Accession Nos Research Articles

First Report of black spot of Shine Muscat fruit caused by Cladosporium allicinum in China.

Shine muscat is a Vitis vinifera hybrid (Akitsu-21 × Hakunan), that has emerged as a popular table grape cultivar in China. In recent years, shine muscat has been widely cultivated with 66,667 ha being cultivated in 2021. In November 2021, symptoms of fruit spot were observed on shine muscat during the storage at 0~3℃ and 85%~90% RH, while stored at National Agricultural Product Preservation Engineering Technology Research Center, in Tianjin (N 116°20', E 39°09'), China. The incidence of this disease was about 35%. Affected grape berries initially had small brown spots. The spots on the fruit expanded to an ellipse or circular sunken area with a black center. The central peel of the diseased spots were ruptured and collapsed. The diseased fruits eventually fell off the vine. To isolate the pathogen, grape peels with typical symptoms were cut into small pieces, sterilized with 75% ethanol for 45-sec, rinsed with sterilized distilled water three times, and then transferred onto potato dextrose agar (PDA) medium.The plates were incubated at 25°C in the dark. After 10 days, 26 single spore isolates with similar morphology were obtained from 30 symptomatic grape berries. Fungal colonies were grayish brown, with abundant conidia on the obverse-side on PDA. Conidiophores were cylindrical, straight with unbranched, solitary or clustered, elongation at the tip and ranged in size from 3.2-6.8 × 35.6-150.9 µm (n=50). Conidia were grew in chains, ovoid, aseptate, and 2.2-6.0 × 8.3-16.8 µm (n=50). The morphological characteristics were consistent with Cladosporium allicinum (Bensch et al. 2012). Molecular data were also used to support the microscopic identification by extracting genomic DNA from 26 isolates using a Plant Genomic DNA kit (Tiangen, China). Amplicons were generated for the internal transcribed spacer (ITS), translation elongation factor 1-alpha(tef1-α), and actin (act) using the following primers ITS1/ITS4, EF1-728F/ EF1-986R and ACT-512F/ ACT-783R, respectively (Bensch et al. 2012). Blast analysis showed that three amplified fragments of 26 isolates were highly similar to C. allicinum, with 98.96~100% sequence identity with Cladosporium allicinum accessions in GenBank (ITS, OK661041; tef1-α, MF473332; act, LN834537). Three amplified fragments of representative isolate YG03 were deposited in GenBank with accession nos. OP799670 for ITS, OP888001 for tef1-α and OP887999 for act, respectively. Neighbor-joining trees based on concatenated sequences of three genes were constructed using MEGA5.2. The results showed that the strain YG03 from shine muscat was closely related to C. allicinum. Pathogenicity tests of 26 isolates were performed on healthy shine muscat berries using pin pricks and a humidor. In each wound, 5 μL of conidial suspension (1×106 conidia/mL) and sterile distilled water were inoculated on 30 berries, and maintained in a dark incubator at 25°C, 90% relative humidity. Each treatment was repeated twice. After 10 days, the wounded berries inoculated with the spore suspension showed dark brown spots, similar to the original diseased fruits, while no symptoms were observed on the control treament. Pathogen re-isolated from inoculated fruits were identical to the original strains on colony and microscopic morphology, and identified to Cladosporium allicinum based on act gene by molecular method, thereby fulfilling Koch's postulates. C.allicinum has been reported causing leaf spot on 11 host plants around the world (Bensch et al. 2012, 2015; Quaedvlieg et al. 2014; Jurisoo et al. 2019). To our knowledge, this is the first report of C. allicinum causing black spot on fruit of Vitis vinifera worldwide. The identification of this disease could establish a foundation for developing management strategies to reduce losses in storage period.

First Report of Calystegia hederacea as a new host of sweet potato leaf curl Hubei virus.

Calystegia hederacea (Convolvulaceae) is one of the most problematic perennial weeds widely distributed around or in crop fields. Our previous studies showed that C. hederacea is natural reservoir of sweet potato chlorotic stunt virus isolate CH (SPCSV-CH) and sweet potato latent virus (SPLV) (Liu et al. 2020; Zhao et al. 2022). To shed further light on the role of C. hederacea in the epidemiology of sweet potato viruses, in May 2021, a total of seven C. hederacea plants (five asymptomatic, one curling and one mild vein-clearing) were collected from two different sweet potato fields in Xinxiang city of Henan Province in China. Total RNA was prepared from a pool of the seven leaf samples using the EZNA Plant RNA Kit (Omega Bio-Tek, Norcross, GA). A library was constructed from the ribosomal-depleted RNA using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, MA, USA) and sequenced using the Illumina HiSeq platform (Novogene, Tianjin, China). A total of 139,057,020 paired-end clean reads of 150 bp were obtained after removing adaptor sequences and low-quality reads and used for de novo assembly using the Trinity (v2.2.0) software. Blast searches of the assembled contigs longer than 200 bp against NCBI nucleotide and protein sequence databases revealed the presence of 37 contigs (237 to 4885 bp) and 19 contigs (261 to 758 bp) with high nucleotide (nt) identity with SPLV and SPCSV-CH, respectively. The occurrence of SPLV and SPCSV-CH on C. hederacea was previously reported, and thus the contig sequences related to SPLV and SPCSV-CH were not subjected to further verification in this study. In addition, one contig (2,827 bp) with the highest nt sequence identity of 94.94% with sweet potato leaf curl Hubei virus (SPLCHbV, genus Begomovirus, family Geminiviridae, accession no. MK931304) was assembled from 16,592 reads, with average coverage depth of 740.5X. These results suggested the presence of SPLCHbV in C. hederacea. To further confirm the RNA sequencing result, each of the seven samples was tested by PCR using partially overlapping (italicized nucleotides) forward and reverse primers (SweeIn-F1, 5`-GGAGGAAGCTAAGTACGAGAATCAGTTAGAG-3`; SweeIn-R1, 5`-GCTTCCTCCTTGTGATTGTAAGTAACATGG-3`) that were designed based on the SPLCHbV-related contig for amplification of circular DNA viral genome (approximately 2.7 kb). Two symptomatic and three symptomless C. hederacea samples were SPLCHbV positive, indicating that virus-like symptoms of the two C. hederacea samples were probably not induced by SPLCHbV. Two of the five amplified products were completely sequenced and deposited to GenBank (accession nos. OQ551733 and OQ551734). Sequences analysis showed that the complete genome sequences of two SPLCHbV C. headrace isolates (2,763 nt and 2,761 nt) had 96.53% nt identity with each other and 95.92 to 97.70% nt identity with that of SPLCHbV isolate Shandong7-2017 (MK931304). In August 2021, fourteen C. hederacea plants (three symptomatic, 11 asymptomatic) collected from natural fields from Zhumadian and Pingdingshan cities in Henan Province, were tested by PCR using SweeIn-F1/R1 primers for SPLCHbV, showing that eight samples were SPLCHbV positive. SPLCHbV belongs to the sweepoviruses, a group of phylogenetically distinct begomoviruses infecting sweet potato, and was reported to infect sweet potato from many provinces of China (Wang et al., 2021). To the best of our knowledge, this is the first report of SPLCHbV infection in C. hederacea, which expands the natural host range of SPLCHbV.

