acute apex, measuring 17 to 23 × 3 to 6 μm (n = 50), and were matched to Colletotrichum species (Damm et al. 2009). To further confirm species, two representative isolates (JFRL 03-2873 and JFRL 03-2874) were selected for molecular identification. The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS), histone 3 (HIS3), actin (ACT) and β-tubulin 2 (TUB2) regions were amplified and sequenced by using primers sets ITS5/ITS4, Gpd1/Gpd2, CHS-79F/CHS354R, CYLH3F/CYLH3R, ACT-512F/ACT-783R and T1/Bt2b (Tan et al. 2022), respectively. These sequences were deposited into GenBank with accession number PP425890-PP425891 (ITS), PP437551-PP437552 (GAPDH), PP437549-PP437550 (CHS), PP480643-PP480644 (HIS3), PP437547-PP437548 (ACT) and PP437553-PP437554 (TUB2). A BLASTN search revealed high similarity of 99%-100% to ITS (GU227807, 518 nt/519 nt), GAPDH (GU228199, 525 nt/526 nt), CHS (GU228297, 251 nt/251 nt), HIS3 (GU228003, 372 nt/373 nt), ACT (GU227905, 236 nt/237 nt) and TUB2 (GU228101, 490 nt/490 nt) sequences of Colletotrichum spaethianum CBS 167.49. A maximum likelihood phylogenetic tree was constructed by combining ITS, GAPDH, CHS , HIS3, ACT and TUB2 sequences in IQtree web server (Ngugen et al. 2015). The result indicated that the two representative isolates were clustered together with Colletotrichum spaethianum in a clade with 100% bootstrap support. Based on morphologicalobservationandsequenceanalysis, the isolates were identified as C. spaethianum. To confirm pathogenicity, six surface-sterilized leaves of Z. candida were wounded and inoculated with 1 × 106 conidia/ml conidial suspension of JFRL 03-2873, and control leaves were inoculated with sterile water. They were incubated at 25 ℃ with 12 h photoperiod and 80% humidity, theexperimentwasrepeatedtwice. After five days, all leaves inoculated with JFRL 03-2873 displayed anthracnose symptom, whereas the control leaves remained unaffected. We re-isolated C. spaethianum from the symptomatic leaves and identified it based on morphological and molecular characteristics. Previous studies reported that C. spaethianum caused anthracnose on various common herbaceous plants in China (Vieira et al. 2014, Guo et al. 2013), but to our knowledge, this is the first report of C. spaethianum causing anthracnose on Z. candida in China. Anthracnose disease caused great economic loss to the cultivation of landscape plant Z. candida. Therefore, it is necessary to pay more attention to the anthracnose disease of herbaceous plants caused by C. spaethianum and develop appropriate control strategies.

First Report of Colletotrichum siamense Causing Anthracnose on Bixa orellana in China.

Annatto (Bixa orellana L.) is widely cultivated in China. Its seed is used as medicine and as an astringent antipyretic. Since 2019, anthracnose-type lesions have been observed on the annatto leaves in the field (about 30 hectares) in Zhanjiang (21˚18'12''N, 110˚17'22''E), Guangdong Province, China. Disease incidence was around 70% (n = 100 investigated plants from about 3 ha). The early symptoms were yellow spots on the edge or tip of leaves. The spots gradually expanded and became dark brown, eventually coalescing into large irregular or circular lesions (Supplemental Figure 1-A). Ten symptomatic leaves from 10 plants were sampled. The margins of the lesions were cut into 2 × 2 mm pieces and the surfaces were disinfected with 75% ethanol for 30 sec and 2% sodium hypochlorite for 60 sec. After that, pieces were rinsed thrice in sterile water, placed on potato dextrose agar(PDA) medium, and incubated at 28 ℃ for 3 days. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty isolates were obtained. Three representative single-spore isolates (BOC-1, BOC-2, and BOC-3) from the twenty isolates were confirmed to be identical based on morphological characteristics and ITS analysis and used for further study. Besides, the three isolates were deposited in the fungus collection at Aquatic Organisms Museum of Guangdong Ocean University. Colonies on PDA were white to gray with cottony mycelia after incubating in the dark for 6 days at 28 ℃. Conidia were one-celled, hyaline, cylindrical, clavate, and obtuse at both ends; they measured 9.6 to 18.5 µm × 3.5 to 5.5 µm (n = 50). Appressoria were oval to irregular in shape and dark brown, and they measured 6 to 9 µm × 4.5 to 8 µm (n = 30) (Supplemental Figure 1-D, E and F). These morphological characteristics matched the description of Colletotrichum siamense (Prihastuti et al. 2009;Sharma et al. 2013). For molecular identification, the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and actin (ACT) loci of the isolates were amplified using primer pairs ITS1/ITS4, GDF1/GDR1, CHS-79F/CHS-354R, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). Sequences were deposited in GenBank under nos. MZ047377-MZ047379 (ITS), MZ126934-MZ1269346 (GAPDH), MZ126904-MZ1269046 (CHS-1), and MZ126844-MZ1268446 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from ITS, GAPDH, CHS-1, and ACT sequences that clustered the three isolates with C. siamense (the type strain MFLU 090230), (Supplemental Figure 2). The pathogenicity of the three isolates was tested respectively in a greenhouse maintained at 25 to 29℃ and 80% relative humidity. Annatto seeding ( n =5, 2-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.. The experient was repeated three times. Anthracnose lesions were observed on the inoculated leaves after 10 days while the control plants remained healthy (Supplemental Figure 1-G, and H). The same pathogen was re-isolated from all the inoculated leaves based on morphology and ITS analysis. C. siamense has been reported to cause anthracnose in a broad range of hosts (Weir et al. 2012; Wang et al. 2017; Liu et al. 2017; Zhuo et al. 2017 ), but not in B. orellana. To our knowledge, this is the first report of C. siamense causing anthracnose on B. orellana in China. Our study provides important reference information for controlling this disease.

First report of anthracnose caused by Colletotrichum grevilleae on apple in Korea.

In October 2022, typical symptoms of anthracnose were observed on apple (Malus ⅹ domestica cv. Fuji) fruits collected from Pocheon in Gyeonggi province, South Korea (N37.98074°, E127.33995°). In the surveyed orchard, the incidence rate of apple anthracnose was less than 1%. The initial symptoms were brown-to-dark brown lesions, and with disease progression, they enlarged and the pulp became soft, forming a brown band. In total 29 apple fruits were collected, and the causal agent was isolated by removing the peel, and the diseased tissues were directly transferred onto potato dextrose agar (PDA), followed by incubation for 7 days at 25°C. As the results, two isolates (GgPc22-1-11 and GgPc22-1-13) were obtained. For describing morphological and cultural characteristics, isolate GgPc22-1-11 was cultured on PDA and synthetic nutrient-poor agar (SNA) at 25°C under near-UV light with a 12-h photoperiod for 10 days. The colonies of GgPc22-1-11 on PDA were initially white and subsequently appeared light gray to olivaceous with white margins. The reverse side of the plates were dark brown and slate blue (Supplementary Fig. S1). Colonies on SNA were flat with an entire margin and short sparse white aerial mycelium. No setae were observed. Conidia on PDA were hyaline, straight, aseptate with a rounded apex, clavate to cylindrical, and measured 16.4 ± 2.4 (10.8-23.8) × 5.5 ± 0.7 (3.6-7.7) μm (n = 200). Appressoria were medium-to-dark brown, aseptate, solitary or in groups with irregular outlines, and lobate or having undulate margins (Supplementary Fig. S1). These morphological and cultural characteristics of GgPc22-1-11 were consistent with those of Colletotrichum grevilleae F. Liu, Damm, L. Cai & Crous, pathogens of Proteaceae and Punica granatum (Liu et al. 2013; Huang et al. 2023). DNA was extracted from GgPc22-1-11, PCR was performed and Phylogenetic analysis of concatenated partial sequences of the internal transcribed spacer (ITS) of rDNA, β-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), and actin (ACT) genes was conducted (Weir et al. 2012). The resulting sequences were deposited in GenBank under the accession numbers LC773710-LC773714. A nucleotide BLAST search revealed that the ITS sequences of the isolates were 98.95% identical to those of C. grossum CAUG7 (KP890165.1). The TUB2, GAPDH, CHS-1, and ACT sequences of the isolates were 99.79%, 99.24%, 100%, and 100%, respectively, identical to those of C. grevilleae WP4. GgPc22-1-11 was clustered with C. grevilleae WP4 using neighbor joining analysis conducted with MEGA X software (Kumar et al. 2018) (Supplementary Fig. S2). Pathogenicity tests were conducted using GgPc22-1-11 and repeated three times. A total of 12 symptomless apples of each variety were selected, including Fuji, Hongro, Tsugaru, and RubyS. The apples were surface-sterilized with 70% ethanol and wounded using a sterile needle. Both wounded and unwounded apples were inoculated with mycelium plugs and paper disks containing a conidial suspension (1 × 106 conidia/ml) and placed in a plastic box with moist paper towels (>90% relative humidity) at 25°C in dark. At 5 days after inoculation, all artificially wounded fruits exhibited symptoms and 30% (4 out of 12) of unwounded inoculated fruits showed symptoms in each apple variety while control fruits were asymptomatic both the unwounded and wounded inoculations (Supplementary Fig. S1). To fulfill Koch's postulates, the fungi were reisolated from symptomatic tissues and were identical to GgPc22-1-11 confirmed by morphological and molecular analysis. To the best of our knowledge, C. grevilleae has been reported in Protea sp. and pomegranate (Liu et al. 2013; Huang et al. 2023) but not in apples to date, and this is the first report of C. grevilleae causing anthracnose in apple fruits. This research of the newly emerged unreported Colletotrichum species can offer valuable information for development of an effective fungicide spray program to control apple anthracnose.