First Report of Root rot Caused by Mucor circinelloides on Fructus forsythiae in Hubei，China.

Fructus forsythiae is a typical traditional Chinese medicinal herb with antibacterial and anti-inflammatory effects. Surveys for root rot of F. forsythiae were conducted from 2021 to 2022 in China's major planting areas (Daweiyuan Village, Sanguandong Forest Area, Yunxi County, Shiyan City, Hubei Province, 32°52'52"N, 110°19'29"E). The disease has occurred in several plantations. A total of 200 F. forsythiae were investigated, among which 112 were diseased, the incidence rate was more than 50%, and all the plants in the plantation were over 3 years old. The roots of diseased plants were fully covered by white mycelia. When the disease was severe, leaves curled and fell, roots withered, and some eventually died. A total of 22 isolates were isolated from the 18 infected tissues ofF. forsythiae and purified by single spore cultures on PDA medium. The 22 isolates similared in morphology to isolate Lianmao (The name is one of the five sequenced samples in the lab) were selected to be representative of the group. The results showed that these samples belonged to the same pathogen. The isolates were characterized by yellowish colonies composed of tall and short sporangiophores 6 to 11 μm in width, terminal and globose sporangia, ellipsoidal sporangiospores with dimensions of 5 to 8 μm in length and 4 to 5 μm in width, and obovoid columellae. It was identified as Mucor circinelloides, based on morphological characteristics (Schipper 1976). The ITS and LSU sequences of the fungus were amplified, and sequenced with the primers ITS1/ITS4 and LROR/LR5 (White et al. 1990; Rehner et al. 1994). Sequences from isolate Lianmao were deposited in GenBank (accession nos. OQ359158 for ITS, OQ359157 forLSU). Analysis using the BLAST algorithm of the two amplified sequences showed 99.69 to 100% similarity with the sequences (KY933391 and MH868051) ofM. circinelloides.The isolated M. circinelloides was prepared into 150ml spore suspension (The method was to filter the PDB after 10 days of culture using gauze to get spore suspension. Then the concentration of spore suspension was diluted to 1.0×106 spores/ml using sterile water). The spore suspension was subsequently inoculated into healthy potted F. forsythiae plants. Uninoculated potted F. forsythiae plants served as controls. All the potted F. forsythiae plants were incubated at 25°C under 12h light and 12h dark conditions. The symptoms of the infected plants were similar to those observed in the field; The control plants were symptomless. The pathogen reisolated from symptomatic roots and morphologically identified as M. circinelloides. M. circinelloides has been reported as a pathogen of Morinda citrifolia, Aconitum carmichaelii and so on (Cui et al. 2021; Nishijima et al. 2011), but it has never been reported on F. forsythiae before. This is the first report of root rot caused by M.circinelloides on F. forsythiae. This pathogen may present a threat to the production of F. forsythiae in China.

First Report of Fusarium oxysporum Causing Root Rot on Pleione bulbocodioides in China.

The members of Pleione (Orchidaceae) are popular worldwide due to their beautiful flowers and medicinal values. In October 2021, we observed the typical symptoms of yellow or brown leaves, rotted root, and plant death on P. bulbocodioides (Sup. S1a). Nearly 30% of the plants showed disease symptom in the farm in Zhaotong city, Yunnan Province, China. Three fresh root samples with typical symptoms were collected from the plants of P. bulbocodioides in the field. The root sections (3mm × 3mm) from the border of the symptomatic tissue were cut and sterilized with 75% ethanol for 30 s, followed by 3% sodium hypochlorite (NaClO) for 2 min, and then rinsed three times with sterile water. The sterilized root tissues were inoculated on potato dextrose agar (PDA) at 28℃ in the incubator for 3 days. The colonies were obtained and sub-cultured from the hyphal tip to the new PDA to further purify. The colonies grew up on PDA at 28℃ for 1 week, the hyphal color from white turned to purple, the center of colony became brick red. The colonies produced abundant microconidia, macroconidia and chlamydospores, but no sporodochia was observed (Sup. S2). The microconidia were oval and irregularly oval, zero to one septate, and measured 2.0 × 5.2 to 4.1 × 12.2 µm (n = 20). The macroconidia were falcate, slender, with a distinct curve to the latter half of the apical cell, three to five septate, and measured 4.0 × 15.2 to 5.1 × 39.3 µm (n = 20). Morphological characterization showed that the three isolates were similar, and appeared to be Fusarium oxysporum (Leslie and Summerell, 2006). For molecular identification, total genomic DNA of two representative isolates DSL-Q and DSL-Y were extracted with CTAB method, and the PCR amplification were performed. The sequence of partial elongation factor (TEF1-α) gene was amplified using the primer pair EF-1/EF-2 (O'Donnell et al. 1998). The sequence of β-tublin gene (TUB2) was amplified using the primer pair T1/T22 (O'Donnell and Cigelnik, 1997). The sequences from the two isolates were obtained and sequenced. Clustal2.1 searches indicated that the sequences of the three loci of the two isolates revealed 97.8% to 100%similarity to F. oxysporum strains and were deposited in GenBank (accession nos. OP150481 and OP150485 for TEF1-α; OP150483 and OP186426 for TUB2). A pathogenicity test was performed to confirm Koch,s postulates. Inoculum was obtained from the two isolates by cultivating in 500 mL of potato dextrose broth on a shaker at 25℃. After 10 days, the hyphae grew into a cluster. The 6 individuals of P. bulbocodioides were divided into two groups. Three individuals grew in the bark substrate containing hyphae cluster, while another 3 individuals grew in the bark substrate containing sterile agar medium. The plants were kept in a greenhouse (constant temperature at 25℃, day and night for 12 h). After 20 days, the group inoculated with F. oxysporum isolates showed the same disease symptoms as observed on the plants in the field, while the control plants remained disease free. F. oxysporum was reisolated from the infected tissues (Sup. S1b, c). Phylogenetic dendrograms of Fusarium oxysporum were grouped by TEF1-α and TUB2 sequence (Sup. S3). The results confirmed that this fungus was identical to those identified by colony morphology, phylogenetic relationship and TEF1-α and TUB2 sequence. To our knowledge, this is the first report of F. oxysporum causing root rot on Pleione species in China. This is a pathogenic fungus in the production of Pleione species. Our study is helpful for the identification of root rot on Pleione species and the development of disease control strategy on cultivation.

First report of jujube anthracnose caused by Colletotrichum siamense in China.

Jujube (Zizyphus jujuba Mill.), a native small deciduous tree of China, is widely cultivated in China, Korea, India, Japan, Europe, and the United States (Chen et al. 2020). The fruit have been commonly consumed as healthy food supplements and traditional Chinese medicine for over 2000 years (Li et al. 2007). In August 2019, anthracnose-like leaf spot symptoms were observed on jujube plants in Xiaomenya Village, Jinan City, Shandong Province, China (36°27'39″N, 117°3'13″E), with over 30% leaf disease incidence. The spots were circular, sunken, brown in the center and with dark brown edges. As the spots enlarged and coalesced, it resulted in leaf perforation and early defoliation. Sometimes acervuli were observed on the lesions (Fig. S1a, b). To identify the causal agent, 20 diseased leaves were sampled, the margins of the lesions were cut into pieces (5 × 5 mm), sterilized and cultured following the protocol described previously (Wan et al. 2020) at 25 ℃ for 5 days. Twelve monospore isolates showing identical colony morphology were obtained. Three representative isolates, JNZG11, JNZG311, JNZG313, were used for further study. When grown on PDA the colony color was initially white and then turned pale-gray to gray in 5-day-old cultures. On the reverse, colonies were brown-black with an orange pigmentation near the center. Aerial mycelium was cottony, dense, white to pale-gray. Conidia were hyaline, 1-celled, smooth-walled, subcylindrical, oblong, attenuated with slightly rounded ends, (11.1-) 12.7-13.3 (-17.8) ×(-4.4) 5.2-5.5 (-6.3) μm (n=50). Appressoria were dark-brown, oval or irregular, (7.3-) 8.6-9.2 (-9.8) ×(-5.1) 5.8-6.9 (-7.0) μm (n=50) (Fig. S1c-g). The morphology resembled those of Colletotrichum gloeosporioides species complex (Cannon et al. 2012). For accurate identification, the sequences of the ribosomal internal transcribed spacer (ITS), actin (ACT), β-tub2 (TUB2), calmodulin (CAL), chitin synthase (CHS-1), and glyceraldehyde-3phosphate dehydrogenase (GAPDH) of the 3 isolates were sequenced (Weir et al. 2012), and deposited into GenBank (Accession Nos. see Table 1). The six loci (ITS, GAPDH, ACT, CHS-1, CAL, and TUB2) were concatenated and the aligned sequences (1904 bp) were 99.7% homologous to ex-type C. siamense ICMP18578. The sequences of 38 Colletotrichum species (44 isolates) were downloaded from GenBank for phylogenetic analyses. In the maximum likelihood phylogenetic tree generated, the highest log likelihood was -8798.90 and the three isolates were all in the C. siamense clade (bootstrap support 94 %) (Fig. S2). To complete Koch's postulates, 60 healthy, mature jujube leaves on 12 branches (5 leaves per branch) (variety 'Zhongqiuhong') were inoculated with 20 μL of spore suspension (106 conidia/mL) or sterile water as a control. The branches were placed in sterile beakers containing a small amount of sterile water sealed with plastic wrap and maintained at 28 °C, 12 h light/dark. Five days after inoculation, all treated leaves showed the typical anthracnose symptom, similar to that observed in the field (Fig. S1h). The same fungus was re-isolated from the margins of the lesions using the aforementioned methods. Whereas no fungus were isolated from the controls. Previously, C. siamense has been reported to infect Z. mauritiana in China (Shu et al. 2020). To our knowledge, this is the first report of C. siamense causing anthracnose on Z. jujuba in China. This finding provides crucial information for the effective management of this disease.

First Report of Colletotrichum godetiae Causing Anthracnose on Walnut (Juglans regia and Juglans sigillata) in China.