First Report of Colletotrichum cordylinicola Causing Anthracnose on Cordyline fruticosa in China.

Cordyline fruticosa is a shrub plant, commonly used in landscape, and distributed in the tropical regions of southern China. In September 2022, anthracnose symptoms were found on this species in Nanning, Guangxi, China. The disease incidence was between 30% to 80% and disease severity was 10% to 30% in five surveyed planting areas. The symptoms initially appeared as small, round, brown spots on leaves. As the disease developed, the lesions turned gray-white with brown borders and yellow halos. Some spots coalesced into larger irregular shapes and even leading to leaf blight. Small segments of the diseased tissues (3×3 mm) were cut from the leaves, surface-sterilized by dipping in a 1% sodium hypochlorite solution for 1 min, rinsed three times with sterile distilled water, and plated on potato dextrose agar (PDA). These plates were incubated at 28°C in the dark for 5 days. Ten fungal isolates with similar morphology were consistently isolated from these diseased tissues. The colonies on PDA were initially white with sparse aerial mycelia and turned pale orange with abundant orange conidial masses on the center after 8 days of culture. The reverse color was pale orange. No sclerotia or setae were found in culture. Conidia were single-celled, hyaline, straight, cylindrical with round ends, and 12.2 to 17.8 µm long (mean 14.9 µm) and 3.9 to 7.3 µm wide (mean 4.8 µm, n=50). The morphological characteristics of these isolates were similar to the Colletotrichum cordylinicola (Sharma et al., 2014). Genomic DNA of two isolates Z3 and Z4 generated from monospore culture was extracted using a fungal DNA extraction kit (Solarbio, Beijing, China). Partial sequences of internal transcribed spacer (ITS), partial actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2) were amplified using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-345R, GDF1/GDR1, and BT2A/BT2B (Lin et al., 2022), respectively. All the sequences (GenBank accession nos. OQ509909, OQ509910, OQ658690, OQ658691, and OK649310 to OK649314) showed 99% to 100% identity with those of C. cordylinicola in GenBank database. A phylogenetic tree based on concatenated sequences of ITS, ACT, CHS-1, TUB, and GAPDH using maximum likelihood analysis by MEGA X software revealed that Z3 and Z4 clade with reference strains of C. cordylinicola (OJX010226 and MK935473). Based on morphological observation and multi-gene sequence analysis, the isolates were identified as C. cordylinicola (Phoulivong et al., 2010). To assess their pathogenicity, conidial suspensions (106 conidia/ml) of C. cordylinicola were inoculated onto 10 healthy living leaves wounded by slight puncturing (10 μl/wounded spot). Control leaves were treated with sterile water. All inoculated and control plants were maintained under high relative humidity (~90%) and 28℃ in a climate chamber. After 8 days, all the inoculated leaves showed brown lesions resembling natural symptoms, whereas the control group remained symptom-free. The same fungus was re-isolated from the symptomatic leaves, thus completing Koch's postulates. C. cordylinicola is a species of the C. gloeosporioides complex (Weir et al., 2012). It has been reported to cause anthracnose on C. fruticosa in USA and Thailand (Phoulivong et al., 2010; Sharma et al., 2014). To our knowledge, this is the first report of C. cordylinicola causing anthracnose on C. fruticosa in China. Knowing the causal agent is essential to control the serious disease effectively.

First Report of Leaf Spot on Taraxacum mongolicum Caused by Alternaria solani in China.

Taraxacum mongolicum is a perennial herbaceous plant in the family Asteraceae, with a high edible and medicinal value and is widely planted in China. In August 2022, leaf spots were found on T. mongolicum in Tianjiazhai Town, Xining City, Qinghai Province, China (36°27'17.65″N, 101°47'19.65E, elevation: 2,408 m). The plants exhibited round or irregular brown spots, and the centers of some of the spots were gray (Fig. S1A). An investigation was performed over a hectare area, and the incidence of leaf spot reached 15%-30%, seriously affecting the quality and yield of T. mongolicum. Eleven T. mongolicum leaf spot samples were collected. To isolate the pathogenic fungus, approximately 0.5 cm×0.5 cm pieces of tissues were obtained using sterile scissors from the junction of infected and healthy tissues. The symptomatic leaves were surface-disinfected with 3% NaClO for 1.5 min and washed three times with sterile water. The disinfected pieces were dried and placed on water agar plates in an incubator for 2 days at 25°C. Subsequently, the leaf surface exhibited conidiophores and conidia. Eleven isolates were obtained by single spore isolation. The sparse aerial mycelia were dark grey to black brown in color on potato dextrose agar (PDA) (Fig. S2A), and produced dark, multi-septate conidia with 7-11 transverse septa and 1-2 longitudinal septa (Fig. S2C). Conidia with one or two beaks were long-ovoid, with an average length and width of 103.4 × 21.2 μm, and 80.7 × 3.9 μm of the beaks. One hundred and ten conidia were measured. The identification of 11 isolates was confirmed by multilocus sequence analyses of the internal transcribed spacer of ribosomal DNA (rDNA ITS) (White et al. 1990), and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Xu et al. 2022), actin (ACT) (Yang et al. 2020), histone 3 (HIS3) (Zheng et al. 2015), translation elongation factor 1-α (TEF1-α) (Carbone. 1999), and the second largest subunit of RNA polymerase II (RPB2) (Liu et al. 1999) genes. The sequences of all the isolates were deposited in Genbank (NCBI Accession Nos. ITS: OR105029-OR105039, ACT: OR135220-OR135230, GAPDH: OR135231-OR135241, HIS3: OR122992-OR123002, TEF1-α: PP055972-PP055982, and RPB2: PP055983-PP055993), and the sequence similarity of ITS, ACT, GAPDH, HIS3，TEF1-α and RPB2 were 100%, 98%, 100%, 99%, 100%, and 99% to the sequences of Alternaria solani, respectively. Combined sequences of ITS, GAPDH, TEF1-α, and RPB2 genes were concatenated and a maximum parsimony tree was constructed with PAUP* v. 4.0 alpha. The results indicated that 11 isolates were clustered together with A. solani (Fig. S2D). Therefore, 11 isolates were identified as A. solani based on their morphological and molecular characteristics. Eleven isolates were inoculated on their host to perform Koch's postulates. The isolates were grown on PDA for six days. Healthy one month old T. mongolicum seedlings were planted in 10 cm flowerpots (Fig. S1B) or the seedlings were moved to Petri dish (Fig. S1C), and their leaves were inoculated with 5 mL of hyphae suspension by smearing method. In addition, seedlings of the same age were treated with sterile water to serve as the control. The inoculated seedlings were moved into an artificial climatic box at 25℃, relative humidity was 70%, with 12 h light/12 h dark condition. Totally 80 seedlings were inoculated with isolates and 15 were used as the control. After 7 days, similar symptoms were observed on the plants inoculated with isolates, while control plants did not produce symptoms. The assays were conducted three times. Furthermore, isolates were re-isolated from the symptomatic leaves, and the colonial morphology was the same as the original isolates (Fig S2 A and B). The recovered isolates were identified as A. solani by amplifying and sequencing a portion of the HIS3 gene. Alternaria solani has been previously reported to cause early blight of potato and other Solanum crops (van der Waals et al. 2004; Zheng et al. 2015). To our knowledge, this is the first report of A. solani causing leaf spot of T. mongolicum in China. This disease must be considered in management practices, and our finding provided a basis for disease prevention and management.