In August 2020, anthracnose lesions were observed on fruits of Juglans regia and J. sigillata in walnut orchards, in Yijun (Shaanxi Province) and Nanhua (Yunnan Province) counties, China. Symptoms on walnut fruits first appeared as small necrotic spots that rapidly enlarged into subcircular or irregular sunken black lesions (Fig. 1a, b). Sixty diseased walnut fruits (30 fruits of J. regia and J. sigillata, respectively) were randomly sampled from six orchards (10-15 ha each orchard, three orchards were selected in each county) with severe anthracnose (incidence rate of fruit anthracnose is over 60% in the orchard.) in two counties. Twenty-six single spore isolates were obtained from diseased fruits as described by Cai et al. (2009). After seven days, isolates formed grey to milky white colony with abundant aerial hyphae on the upper surface of colony, and milky white to light olive on the back of PDA (Fig. 1c). Conidiogenous cells were hyaline, smooth-walled, and cylindrical to clavate (Fig. 1d). Conidia were smooth-walled, aseptate, cylindrical to fusiform, with both ends acute or one end round and one end slightly acute (Fig. 1e), and ranged in size from 15.5-24.3×4.9-8.1 µm (n=30). Appressoria were brown to medium brown, clavate to elliptical, with the edge entire or undulate (Fig. 1f), and ranged in size from 8.0-27.6×4.7-13.7μm (n=30). The morphological characteristics of 26 isolates were similar to those of the species complex Colletotrichum acutatum (Damm et al. 2012). Six representative isolates were randomly selected (three isolates for each province) for molecular analysis. The ribosomal internal transcribed spacers (ITS) (White et al. 1990), beta-tubulin (TUB2) (Glass and Donaldson 1995), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Templeton et al.1992) and chitin synthase 1 (CHS-1) (Carbone and Kohn 1999) genes were amplified and sequenced. Sequences of 6 of 26 isolates were submitted to GenBank (Accession Nos: ITS: MT799938-MT799943, TUB: MT816321-MT816326, GAPDH:MT816327-MT816332, CHS-1: MT816333-816338). Multi-locus phylogenetic analyses revealed that six isolates clustered together with Colletotrichum godetiae ex-type culture isolates CBS133.44 and CBS130251, and the bootstrap support value was 100% (Fig.2). The pathogenicity of two representative isolates (CFCC54247 and CFCC54244) was tested using healthy fruits of the " J. regia cv. Xiangling" and " J. sigillata cv. Yangbi" varieties. Forty sterilized fruits (20 fruits were inoculated with CFCC54247, and 20 fruits with CFCC54244) were wounded by puncturing with a sterile needle through walnut pericarp and inoculated in the wound site with 10 μl of conidial suspension (106 conidia/ml) from seven day old colonies grown on PDA at 25℃. Twenty wounded fruits were inoculated with sterile water as control. Inoculated and control fruits were incubated in containers at 25℃ in a 12/12h light/dark cycle. The experiment was repeated three times. Anthracnose symptoms (Fig. 1g-h) were observed in all inoculated fruits after 12 days, whereas controls showed no symptoms. Fungal isolates from inoculated diseased fruits showed the same morphological and molecular characteristics as the isolates obtained in this study, confirming Koch's postulates. To our knowledge, this is the first report of C. godetiae causing anthracnose on the two walnut species in China. The result will be helpful for providing a basis for further research on the control of the disease.

First report of Dickeya dadantii causing bacterial soft rot of Scindapsus pictus in Taiwan.

Scindapsus pictus (satin pothos or silver vine) is an evergreen climbing plant belonging to the Araceae family, subfamily Monstereae (Bown, 2000), which is also cultivated as a foliage ornamental (Masnira et al. 2019). In September of 2022, soft rot symptoms were observed on potted S. pictus plants grown in a greenhouse in Nantun District, Taichung, Taiwan, in which soft rot of another aroid (philodendron) has also been reported (Wu et al. 2023). The symptoms appeared on the petioles and most of them tended to extend to the leaf blades; the colors of leaf lesions ranged from dark brown to gray (Fig. S1). Some 70% of the plants in the greenhouse showed similar symptoms and losses were estimated to be 15-30%. Four symptomatic plants were sampled. Macerated tissues from rotting petioles were soaked in 10 mM MgCl2 and observed under a light microscope (Nikon, Japan) at 400 x magnification. Motile, rod-shaped bacteria were observed, and 1-2 loopfuls of undiluted sample suspension were streaked onto nutrient agar (NA; Gibco, USA). After culturing at 28°C for 1 day, all samples yielded round, creamy-white colonies (0.9 mm in diameter) and from each of the four samples a pure culture was obtained (Spi1-Spi4). All isolates exhibited oxidative and fermentative metabolism of glucose (Schaad et al. 2001). They caused pitting on crystal violet pectate agar, induced maceration on potato tuber and were tested positive for phosphatase activity and indigoidine production (Lee and Yu 2006; Schaad et al. 2001). Polymerase chain reaction tests using Dickeya-specific primers 5A and 5B (Chao et al. 2006) amplified the expected amplicon (0.5 kb) in extracted DNA samples of all isolates. Identification of the strains was achieved by amplifying and sequencing fragments of the housekeeping genes gyrB, recN, dnaA, dnaJ, and dnaX (Marrero et al. 2013); the lengths of the five gene fragments analyzed were 822, 762, 720, 672, and 450 bp, respectively (accession nos. OP985528-OP985532). The five sequences were concatenated for every isolate; the resulting 3,426 bp sequences were aligned with ClustalW and found to be identical. A maximum-likelihood analysis was conducted using the 3,426-bp sequences and those of known Dickeya species' type strains. Spi1 to Spi4 clustered with D. dadantii subsp. dieffenbachiae NCPPB 2976T and D. dadantii subsp. dadantii CFBP 1269T (Fig. S2) with sequence identities of 98.4 and 98%, respectively. To fulfil Koch's Postulates, stab inoculations of the four isolates into the petioles of cutting propagated, 38-day-old S. pictus plants (3 plants per isolate) were conducted using sterile toothpicks. The amounts of bacteria used was approximately 106 cfu per toothpick; the bacterial loads were estimated by suspending the cells in 10 mM MgCl2 and spread-plating diluted suspensions on NA. Sterile toothpicks were used as control. All tested plants were sealed in plastic bags (containing wet paper towel) and kept in a growth chamber (28°C; 12-h photoperiod). After 1 day, all isolates induced soft rot symptoms resembling those observed under natural conditions in the greenhouse. Bacteria were re-isolated, and they all shared the same dnaX sequence with strains Spi1 to Spi4. This is the first report of S. pictus affected by D. dadantii in Taiwan. Further investigation is needed to determine whether Spi1-Spi4 belong to D. dadantii subsp. dieffenbachiae. Dickeya dadantii has been found infecting different aroids (Lee and Chen 2021; Lin et al. 2012). The species has also been reported in Taiwan on poinsettia (Wei et al., 2019) and philodendron (Wu et al. 2023). Because these plants are often grown closely in the same facilities, growers should be wary of D. dadantii's spread among these plants. Reduction of environmental humidity and avoiding overhead irrigation may be effective in preventing the pathogen's transmission.