First Report of Sclerotinia Rot Caused by Sclerotinia sclerotiorum on Artemisia capillaris in China.

Artemisia capillaris (Asteraceae) is an annual herb found in ˃10 provinces in China. It is cultivated on ˃670 ha, with annual production around 2,500 tons. Its shoot is used in traditional Chinese medicine (Liu et al. 2021). From April to May 2023, Sclerotinia rot symptoms were seen at the Institute of Medicinal Plant Development (40.04°N, 116.28°E), Beijing, China. Disease incidence was up to 10% in the field through investigation of 300 plants. Initial symptoms were irregular tan-brown lesions (0.5 to 5.0 mm) that expended to circumferential necrosis on the roots and basal stem, aerial mycelia and sclerotia were developed on them. The leaves and stem tips were withered and droopy in severe cases. Twelve symptomatic primary roots of 12 plants from two sites were cut into 5 × 5 mm pieces, surface sterilized with 75% ethanol for 30 s and 5% NaClO for 60 s, rinsed with distilled water for three times, dried with sterile filter paper, put on potato dextrose agar (PDA), and incubated at 25°C in the dark for 2 days. Two Sclerotinia-like isolates were obtained using the hyphaltip method. White aerial mycelia were sparse and appressed for isolate YC1-3 and dense for isolate YC1-7. After incubated at 25°C in the dark for 15 days, 10 to 25 sclerotia were developed near the colony margin. Sclerotia of isolate YC1-3 were 1.0 to 3.9 × 1.2 to 4.5 (mean 1.8 × 2.2) mm (n = 60), ovoid or arc-shaped. Sclerotia of isolate YC1-7 were 1.5 to 3.4 × 2.7 to 9.2 (mean 2.3 × 4.3) mm (n = 60), ovoid, dumbbell shaped or curved. The isolates were identified as Sclerotinia sclerotiorum based on morphology (Maas 1998). To further identify the pathogens, molecular identification was performed with isolates YC1-3 and YC1-7. DNA of the two isolates were extracted by the cetyltrimethylammonium bromide (CTAB) method. Polymerase chain reaction was performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) region (Choi et al. 2020; White et al. 1990) and primers G3PDHfor/G3PDHrev for the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene (Garfinkel. 2021). BLAST search analysis revealed that the ITS sequence (GenBank OR229758 and OR229762) was ≥99% similar to S. sclerotiorum (MN099281, MZ379265, KX781301, etc.), and the G3PDH sequence (OR778388 and OR761975) was too (MZ493894, JQ036048, OQ790148, etc.). Phylogenetic trees were computed with ITS and G3PDH sequences using the Maximum Likelihood in MEGA 11. Nine two-month-old seedlings of A. capillaris were used to test pathogenicity. The epidermis layer of each primary root was slightly wounded (2 × 2 mm, 1 mm deep) using a sterile dissecting blade. Three plants were inoculated with mycelial plugs (5 mm in diameter) of YC1-3 and YC1-7 that cultured on PDA for 7 days. Control plants were inoculated with sterile PDA plugs. All seedlings were then incubated at 25oC and 90% relative humidity. After isolate YC1-7 inoculation 3 days and isolate YC1-3 inoculation 5 days, inoculated roots had symptoms like those in the field, controls had no symptoms. S. sclerotiorum was consistently re-isolated from diseased roots, fulfilling Koch's postulates. Diseases caused by S. sclerotiorum have been reported threatens several important economical crops (Marin and Peres 2020; Guan et al. 2022). To our knowledge, this is the first report of S. sclerotiorum causes Sclerotinia rot on A. capillaris. To avoid of significant economic losses, it is urgent to establish an effective disease-management strategy.

Leaf Spot of Hedychium coronarium Caused by Colletotrichum gloeosporioides in China

Hedychium coronarium (white ginger) is widely cultivated for garden decoration and folk medicine. Since 2020, symptomatic leaves showed brown necrosis and yellow borders on H. coronarium in the field (approximately 200 m2) at Southwest University, Rongchang District, Chongqing City. Small brown-yellow spots gradually enlarged and caused withering in severe cases with a mortality rate of around 10%. Disease incidence and severity varied from 55 to 65% and from 30 to 40%, respectively. Infected tissues (5 mm in diameter) were cut from lesion margins, surface sterilized in 70% ethanol for 10 s and 0.1% acidic mercuric chloride for 3 min, followed by rinsing in sterile water three times, and then were cultured onto potato dextrose agar (PDA) at 25 °C. Five isolates were transferred onto fresh PDA and purified by single-spore culturing. The colonies were initially white and turned hoary, and the diameter reached 32.95 to 38.37 mm × 32.42 to 38.61 mm after 3 days of incubation. Pale gray abundant fluffy aerial mycelia were arranged irregularly and densely. Hyphae were septate and branched, 2 to 5 µm in width. Conidiophores were pale brown, septate, branched, cylindrical to ampulliform. Conidia were hyaline, smooth-walled, cylindrical with obtuse ends, and 8.1 to 13.3 μm × 2.4 to 5.8 μm (n = 50) in size. Appressoria were medium brown to dark brown, aseptate, in irregular shape, solitary or in groups, and measured 1.5 to 12.5 μm × 2.1 to 13.3 μm. Morphological characteristics of isolates agreed with the description of Colletotrichum (Liu et al. 2015). Genomic DNA was extracted from fungal colonies incubated on PDA for 7 days following the instructions from the PlantGen DNA Kit (CWBIO, China). The internal transcribed spacer (ITS) region, and fragments of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), beta-tubulin (TUB2) and large subunit ribosomal DNA (LSU rDNA) genes were amplified by primer pairs ITS1/ ITS4, GDF1/GDR1, ACT512F/ACT783R (Naz et al. 2017), T1/Bt2b (Glass et al. 1995) and LROR/LR7 (Castlebury et al. 2002), respectively. The sequence of representative isolate CG-H (GenBank accession nos. OM010355, OM238213, OM238214, OM045778 and OM010358 for ITS, GAPDH, ACT, TUB2 and LSU rDNA, respectively) exhibited 99 to 100% identity to Colletotrichum gloeosporioides. A multi-locus phylogenetic tree with concatenated sequences of ITS, GAPDH, ACT and TUB regions was constructed using the maximum likelihood method by MEGA7, which revealed that strain CG-H was grouped with C. gloeosporioides. To confirm the pathogenicity, six healthy H. coronarium plants were surface sterilized, and conidial suspension (1 × 106 conidia/mL) was sprayed onto the leaves. Six plants were inoculated with sterile distilled water as controls. All the plants grew in a greenhouse at 25 °C under 12 h/12 h photoperiod. The experiment was repeated four times. Yellow lesions appeared after 7 days of inoculation, irregular-shaped brown spots were formed and slightly sunken within 14 days, and the whole leaf gradually became withered in 50 days. All inoculated plants exhibited leaf spot symptoms while the control plants remained asymptomatic. C. gloeosporioides was re-isolated from lesions of leaves and identified by morphology and sequence analysis, fully confirming Koch’s postulates. This is the first report of C. gloeosporioides associated with H. coronarium leaf spot in China and worldwide. Further studies will be conducted on the sensitivity of C. gloeosporioides to various fungicides.