First report of Neopestalotiopsis rosae causing leaf blight on Shatangju in southern China.

Shatangju (CitrusreticulataBlancocv.Shatangju) belongs to genus Citrus and was cultivated extensively in southern China. In April 2022, a leaf blight-like symptom (firstly brown spots appeared on infecting leaves, then these brown spots extended, finally the whole leaves displayed blight-like symptom) was observed on 5%~10% of Shatangju seedlings (around five hundreds in total) in an orchard located in Wuhan city, Hubei, China. Diseased leaves from three seedlings were collected and cut into pieces (0.2 to 0.5 cm). These pieces were surface-sterilized using 75% ethanol for 3 min, rinsed with sterile distilled water for several times, then placed on potato dextrose agar (PDA) and incubated at 26°C with 12-h light/dark cycle. Over 20 pieces plated, wherein 30% were identified as Colletotrichum fructicola, 60% as Neopestalotiopsis spp., and 10% developed saprophytes. C. fructicola was a known pathogen on citrus, thus Neopestalotiopsis spp. was further investigated. Eight single-conidium colonies of the Neopestalotiopsis spp. were obtained, wherein STJ-8 was chosen as a representative for further study. The average growth rate of STJ-8 was 15.1±0.5 mm/day (n=5). Fungal colonies produced white cottony mycelium with abundant black acervuli distributed in concentric rings 6-8 days after planting, which ranged from 342.3 to 710.5 μm in diameter (n=100). Conidia were fusoid, five cells, four septa with average dimensions of 25.36×5.47 μm (n=100). Basal and apical cells were hyaline, wherein three middle cells were brown with darker septa. The apical cell was cylindrical with two to three transparent accessory filaments (13.7 to 30.5 µm in length, n=80). Basal cell was conic with an appendage (4.1 to 8.8 µm in length, n=40). Partial sequences of internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α), and β-tubulin (TUB2) were amplified with reported primers (White et al. 1990; Lee et al. 2006; Maharachchikumbura et al. 2014), sequenced, and submitted to GenBank (accession nos. ITS: OP236541; TEF-1α: OP250124; TUB2:OP263094). BLASTn results showed 100% identity with the corresponding sequences of Neopestalotiopsis rosae. A multilocus phylogenetic analysis showed STJ-8 was closest to N. rosae. Thus, STJ-8 was identified as N. rosae. Pathogenicity tests were performed on one-year-old Shatangju seedlings and detached primary leaves by inoculating needle-wounded leaves with seven days old 5-mm mycelial plugs/acervuli (about 5000 spores) of STJ-8. Control seedlings/leaves were inoculated with 5-mm PDA plugs/sterile water drops. All inoculated detached leaves were cultured at same the place with STJ-8 cultured, while inoculated seedlings were put in a growth chamber at 26°C under a 16-h light/dark cycle (60% humidity). Symptoms developed on all inoculated leaves (except healthy control) 2 and 4 days post-inoculation by mycelial plugs and acervuli, respectively. N. rosae was re-isolated from the inoculated leaves, confirming Koch's postulates. N. rosae has been reported to cause diseases on various plants worldwide (Rebollar-Alviter et al. 2020; Xavier et al. 2021; Lawrence et al. 2022). In China, N. rosae has been reported to cause leaf spot/blight on pecan and strawberry (Wu et al. 2021; Gao et al. 2022), which caused great loss on these crops. To our knowledge, this is the first report of N. rosae causing leaf disease on citrus. Our study is important for developing control strategies against N. rosae in future.

First report of cobweb disease of Auricularia heimuer caused by Hypomyces mycophilus in Fujian Province, China.

Auricularia heimuer is a gelatinous fungus with great edible and medicinal values. In September 2021, a suspected cobweb disease was found in some A. heimuer farms in Fujian Province, China. The disease caused white cottony mycelium to grow on the basal surface of the A. heimuer at the beginning of infection and gradually spread along the outer edge of the fruiting body, and eventually the white pathogen mycelium covered the entire fruiting body, which eventually led to the wilting and death of about 35% of A. heimuer . Two heavily infected tissues of A. heimuer were collected and two isolates were obtained by single spore isolation and purification technique. The pathogen colonies grew 10 to 12 mm per day on potato dextrose agar (PDA), and the colonies were initially white in color and gradually changed to yellowish brown with neat margins. Well-developed mycelium with septum, Conidiophores are bottle-stemmed and whorl-shaped branches, Conidia solitary, as ovoid, colorless singletons or doublets. The chlamydospores are yellowish, smooth surface, with 2-3 septa, size 9-22 μm × 30-38 μm. These morphological features are consistent with the Hypomyces mycophilus (Gea et al. 2019;Wang et al. 2021). For molecular identification, genomic DNA of the two isolates was obtained using an extraction kit (Biocolor, Shanghai, China), internal transcribed spacers (ITS) regions and 5SrRNA were amplified using ITS1 and ITS4 primers (White et al. 1990). A 590 bp DNA fragment was obtained and the sequences were deposited in GenBank (Accession Nos. OP715875 and OP782039),A BLAST search in GenBank revealed the highest similarity (≥99%) toH. mycophilus (GenBank Accession Nos.MH857567 and KU937111) .To fulfill Koch's postulates, the isolates cultured on PDA plates for 10 days were made into a spore suspension with sterile water at a concentration of 5 × 106conidia/ml and sprayed onto twenty healthy fruiting bodies grown to about 2 cm in diameter, and another ten healthy fruiting bodies sprayed with sterile water as control, and incubated in an artificial climate chamber at 25℃ and relative humidity of 90%-95% (Back et al. 2012). After 4 days of inoculation, the pathogen started to germinate and slowly grew white mycelium, then the white mycelium multiplied at the base of the fruiting bodies and spread from the base to the periphery, and finally the fruiting bodies were completely covered by the pathogenic mycelium and gradually wilted. The symptoms were consistent with the natural disease symptoms under cultivation conditions, while the control group had normal growth of the seeds and no disease symptoms. H. mycophilus was reisolated and purified from symptomatic cotyledons and identified by the above method, and the results of the two experiments were consistent. To our knowledge, this is the first report of H. mycophilus causing cobweb disease in A. heimuer.

First Report of Black Rot on Persimmon Fruit Caused by Diaporthe eres in China.

The fruit of persimmon (Diospyros kaki L.) is popular with consumers for its delicious taste and is widely grown in China. In September 2022, fruit rot symptoms were found on persimmons grown at the Tobacco Research Institute of Chinese Academy of Agricultural Sciences (120°26'43.879″E, 36°7'59.794″N) in Qingdao City, Shandong Province, China. Diseases incidence on fruit is 70%. Typical symptoms were small, black spots, which gradually expanded, and became rot lesions, causing fruit rot and serious losses. To isolate the pathogen, small sections (approximately 2 × 2 mm) of symptomatic tissues from five persimmon fruits were surface-sterilized in 5% NaClO, followed by 75% ethanol, rinsed with sterile distilled water, and then incubated on potato dextrose agar (PDA) plates incubated at 28℃ for 5 days. By single spore isolation method, seven dominant fungal isolates developed with similar colonies. They were initially white and gradually turned to gray after 7 d. The alpha conidia were unicellular, colorless, elliptic or fusiform, and 5.0 to 8.0 μm × 2.5 to 3.5 μm in size. Beta conidia were unicellular, colorless, filiform, often hooked at one end and 18 to 28 μm × 1.3 to 1.5 μm in size. These morphological features were consistent with Diaporthe spp (Udayanga et al. 2014). To confirm identity of the causal fungus, the internal transcribed spacer (ITS) rDNA, translation elongation factor 1-a (TEF1-a), b-tubulin (TUB), and histone H3 (HIS) genes of three representative isolates (De2, De3 and DeR2) were amplified and sequenced using primers ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b and CYLH3F/H3-1b as described by Chaisiri et al. (2021). The resulting sequences were deposited in GenBank (accession nos. OP963195, OP963386 and OP963389 for ITS; OP968958, OP968959 and OP9689600 for EF1-α; OP978141, OP978142 and OP978143 for TUB; OP968955, OP96896 and OP968957 for HIS), and showed 99-100% similarity with D. eres by NCBI blast. Phylogenetic analysis was conducted using MEGA-X software, with the NJ method. In the phylogenetic tree, the combined sequences attributed the three isolates to the D. eres. On the basis of morphological and molecular characteristics, the pathogen was identified as D. eres. The strain De2 was preserved in the Agricultural Culture Collection of China (preservation number: ACCC 35482). Pathogenicity tests were performed by wound inoculation on healthy persimmon fruit. These were surface sterilized with 75% ethanol for 1 min, rinsed in sterile water three times, and allowed to dry. A 5-mm agar disc with mycelium of the causal pathogen was placed on the surface of fruit, which was wounded with a sterile needle in advance, with the sterile PDA plugs used as control. Three persimmons were used for each treatment, repeat three times. All the treated fruits were incubated in the sterilized transparent plastic cans at 28°C and 90% RH. After 3 days inoculation, the first symptom of rot lesion appeared on the fruit with mycelial plugs, whereas the control fruit remained symptomless. The same causal pathogens were re-isolated from the lesions and identified by morphologyand confirmed to fulfill Koch's postulates. This is the first report of D. eres causing fruit rot on persimmon. This finding showed that Diaporthe eres is a causal agent of persimmon fruit rot in China.

First report ofFusarium meridionalecausing Fusarium head blight of wheat in Henan Province, China.

Fusarium graminearum and F. asiaticum have been found as a major cause of Fusarium head blight (FHB) of wheat (Triticum aestivum L.), especially in Henan Province of China (Zhang et al. 2014; Xu et al. 2021). In May 2021, a survey to determine the composition of Fusarium species infecting wheat heads was conducted in commercial fields in Henan. A total of 395 diseased spikes with premature whitening symptom were collected from 31 commercial fields in Henan. Symptomatic spikelets were excised, surface-sterilized for 10 s in 70% ethanol followed by 1 min in 3% sodium hypochlorite, rinsed three times with autoclaved distilled water, and then plated onto potato dextrose agar (PDA) medium. Isolated colonies that resembled Fusarium species were transferred to fresh PDA plates and purified using a single spore method. Species were identified based on sequence analysis of the translation elongation factor-1α (TEF) and trichothecene 3-Oacetyltransferase (Tri 101) gene (Proctor et al. 2009). The results indicated that F. graminearum (43.3%), F. asiaticum (47.8%), F. pseudograminearum (6.6%) were the main causal agents of FHB in Henan. However, nine isolates (2.3%) were found to be identical to F. meridionale by sequence comparison in GenBank, and eight isolates of which came from three fields with 1% to 2% diseased spikes near Reservoir Luhun (34.1255° N, 112.1111° E, altitude: 388 m above sea level), Songxian County of Henan. The isolates of F. meridionale were transferred onto carnation leaf agar (CLA) and incubated at 20℃ under black light blue illumination. Macroconidia were abundant, relatively slender, curved to almost straight, commonly six- to seven-septate, and 27.0 to 61.0 (average 44.0) μm × 3.2 to 6.8 (average 5.3) μm. Microconidia were not observed. The TEF sequences (Accession nos. OM460748 to OM460756) and the Tri 101 sequences (OM460759 to OM460767) of the nine isolates showed 99 to 100% similarity with the TEF and Tri 101 sequences of F. meridionale NRRL 28436 and NRRL 28723 (AF212435 and AF212436 (TEF); AF212582 and AF212683 (Tri 101)). To complete Koch's postulates, the pathogenicity of the fungus was tested by using the single floret inoculation method by injecting 20-μl conidial suspension (5 × 105 conidia per milliliter) into healthy inflorescences of wheat cultivar Bainong 207 at anthesis in the field. Another 30 healthy inflorescences were injected with sterile distilled water. The heads were covered with polyethylene bags that were removed after 2 days. Twenty days after inoculation, while control inflorescences were asymptomatic, the F.meridionale-inoculated inflorescences showed 12% bleached spikelets per spike. By using the methodology described above, the fungus was re-isolated from infected spikelets of inoculated wheat heads but not from the controls. Although F.meridionalehas frequently been reported in association with Fusariumear rot (FER) of maize in Chongqing City and Gansu Province (Zhang et al. 2014; Zhou et al. 2018), and riceFER in Sichuan Province (Dong et al. 2020), to our knowledge, this is the first report of F. meridionale from diseased wheat heads in Henan, China. Further investigation is needed to gain a better understanding of this species by collecting isolates from different cropping system in Henan, which maize-wheat and rice-wheat rotation fields have coexisted in the region.