First Report of Verticillium Wilt caused by Verticillium dahliae on Chilli in South Korea.

Chili pepper (Capsicum spp.) is an important crop in South Korea and is widely used in Korean cuisine, cultivated across a land area of roughly 29.8 thousand hectares, with a total production of 69 thousand tons (Lee et al., 2005; Statista, 2022). In September 2020, two farmer fields in Samcheok (37.444039°N, 129.135875°E; 37.633738°N, 128.911731°E), Gangwon province, South Korea, it is observed that chili pepper leaves showing yellowing and wilting symptoms, with an estimated disease incidence of approximately 10-15%. To identify the causal agents six affected plants were brought to lab. All the plants exhibited vascular discoloration in stem and root. After surface sterilizing small pieces of discolored tissue in 1% NaOCl for 30 s and rinsing twice in sterile distilled water, the tissue pieces were placed on water agar and incubated at 25°C for 10 days. Six pure isolates with consistent morphological characteristics were obtained by single spore isolation. Two representative isolates, NC17601 and NC20845 were selected for identification based morphological and molecular characters. Colonies on potato dextrose agar (PDA) during 10 days of incubation at 25°C in the dark were cottony white initially but progressively became dark as the formation of melanized microsclerotia. Conidiophores were hyaline, vertically branched or not, and had 2 - 4 phialides per node. Phialides were subulate and tapering from base to tip. The colonies produced abundant conidia, which were hyaline, single celled, smooth walled, cylindrical to oval, clustered on phialides, and 3.8 - 7.2 ×2.1 - 3.9 ㎛(mean ± SD: 5.2 ± 0.7 × 2.8 ± 0.5). Microsclerotia were aggregated form, various size and shape, and brown. These are the typical morphology of Verticillium dahliae (Inderbitiz et al. 2011; Yu et al. 2016). The molecular identification was later confirmed through PCR amplification, and sequencing targeting the translation elongation factor 1 alpha (TEF), actin (ACT), tryptophan synthase (TS), and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes using the primer sets described by Inderbitiz et al. (2011). The resulting sequences were deposited in GenBank with accession numbers LC761935 to LC761942. The maximum likelihood tree based on combined sequences of ACT, GPD, TEF and TS was inferred using RAxML- HPC2 on XSEDE as implemented on the CIPRES web server. The phylogenetic tree showed that the isolates were sit together with V. dahliae isolates (Ex-type PD322, PD227 and PD502). Pathogenicity tests using two isolates (NC17601 and NC20845) were conducted in the greenhouse, where 10 two weeks old seedlings per isolates (cv. Bigstar) were root-tip cut and then soaked in a fungal spore suspension of 107 conidia ml-1 for 1 h, while 10 seedlings were treated with sterile distilled water as a control. All the treated plants were maintained at 25°C (night)/ 25°C (Day) under natural light. After three weeks, all inoculated plants exhibit growth stunting with vascular discoloration in the stem and roots as compared to asymptomatic control plants. The isolates of V. dahliae were consistently re-isolated from discolored root tissues and identified based on morphological characteristics, thus fulfilling Koch's postulates. In South Korea, V. dahliae has been reported to cause wilt disease in various crops, including Kimchi cabbage and radish (Dumin et al. 2020; Choi et al. 2023). To the best of our knowledge, this is the first report that V. dahliae causing Verticillium wilt of chili pepper in South Korea. Overall, Verticillium dahliae is considered to be a significant threat to agriculture in South Korea, and efforts are being made to develop effective control strategies to mitigate its impact on crops.

First Report of Colletotrichum truncatum Causing leaf spot on Clausena lansium in China.

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.

First report of Colletotrichum grossum causing apple bitter rot worldwide (Italy).

Apple bitter rot is a globally widespread disease that is observed on fruits both pre-harvest and post-harvest, contributing to considerable economic losses. While the Colletotrichum acutatum species complex are predominant in Europe (Baroncelli et al. 2014; Amaral Carneiro and Baric 2021), in recent years, the Colletotrichum gloeosporioides species complex are emerging, raising many concerns (Amaral Carneiro et al. 2023). Circular, slightly sunken, brown lesions with acervuli produced in concentric spots were observed on 'Story® Inored' cultivar harvested in September 2022 from an organic orchard in Masi (Padova province, Italy), with a disease incidence close to 30%. From ten diseased apples, tissue samples were excised under aseptic conditions from surface-cleaned fruit at the margin between healthy and diseased pulp tissue, transferred to potato dextrose agar medium and incubated in the dark at 25 °C for 7 days, whereafter five single-spore cultures were obtained. Pure colonies grown at 25 °C for 7 days appeared light gray-white on the upper side with floccose aerial mycelium, while the reverse side was dark gray with a distinct margin. Conidia were hyaline, cylindrical in shape with both ends rounded or one end acute and measured 16.6 ± 1.4 × 6.1 ± 0.5 μm [mean ± SD] (n=50) as described by Diao et al. 2017. To identify the species, genomic DNA of a representative isolate (C38) was extracted, beta-tubulin (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), Apn2-Mat1-2 intergenic spacer (ApnMat) genes and the internal transcribed spacer (ITS) region, were amplified by PCR and Sanger sequenced (Rojas et al. 2010; Weir et al. 2012). The obtained DNA sequences of, TUB2, CAL, GAPDH, GS, ApnMat and ITS were submitted to GenBank under the accession numbers OR025589, OR025586, OR025587, OR025588, OR025585 and OR004800, respectively. A MegaBLAST analysis resulted 100 % identity to the epitype CAUG7 of Colletotrichum grossum (Diao et al. 2017) for GAPDH (KP890159), for TUB2 (KP890171), 99.85% for CAL (KP890147) and 99,5 % for ITS (KP890165). The phylogenetic tree constructed by concatenation with the obtained sequences, as well as references, revealed that the C38 isolate clustered within C. grossum, confirming the BLAST approach. Pathogenicity tests were performed on 40 'Story® Inored' apples cleaned and wounded with a sterilized needle and exposed to two different conditions: 20 apples (10 inoculated with 20 μl of spore suspension (105 ml-1) and 10 with sterile water as control) were incubated at 20°C with a 12-hour photoperiod for 14 days, while the remaining 20 apples, prepared with same approach, were placed at 1°C for 3 months, then at room temperature for 14 days. Symptoms appeared after 6 days on apples incubated at 20°C, whereas those stored at 1°C displayed symptoms at 11 days after being placed at room temperature. In both conditions, lesions were similar to those observed on the original fruits; while the controls remained asymptomatic. Identity of reisolated fungal colonies was confirmed by CAL, GAPDH and GS region sequence analysis. C. grossum has been reported rarely: in 2017 on Capsicum annuum var. grossum in China, in 2018 on Mangifera indica leaves in Cuba, and in 2021 on Rhyncospermum jasminoides in Italy (Diao et al. 2017; Manzano León et al. 2018; Guarnaccia et al. 2021). To the best of our knowledge, this is the first report of apple bitter rot caused by Colletotrichum grossum worldwide.

First Report of Anthracnose Caused by Colletotrichum nymphaeae on Strawberry Fruits in Central Argentina.