First report of Athelia rolfsii (=Sclerotium rolfsii) associated with foot rot disease of Chrysanthemum morifolium in India.

Chrysanthemum morifolium L. is an important flower crop grown in different parts of Karnataka for its striking cut flowers and international market value. During a field survey (Mysore district, Karnataka, February, 2022), chrysanthemum fields were found infected with foot rot disease. The presence of white mycelial structures with sclerotia were recorded near the stem-soil interface. The disease incidence ranged 10-12% measured in an area of approximately 10 hectares. The infected plants showed quick wilt, yellowing and toppling of the entire plant. Infected plants from Doddamaragowdanahally and Rayanahally (n=15) were collected and associated fungal pathogen isolated after surface sterilization with NaOCl (1%) on potato dextrose agar (PDA) amended with chloramphenicol (50 mg/L). Fungal mycelia developed from the infected tissues were inoculated on to fresh PDA plates to obtained pure cultures for further identification. Fungal colonies with dense, aerial whitish-cottony mycelia with uniformly globoid sclerotia (0.284.2 mm) were observed after 15 days of incubation (28 ± 2°C). Sclerotia were white in the beginning and turned brown at maturity. The average number of sclerotia produced per plate ranged from 240 to >480 (n = 10). To further to confirm the identity of the isolates, two representative isolates (CmSr1 and CmSr2) was subjected to molecular identification based on ITS-rDNA sequences. Briefly, genomic DNA was isolated from 12 day old cultures using the CTAB method and ITS-rDNA was amplified using ITS1-ITS4 primers (White et al., 1990). An expected amplicon of >650 bp (ITS) was obtained and later sequenced from both the directions. The consensus sequences were analysed through nBLAST search which revealed that 100% sequence similarity with reference sequences of Athelia rolfsii (S. rolfsii) from GenBank database (MT127465, MN974137, KC292637; identity 656/656; 0 gaps). A phylogenetic tree obtained by the neighbor-joining method using MEGAX shared a common clade with the reference sequences retrieved and computed, thus confirming the identification based on sequence analysis and molecular phylogeny. The representative sequence of A. rolfsii isolates CmSr1 and CmSr2 isolates deposited in GenBank with Accession nos. ON456153 and ON456154, respectively. Based on etiology, morphological, cultural and molecular data the pathogen was identified as Athelia rolfsii (Curzi) Tu & Kimbrough (Syn: Sclerotium rolfsii Sacc.) (Mordue, 1974; Mahadevakumar et al., 2016, 2018). Plants (n=60) were inoculated with sclerotial bodies (2 sclerotia/plant) near stem soil interface under green house and covered with polythene bags (at 27 ± 2°C and 80% RH). Non-inoculated plants (n=20) served as controls. The development of foot rot disease was observed eight days after inoculation. A total of 48 plants showed the foot rot symptoms and 12 inoculated plants and control plants remained healthy. The identity of the fungus was confirmed by morphological and cultural characters after re-isolation. C. morifolium is an important flower crop in Karnataka. S. rolfsii is known to be associated with blight and collar rot of Chrysanthemum spp. from Kerala (Beena et al., 2002) but no species (host) identity provided. Therefore, to the best of our knowledge, this is the first report of foot rot disease caused by Athelia rolfsii on C. morifolium in India. Early diagnosis of this disease will help the farmers to adopt suitable management practices to avoid loss.

First report of Phytophthora gonapodyides causing root rot on raspberry in Canada.