Strawberry (Fragaria x ananassa) production in Argentina extends to around 1700 hectares. Coronda city, located in Santa Fe province, is an important strawberry producer due to ideal agroecological conditions for culture and a high specialization for production. In November 2021, anthracnose symptoms were observed on strawberries cvs. 'San Andreas' and 'Splendor' in Coronda (31°58'S, 60°55'W), central Argentina. During these years, the incidence of the disease reached 40% of the production. Symptoms included 2-3 mm circular to irregular dark brown spots which enlarged rapidly and became sunken. Under high humidity conditions, concentric rings of pinhead-size salmon-colored acervuli developed on the lesions. The causal agent was isolated by touching acervuli with a sterile needle and monosporic cultures were obtained on PDA after 10 days at 25°C, with a 12-h light period. Colonies were white to gray on the top and orange on the underside, where concentric rings of salmon acervuli were clearly distinguished. The width and length of one hundred conidia were examined in three isolates (CF1, CF2, and CF3), ranging from 3.27 to 5.53 μm (avg.= 4.3 μm), and from 10.37 to 19.52 μm (avg.= 14.27 μm), respectively. The conidia were hyaline, smooth-walled, aseptate, and cylindric-clavate with one end round and one end acute. These morphological characteristics correspond to species belonging to the C. acutatum complex (Damm et al. 2012; Liu et al. 2022). To accurately identify the species, DNA was extracted from isolates, and ß-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and histone (HIS3) genes were partially amplified and sequenced (Vieira et al. 2020). TUB2, GAPDH, and HIS3 sequences presented a 100% of identity with species of Colletotrichum nymphaeae. The nucleotide sequences were deposited in GenBank (OR271556-OR271558, TUB2; OR271559-OR271561, GAPDH; and OR271562-OR271564, HIS3). Multilocus phylogenetic analyses performed with reference sequences (Damm et al. 2012) showed that the three isolates clustered with C. nymphaeae, in accordance with BLAST results. To confirm pathogenicity, each isolate was inoculated in eight detached fruits of the cultivar from which it was originally obtained. Two drops of 10 μl of conidial suspension (1x105 conidia per ml) were deposited in non-wounded areas on fruits previously disinfested with 1% sodium hypochlorite solution for 1 min and rinsed twice with sterile distilled water. Drops of sterile water were deposited in eight fruits as control. Pathogenicity tests were repeated twice. Fruits were kept in moist chamber (80+5% relative humidity) at 25°C for ten days. First symptoms appeared 4 days after inoculation. After that, all of the isolates produced symptoms identical to those previously described, whereas the controls remain symptomless. The pathogen was re-isolated from lesions, and identified as C. nymphaeae by morphological characteristics and based on the TUB2 sequences, as previously described. Strawberry anthracnose in Argentina was previously associated with Colletotrichum acutatum, C. gloeosporioides and C. fragariae species based on morphological characteristics (Ramallo et al. 2000; Monaco et al. 2000) but molecular identification was not performed until today. To our knowledge, this is the first report of C. nymphaeae causing anthracnose on strawberry in Argentina. This accurate identification will help to develop more efficient management strategies.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Xanthium orientale in Korea.

Xanthium orientale L. (syn. Xanthium canadense Mill., Asteraceae), known as cocklebur, is an annual weed native to North America, which is now a neophyte distributed throughout the world. This plant was accidentally introduced to Korea in the late 1970s ( So et al. 2008) and is considered a problematic exotic weed in orchards, for which many herbicides are ineffective (Kim et al. 2020). In September 2018, powdery mildew was observed on X. orientale in Jeju, Korea. The disease incidence ranged from 40 to 60%. Voucher specimens were deposited in the Korea University Herbarium (Accession No. KUS-F30795) and Kunsan National University Herbarium (KSNUH1988). Symptoms appeared as round to irregular white patches with abundant hyphal growth on the leaf surface. Hyphal appressoria were nipple-shaped, and 3 to 6 μm diam. Conidiophores (n = 30) were 145 to 206 × 9 to 11.6 µm and produced 2 to 5 immature conidia in chains with a sinuate outline. Foot-cells of the conidiophores were straight, cylindrical, and 43 to 100.9 μm long. Conidia (n = 30) were ellipsoid-ovoid, doliiform to somewhat limoniform, 25.2 to 31.8 × 13.6 to 16.8 μm (l/w 1.6 to 2.1), and devoid of distinct fibrosin bodies. The morphological characteristics corresponded to those of Golovinomyces ambrosiae (Schwein.) U. Braun & R.T.A. Cook (Braun and Cook 2012, under Golovinomyces spadiceus (Beck. & M.A. Curtis) U. Braun; Qiu et al. 2020). To confirm the identity of the causal fungus, the internal transcribed spacer (ITS), large subunit (LSU) (Bradshaw and Tobin 2020), the intergenic spacer (IGS) of rDNA, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (Bradshaw et al. 2022) were amplified for a herbarium specimen (KUS-F30795). A BLASTn search of these sequences revealed 100% identity with reference sequences of G. ambrosiae on diverse Asteraceae plants (AB077644 for ITS, AB077643 for LSU, ON361171 for IGS, and ON075648 for GAPDH). However, there was a single nucleotide difference on both the IGS and GAPDH sequences when compared to the closely related species Golovinomyces latisporus. The sequences were deposited in GenBank (Accession No. OQ165157 (ITS), OQ165164 (LSU), OR050524 (IGS), and OR086076 (GAPDH)). Phylogenetic analyses of ITS, LSU, IGS, and GAPDH sequences revealed the Korean sample formed a well-supported group with other G. ambrosiae sequences, confirming its identity. A pathogenicity test was performed through inoculation by gently pressing diseased leaves onto the leaves of five healthy plants. Five non-inoculated plants served as controls. All plants were maintained in a greenhouse at 25±2°C. Powdery mildew colonies developed on the inoculated plants after ten days, whereas the control plants remained symptomless. The fungus present on the inoculated leaves was morphologically identical to that observed on the initially diseased leaves, fulfilling Koch's postulates. Powdery mildew on X. orientale has previously been reported as Golovinomyces cichoracearum (≡ Erysiphe cichoracearum) sensu lato in the USA, G. ambrosiae (= G. spadiceus) throughout all continents, and Podosphaera fusca sensu lato (now P. xanthii) in Korea (Braun and Cook 2012; Farr and Rossman 2023). To date, powdery mildew in Korea has been reported only on Xanthium strumarium as G. cichoracearum s. lat. and Podosphaera xanthii (KSPP 2022). To our knowledge, this is the first report of powdery mildew caused by G. ambrosiae on X. orientale in Korea.

First Report of Anthracnose Caused by Colletotrichum spaethianum on Iris lactea in Daqing, China.

Iris lactea Pall. has high ornamental value and strong drought resistance, which can be used as a useful sand-stabilization and ornamental plant. In September 2021, typical anthracnose symptoms on I. lactea were found in the campus of Heilongjiang Bayi Agricultural University (125°10'45.31"E, 46°35'36.88"N), Daqing, China. Disease incidence varied from 37 to 51% at four survey sites (~20 m2 per site) of approximately 1,000 plants. Initially brown lesions with a yellow halo, and gray in the center were observed on the leaves. As disease progressed, the lesions expanded rapidly, resulting in dieback. To identify the pathogen, symptomatic tissues were excised from four infected leaves of four individual plants, surface sterilized for 1 min in 75% ethanol, washed twice with sterile water, plated on potato dextrose agar (PDA) contain 0.5 g/L streptomycin sulfate, and incubated at 25℃ for 3 to 4 days under dark conditions. Four morphologically similar fungal isolates (Irs-1 to -4) were obtained by using a single-spore isolation. After growth on PDA for 17 days at 25℃, a circular like zonation colonies with a color of greyish to gray green were observed. On the reverse side, circular like with zig-zag zonation colonies light brown to light black from the margin to the center. Conidiophores were pale brown, septate. Conidia were hyaline, aseptate, curved or slightly curved, round, or somewhat acute apex, base truncate, unicellular, whose sizes ranged at 18.5 ± 2.3 × 4.6 ± 0.7 μm (n = 170), with length/width ratio 4.04. Appressoria were in oval, star, or irregular shape, with brown color with sizes approximately at 13.3 ± 2.1 × 10.4 ± 2.1 μm (n = 30). These morphological characteristics are consistent with Colletotrichum spaethianum with curved conidia (Damm et al. 2009). For molecular identification, primers ITS1/ITS4 (Schoch et al. 2012), GDF1/GDR1 (Guerber et al. 2003), ACT-512F/ACT-783R and CHS-354R/CHS-79F (Carbone and Kohn 1999) were used to amplify the partial region of rDNA-ITS, a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a partial sequence of the actin (ACT), and chitin synthase 1 (CHS-1) from fresh mycelia of the four isolates (Irs-1 to -4), respectively. The sequences were submitted to GenBank (ITS: OM004553, OP804624, OR029392, and OR029391; GAPDH: OP883938, OP883939, OR060963, and OR060962; ACT: OP883940, OP883941, OR060965, and OR060964; CHS: OP868844, OP868845, OR046504, and OR046503). Phylogenetic analysis based on the ITS, GAPDH, ACT and CHS gene sequences indicated that isolates obtained in this study were all clustered with C. spaethianum. Pathogenicity test was conducted by inoculating mycelial plugs on the I. lactea seedlings (2 seedlings with 4 to 5 leaves per isolate). Two seedlings inoculated with sterilized PDA plugs were used as control. All inoculated plants were maintained in humid chamber at 25℃ under dark, and typical anthracnose symptoms were observed up to 6 days after inoculation, while the control leaves were asymptomatic. The same pathogen was successfully re-isolated and phenotypically identical to the original isolates to fulfill Koch's postulates. C. spaethianum has been described on Hemerocalis flava (Vieira and Michereff 2014) and Allium fistulosum (Santana et al. 2016) in Brazil, Polygonatum cyrtonema in China (Ma et al. 2020), Iris germanica in Japan (Sato et al. 2012). To our knowledge, this is the first report of C. spaethianum causing anthracnose disease on I. lactea in Daqing, China.

First Report of Stemphylium lycopersici Causing Leaf spot on Sedum plumbizincicola in Hunan Province of China.

Sedum plumbizincicola is a perennial succulent herb that can hyperaccumulate high concentrations of cadmium and zinc (Liu et al. 2017). In October 2021, a leaf spot disease occurred on S. plumbizincicola seedlings in a nursery in Changsha (28°13' N; 112°56'E), the Hunan Province of China. Almost 30% of the nearly 1 million seedlings were infected. Symptoms initially appeared as small brown spots on the leaf surface or edges, gradually enlarged, becoming oval, and bearing chlorotic lesions with dark brown borders. Eventually, the center of the lesions became sunken and then fell off. Eight symptomatic plant samples were collected by five-point sampling method (Zheng et al. 2018). Small pieces of 5×5 mm were excised from the lesion margins, sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 40 s, rinsed with sterile distilled water three times, and then cultured on potato dextrose agar (PDA) at 26 °C for 5 days in the dark. Fungal colonies showing similar morphology were observed from all the isolated samples and, in total, eight fungal strains were obtained. On PDA, fungal colonies were initially white, and later become light gray. After cultured on V8 juice agar (V8A, each litre of medium contains 200 mL of V8 juice, 3 g of CaCO3 and 15 g of agarose) for 14 days (Hyowon et al. 2016), conidia of a representative isolate SY-1 were produced, which were oblong, muriform, with blunt ends and conical apex, pale to light brown, and constricted at the 1 to 3 major transverse septa, 38.34-46.68 μm×11.67-18.34 μm (n=50). These morphological characteristics were consistent with that of Stemphylium lycopersici (Nasehi et al. 2016). The internal transcribed spacer (ITS) region of rDNA and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of representative isolates SY-1 to SY-3 were amplified and sequenced using the primer pairs ITS4/ITS5 and gpd1/gpd2 as described previously (Woudenberg et al. 2017). BLASTn analysis showed that ITS sequences of isolates SY-1, SY-2 and SY-3 (accession nos. OP317641, OQ852042 and OQ852043) had more than 99% identity with Stemphylium sp, while GAPDH sequences (OP331223, OQ858620 and OQ858621) had 100% identity with S. lycopersici KR911813 (Sun et al. 2016). A concatenated ITS-GAPDH phylogenetic tree grouped our isolates within the S. lycopersici clade. For the pathogenicity test, one-month-old potted S. plumbizincicola seedlings were inoculated with conidia suspension (105 conidia/ml), which was induced on V8A. Four sites of each leaf of the potted S. plumbizincicola plants were dropped with a conidia suspension of strain SY-1, with 10 μL per site. Leaves treated with sterile water were served as controls. All of the inoculated seedlings were placed in a growth chamber at 26°C with a photoperiod of 12 h. The pathogenicity tests were repeated twice, with each had three replicative plants. After 7 days, all the inoculated leaves developed brown spots resembling those observed in the nursery, whereas the control plants remained symptomless. Stemphylium lycopersici was specifically re-isolated and identified by morphological and molecular methods (accession nos. OQ852045 for ITS and OQ858622 for GAPDH, respectively), thus fulfilling Koch's postulates. To our knowledge, this is the first report of S. lycopersici causing leaf spot on S. plumbizincicola in China. Since S. plumbizincicola played an important role and widely planted for heavy metal pollution treatment (Jiang et al. 2010), and this disease might seriously influence the S. plumbizincicola seedling breeding, identification of the pathogen might provide a foundation for the diagnosis and control of the disease.

Occurrence of Colletotrichum truncatum Causing Foliar Spot on Sesame (Sesamum indicum) in Mexico.

Sesame (Sesamum indicum L.) is an oilseed crop that present agronomic advantages and nutritional contributions in regions where water and soil fertility are limiting. In September 2020 and October 2022, anthracnose symptoms were observed on sesame fields in Mocorito (25°29'04"N;107°55'03"W) and Guasave (25°45'40"N;108°48'44"W), Sinaloa, Mexico. The disease incidence was estimated at up to 35 % (10 has) in five fields. Twenty samples were collected with symptoms on the leaves. On leaves, lesions were irregular and necrotic. Colletotrichum-like colonies were consistently isolated on PDA medium and five monoconidial isolates were obtained. One isolate was selected as a representative for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi of the Biotic Product Development Center at the National Polytechnic Institute under the accession number IPN 13.0101. On PDA, colonies were flat with an entire margin, initially white, then dark gray with black acervuli and setae. The growth rate was 9.3 mm/day. Conidia (n=100) on PDA were hyaloamerosporae, 17.5- 22.7 × 3.6-4.5 μm, smooth-walled, falcated and pointed at both ends, with granular content. Acervuli showed setae acicular (2-3 septate setae) tapered to the apex. The mycelial appressoria were brown, obclavate and irregular. Morphological features matched those of the Colletotrichum truncatum species complex (Damm et al. 2009). For molecular identification, total DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR (Weir et al. 2012) and sequenced. The sequences were deposited in GenBank under accession nos. OQ214919 (ITS), OQ230773 (ACT), and OQ230774 (GAPDH). BLASTn searches in GenBank showed 100%, 100%, and 100% identity to MN842788 (ITS), MG198003 (ACT), and MF682518 (GAPDH) of C. truncatum, respectively. A phylogenetic tree based on the Maximum Likelihood method and Bayesian Inference including published ITS, ACT, and GAPDH sequence data for C. truncatum species complex was generated (Talhinhas and Baroncelli 2021). In the phylogenetic tree, the isolate IPN 13.0101 was placed in the same clade of C. truncatum. Pathogenicity of the isolate IPN 13.0101 was verified on 15 sesame seedlings leaves (Dormilon variety) (15-day-old) disinfected with sodium hypochlorite and sterile water. Each leave was inoculated with 200 µL of a conidial suspension (1 × 106 spores/mL). Five plants non inoculated served as controls. All plants were kept in a moist chamber for two days, and subsequently transferred to a shade house where the temperature ranged from 25 to 30°C. All inoculated leaves developed irregular and necrotic lesions ten days after inoculation, whereas no symptoms were observed on the control leaves. The fungus was consistently re-isolated from the diseased leaves, fulfilling Koch´s postulates. The experiment was conducted twice with similar results. Colletotrichum spp. has been previously reported (Farr and Rossman, 2023) to cause sesame anthracnose in Mexico (Alvarez, 1976), Thailand (Giatgong, 1980) and Cuba (Arnold, 1986), but this is the first report of C. truncatum causing sesame anthracnose in Mexico. This disease is a recurrent problem in sesame fields in Sinaloa, therefore further studies are required to understand its impact.

First Report of Colletotrichum siamense Causing Anthracnose on Dioscorea alata in China.

Dioscorea alata is an annual or perennial dicotyledonous plant which is a vegetatively propagated tuberous food crop (Mondo et al. 2021). In 2021, symptoms of leaf anthracnose occurred on D. alata plants at a plantation in Changsha, the Hunan Province of China (28°18' N; 113°08'E). Symptoms initially showed as small, brown water-soaked spots on the leaf surface or margins, and enlarged to irregular, dark brown or black, necrotic lesions, with a lighter center and darker edge. At latter, lesions extended to most of the leaf surface causing leaf scorch or wilting. Almost 40% of the plants surveyed were infected. Symptomatic leaf samples were collected, and small pieces were taken at their disease-healthy junction, sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 40 s, rinsed three times with sterile distilled water, and then placed on potato dextrose agar (PDA) for incubation at 26 °C for 5 days in the dark. Fungal colonies with similar morphology were observed and, in total, 10 isolates were obtained from 10 plants. On PDA, colonies were initially white with fluffy hyphae, and later became light to dark gray, showing faint concentric rings. Conidia were hyaline, aseptate, cylindrical, and rounded at both ends, measuring 11.36 to 17.67 × 3.45 to 5.9 μm (n = 50). Appressoria were dark brown, ovate, globose, measuring 6.37 to 7.55 × 10.11 to 12.3 µm. These morphological characteristics were typical of Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular identification, the internal transcribed spacer (ITS) region of rDNA, and partial sequences of actin (ACT), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes of a representative isolate Cs-8-5-1 were amplified and sequenced using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and GDF/GDR as described previously (Weir et al. 2012). These sequences were deposited in GenBank (accession nos. OM439575 for ITS, OM459820 for ACT, OM459821 for CHS-1, and OM459822 for GAPDH). BLASTn analysis showed 99.59 to 100 % identity to the corresponding sequences of C. siamense strains. A Maximum likelihood phylogenetic tree based on the concatenated ITS, ACT, CHS-1 and GAPDH sequences was generated by MEGA 6. It revealed that the Cs-8-5-1 was clustered with the C. siamense strain CBS 132456 with 98% bootstrap support. For pathogenicity test, conidia suspension (105 spores/ml) was prepared by harvesting conidia from 7-day-old cultures growing on PDA, and 10 uL was dropped onto leaves of potted D. alata plants (8 droplets per leaf). Leaves treated with sterile water were served as controls. All the inoculated plants were placed in humid chambers (with 90% humidity) at 26°C with a photoperiod of 12 h. The pathogenicity tests were performed twice, with each had three replicated plants. Seven days after inoculation, the inoculated leaves showed symptoms of brown necrosis resembling that observed in fields, however, the control leaves remained symptomless. The fungus was specifically re-isolated and identified by morphological and molecular methods, thus fulfilling Koch's postulates. To our knowledge, this is the first report of C. siamense causing anthracnose on D. alata in China. Since this disease might seriously affect the photosynthesis of the plants, which can influence the yield, prevention and management strategies should be adopted to control this new disease. Identification of this pathogen will provide a foundation for the diagnosis and control of this disease.

First report of melting out on creeping bentgrass caused by Bipolaris sorokiniana in China.

Creeping bentgrass (Agrostis stolonifera L.), is one of the major cool-season turfgrass species, which is widely planted in putting greens at golf courses in China (Zhou et al. 2022). In June 2022, an unknown disease with reddish-brown spots (2-5 cm in diameter) were observed on 'A4' creeping bentgrass putting greens at Longxi golf course in Bejing. As the disease progressed, the spots coalesced and formed irregular patches (15-30 cm in diameter). When looking closely, the leaves were wilting, yellowing and melting out from the foliar tips to the crowns. The disease incidence was estimated up to 10-20 % of each putting green and a total of five putting greens had similar symptoms as described above. Three to five symptomatic samples were collected from each green. Diseased leaves were cut into pieces, surface sterilized in 0.6% sodium hypochlorite (NaClO) for 1 min, washed three times with sterilized water, air dried and placed on potato dextrose agar (PDA) containing 50 mg L-1 streptomycin sulfate and tetracycline. Plates were incubated in the dark at 25 °C for 3 days, and fungal isolates with similar morphology (irregular cultures with dark-brown back and light-brown to white surface) was consistently recovered. Pure cultures were obtained by repeat hyphal-tip transfer. The fungus did not grow well on PDA medium, radial growth was estimated 1.5 mm/d, and dark-brown colony was surrounded by light-white margin. However, it grew fast on creeping bentgrass leaf extract (CBLE) medium, CBLE medium was produced by adding 0.75 g potato powder, 5 g agar and 20 mL creeping bentgrass leaf juice (with 1 g fresh creeping bentgrass leaf) into 250 mL sterile water. The colony was sparse and light-white, and radial growth was roughly 9 mm/d on CBLE medium. Conidia were spindle-shaped, olive to brown in color, pointy or obtuse at both ends, 4 to 8 septa, and a size range of 9.85 to 20.20 × 26.26 to 45.64 μm (14.85 μm × 40.62 μm average, n = 30). Genomic DNA of two representative isolates (HH2 and HH3) was extracted, the nuclear ribosomal internal transcribed spacer (ITS) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) region were amplified with primers ITS1/ITS4 (White et al. 1990) and gpd1/gpd2 (Berbee et al. 1999), respectively. The ITS (OQ363182 and OQ363183) and GAPDH (OQ378336 and OQ378337) sequences were deposited in GenBank. BLAST analyses revealed that the sequences shared 100% and 99% similarity with the published ITS (CP102792) and GAPDH (CP102794) of B. sorokiniana strain LK93, respectively. To complete Koch's postulates, 3 plastic pots (15 cm height × 10 cm top diameter × 5 cm bottom diameter, three replicates for the isolate HH2) were seeded with creeping bentgrass and inoculated with a spore suspension (1×105 conidia/mL) after two months of plant growths. Healthy creeping bentgrass inoculated with distilled water served as controls. All pots were covered with plastic bags, placed in a growth chamber with a 12-h day/night cycle at 30/25°C and 90% relative humidity. Disease symptoms (yellowing and melting out leaf) were noted after 7 days. B. sorokiniana was recovered from the diseased leaves and identified morphologically and molecularly as described above. To our knowledge, this is the first report of melting out on creeping bentgrass caused by B. sorokiniana in China. The report here will provide a scientific basis for developing management strategies on this disease in the future. Additional study is needed to investigate the prevalence of the disease on putting greens from golf courses in larger regions of China.

First report of leaf anthracnose caused by Colletotrichum camelliae on tea plants (Camellia sinensis) in South Korea.

The tea plant (Camellia sinensis (L.) O. Kuntze) is a popular non-alcoholic beverage crop worldwide. The tea market in South Korea is projected to increase annually by 4.59% (Statista, 2022). Boseong, Hadong, and Jeju Island are the main tea-growing regions in South Korea. Anthracnose is one of the major diseases of tea plants and is responsible for substantial yield loss and poor tea quality. In 2021, anthracnose of tea (disease incidence of 30%) was observed in a garden (33°28'45.5"N 126°42'02.2"E) at Jeju Island, where the Yabukita cultivar has been cultivated. The typical symptoms consisted of round or irregularly shaped lesions with gray-white centers and purple-brown borders. Twelve morphologically similar isolates were recovered from 12 infected leaves using the single spore isolation method on solid potato dextrose agar (PDA) (Cai et al. 2009). Four representative isolates (GT6, GT7, GT8, and GT11) were identified based on morphology, molecular analysis, and pathogenicity tests. The upper side of seven-day-old colonies on PDA (incubated at 25 °C in the dark) was off-white with white aerial mycelia and gray-white with black zonation on their reverse side. Conidia were hyaline, aseptate, cylindrical, with both obtuse ends, and measuring 12.3 - 25.8 µm × 4.4 - 9.3 µm (n = 50). Appressoria were dark brown, irregularly shaped with a smooth edge, and measuring 7.3 -18.8 µm × 6.9 - 11.3 µm (n = 50). According to morphological characteristics, the fungal isolates were tentatively identified as the Colletotrichum gloeosporioides complex, including C. caelliae (Wang et al. 2016; Weir et al. 2012). The genomic DNA was extracted, and the ribosomal internal transcribed spacer (ITS), β-tubulin-2 (TUB2) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, actin (ACT), calmodulin (CAL), and the Apn2-Mat1-2 intergenic spacer and partial mating type (ApMat) genes were amplified and subsequently sequenced using primer sets ITS1/ITS4, BT2a/BT2b, GDF1/GDR1, ACT-512F/ACT-783R1, CL1C/CL2C, and AM-F/AM-R, respectively (Silva et al. 2012; Weir et al. 2012). The resulting sequences were deposited in GenBank accession numbers (LC738932-LC738959). All the representative isolates were identified as C. camelliae by constructing the 50% majority rule consensus and maximum likelihood phylogenetic treebased on the combined ITS, TUB2, GAPDH, ACT, CAL, and ApMat sequences using MrBayes v. 3.2.2 and Mega X, respectively (Kumar et al., 2018; Ronquist et al. 2012). The pathogenicity of these isolates was tested on healthy leaves of 2- years-old tea seedlings (the Yabukita cultivar). Onside of unwounded or wounded leaves of seedlings were inoculated with 20 µL of conidial suspension (1 × 106 conidia or spores/ml) per spot (3-4 wounded or unwounded spots per side per leaf). Another side of the leaves received sterile distilled water and served as a control. Each treatment was replicated three times (three seedlings/isolate and four leaves per seedling) and this experiment was repeated twice. All plants were covered with plastic bags, placed in a growth chamber, and incubated at 25 °C with a 12-h photoperiod and 90% relative humidity. Typical anthracnose symptoms appeared on wounded leaves after two days of inoculation. Unwounded and controlled leaves remain asymptotic. To confirm Koch's postulates, fungal isolates were re-isolated from inoculated leaf lesions and identified as C. camelliae based on morphology and ITS sequences. Colletotrichum camelliae is a very common pathogen associated with tea anthracnose worldwide, including China (Liu et al. 2015; Wang et al. 2016).To the best of our knowledge, this is the first report of anthracnose in tea trees caused by C. camelliae in South Korea. The results of this study could help come up with better ways to keep an eye on and deal with this devastating on tea plants. Key words: Tea anthracnose, Colletotrichum camelliae, pathogenicity References Cai, L., et al. 2009. Fungal Divers. 39:183. Kumar, S., et al. 2018. Mol. Biol. Evol. 35:1547. Liu, F. et al. 2015. Persoonia. 35: 63-86. Ronquist, F. et al. 2012. Syst. Biol. 61:539-542. Silva, D. N. et al. 2012. Mycologia. 104:396-409. Statista 2022. Statista Digital Market out Look. Available at www.statista.com. Wang, Y.-C. et al. 2016. Sci. Rep. 6: 35287. Weir, B. S., et al. 2012. Stud. Mycol. 73:115.

First report of Colletotrichum grevilleae causing fruit anthracnose of pomegranate in Taiwan.

Pomegranate (Punica granatum L.), native to northern India, is a popular fruit crop. In Taiwan, pomegranate is mainly cultivated in the mid and southern regions including Waipu, Taichung and Neipu, Pingtung. However, diseases affecting production and quality of pomegranate are largely understudied in Taiwan where gray mold and Cercospora leaf spot of pomegranate were ever reported (Hsieh, 1978; Hsieh and Wu, 1989). Pomegranate fruit (cv. Tunisia) showing anthracnose symptoms were found in a commercial pomegranate farm of 0.4 ha in Waipu (N24.33111, E120.68978) in August 2020. Around 40% fruit were infected and displayed symptoms of brown sunken lesions with indistinct edges typically occurred at the fruit ripening stage (supplementary Fig. S1). To isolate the pathogen, diseased exocarps were surface sterilized with 1% NaOCl followed by 70% ethanol. Surface-sterilized tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. A fungus was consistently isolated from diseased fruit, and five isolates (PGCo1, WP1, WP2, WP3, and WP4) were obtained. When cultured on PDA 25℃ under near UV light (360 nm) with a 12-h:12-h light/dark cycle for 10 days, the isolates initially produced white hyphae and subsequently formed dark gray to olivaceous black colonies with white edge, and no conidiomata was observed (supplementary Fig. S1). Conidia were usually aseptate, hyaline, cylindrical to ellipsoid and obtuse at either one or both ends (supplementary Fig. S1), and measured 16.0 ± 1.6 (11.4-18.9) × 5.6 ± 0.5 (4.5-6.9) μm (n = 100). The isolates were further identified by sequence comparison and phylogenetic analysis of concatenated partial sequences of the internal transcribed spacer (ITS) of the rDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUB2), chitin synthase 1 (CHS-1), actin (ACT) and calmodulin (CAL) genes (Weir et al., 2012). The sequences were deposited in the GenBank under accession numbers ON849040-ON849044 and ON862106-ON862130. BLASTn searches showed the ITS sequences of the pomegranate isolates were 100% identical to that of C. grevilleae CBS 132879T (NR_163525). The ACT, GAPDH and TUB2 sequences of the isolates were 100%, 100% and 99.57%, respectively, identical to those of C. grevilleae CBS 132879T (KC296941, KC297010 and KC297102). The CHS-1 sequences were 99.67-100% identical to that of C. grevilleae CBS 132879T (KC296987). The CAL sequences showed 91.50% and 100% identity with those of C. grevilleae CBS 132879T (KC296963) and C. grossum CAUG7T (KP890147), respectively, both members of the C. gloeosporioides species complex. Multilocus sequence analysis of the combined data was conducted using MEGA X software (Kumar et al., 2018). The five isolates were clustered with C. grevilleae CBS 132879T, indicating that the pomegranate isolates are C. grevilleae (Supplementary Fig. S2). For pathogenicity test, 6 symptomless ripe pomegranate fruit (cv. Tunisia) were surface sterilized, wounded by stabbing a sterile needle, and inoculated with a conidial suspension (10 μl each, 1×106 conidia/ml) of PGCo1. Sterile water was used as control. The inoculated fruit were kept in a moist plastic chamber (>95% relative humidity, 25 ± 2°C) in darkness. The experiment was performed twice. Seven days post inoculation, the artificially inoculated fruit showed anthracnose symptoms of sunken brown lesions indistinguishable from the natural infection. The control remained symptomless. The same fungus was repeatedly isolated from the symptomatic fruit, fulfilling Koch's postulates. Prior to this study, only the ex-type strain of C. grevilleae was collected from root and collar rot of Grevillea sp. (Liu et al., 2013). This is the first report of C. grevilleae causing anthracnose on pomegranate in Taiwan. Since no fungicide has been approved for pomegranate cultivation in Taiwan, future study is necessary to develop a management strategy against this disease.