Raspberry (Rubus idaeus L.) is an economically important fruit crop in Canada and about 80% of red raspberries are cultivated in British Columbia. In 2018, foliar symptoms associated with root rot and wilting complex disease were observed in raspberry field of Fraser Valley areas of British Columbia. Plants were stunted with reduced numbers of primocanes. Chlorosis and necrosis on leaves and partial wilting of branches were observed. When plants were uprooted, necrosis and browning on roots were observed. Two isolates of oomycetes pathogen were isolated using baiting with rhododendron leaves and pear fruit as described in Sapkota et al. 2022. Using FastDNA Spin kit (MP Biomedical, Burlingame, CA), genomic DNA of pathogen isolates was extracted from mycelia cultured on 20% clarified V8 agar medium amended with 10 mg pimaricin, 250 mg ampicillin, 10 mg rifampicin (V8PAR) per liter following the manufacturer's standard protocol. Pathogens were identified using colony morphology on 20% clarified V8 PAR as well as internal transcribed spacer (ITS) sequencing with ITS1 primers (White et al. 1990) and multiplex targeted-sequencing with degenerate primers of three nuclear genes: heat shock protein90 (HSP90), elongation factor 1 alpha (EF1α) and beta tubulin (βtub). BLAST searches of ITS sequences of isolates of this study (accession nos. OP180065, OP180066) in NCBI GenBank showed 98.5 to 99.6% identity with the ITS sequence of P. gonapodyides (accession nos. MN513238.1, MG753496.1). Multiplex targeted sequencing also identified both isolates as a P. gonapodyides (accession nos. SRR20227809, SRR20227807) when mapped with the reference sequences (accession nos. HSP90: KX251233.1, EF1α: KX251231.1, β-tub: KX639710.1). Pathogenicity was confirmed by inoculating mycelial suspension of one isolate of P. gonapodyides on root of intact plants and mycelial plugs of two isolates on detached stems of the raspberry plants, 'Chemainus' in the greenhouse using methods described in Sapkota et al. 2022. Two experiments were conducted with three replicates in each test. Experiments were arranged using completely randomized design. In detached stem assays, distinct dark-lesion symptom appeared at 7 to 9 days after inoculation while uninoculated control stems remained asymptomatic. Intact plants showed wilting and foliar symptoms 15 days after inoculation and progressed higher at 4 to 5 weeks after inoculation. Root infection with dark brown to black color was observed when roots were assessed at 5 weeks after inoculation. The diseased root and crown tissues tested positive for Phytophthora in Agdia ImmunoStrip and P. gonapodyides was re-isolated and confirmed with multiplex-targeted sequencing. Phytophthora gonapodyides was previously reported from raspberry in Chile (Wilcox and Latorre 2002). To our best knowledge, this is the first report of P. gonapodyides infecting red raspberry in British Columbia, Canada. The detection of new Phytophthora species on raspberry may become a new potential problem to growers in addition to P. rubi, which is already a major cause of raspberry decline in the region.

First Confirmed Report of Rhizoctonia solani AG-7 Causing Potato Stem Canker in Mexico.

Rhizoctonia solani, is the causal agent of black scurf and stem canker of potatoes (Solanum tuberosum L.) throughout the world. In November 2021, stem canker symptoms were observed in two potato fields located in Ahome, Sinaloa, Mexico. The disease incidence was estimated up to 15%. For fungal isolation, fragments of symptomatic stems were surface sterilized with 2% sodium hypochlorite for 2 min, rinsed with sterilized distilled water, and blotted dry on sterile filter paper. Fragments were placed on PDA medium and incubated at 25°C in darkness for 4 days. Rhizoctonia-like colonies were consistently obtained and 12 isolates were purified by the hyphal-tip method. Fungal colonies on PDA were white initially and then turned brown, raised, and with entire or undulate edges. Septate hyphae were hyaline, smooth, and branched at right angles with a septum near the point of branching. Microscopic examination by staining with 1% safranin O and 3% KOH solution showed multinucleate cells. The morphological features of the isolates resembled those of Rhizoctonia solani (Sneh et al. 1991). Four representative isolates were selected for molecular analysis and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi at the Research Center for Food and Development (Culiacán, Sinaloa) under accession nos. CCLF267, CCLF274, CCLF277, and CCLF279. For molecular identification, genomic DNA from each of the four isolates was extracted, and the internal transcribed spacer (ITS) region was amplified, and sequenced with the primer pair ITS5/ITS4 (White et al. 1990). The sequences were deposited in GenBank (accession nos. OP784258 to OP784261). Phylogenetic analyses were performed using the Maximum Likelihood method with ITS sequences for anastomosis groups (AG) of Rhizoctonia solani. The phylogenetic tree grouped the four isolates within the R. solani AG-7 clade with high bootstrap support (100%). For pathogenicity tests, certified pathogen-free potato mini-tuber (cv. Fianna) were placed in a polystyrene pot (1 L) filled with a 5 cm layer of a sterile substrate composed of soil and peat moss (2:1 w/w). One rice grain (20 mg) colonized with each isolate was placed 10 mm above the uppermost sprout tip and covered with the sterile substrate (Inokuti et al. 2019). Control plants were inoculated with sterile rice grains. All pots were transferred to a greenhouse where the temperature ranged from 20 to 32°C. Stem necrosis symptoms were observed on all inoculated plants 25 days after emergence, whereas control plants remained symptomless. Pathogenicity test was performed twice with similar results. Fungi were reisolated from the infected stems and found to be morphologically identical to the isolates used for inoculation, thus fulfilling Koch's postulates. The AG-7 has been previously reported to cause potato diseases in South Africa (Truter and Wehner 2004). In Mexico, Carling et al. (1998) reported the presence of an isolate of R. solani AG-7 obtained from a potato tuber-borne sclerotium in Toluca; however, there is no information about the methodology used for the characterization of that isolate. To our knowledge, this is the first confirmed report of R. solani AG-7 causing potato stem canker in Mexico. Our findings improve knowledge about R. solani AGs occurring in potato fields in Mexico. So, further studies should be conducted to investigate the diversity, prevalence, and fungicide sensitivity of AGs distributed in the main potato-producing states in Mexico.

First Report of Leaf Spot Caused by Lasiodiplodia theobromae on Kadsura coccinea in China.

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

First Report of Botrytis cinerea Causing Stem Rot on Pea (Pisum sativum L.) in China.

Pea (Pisum sativum L.) is one of the most important cool season legumes consumed as vegetable in the world. In March 2022, a severe stem rot was observed on pea cultivars in vegetative stage in Wuhan, Hubei Province, China (30°39' N, 114°66' E). The infection started on the lower stems, and the lesions were water soaked, then girdled the stem, resulting in wilting of the leaves. Eventually, the entire plant died, and some necrotic stems were covered with gray conidia. To investigate the causal agent, small pieces cut from diseased stems were surface sterilized with 2% NaOCl for 1 min, then incubated on potato dextrose agar (PDA) at 25°C for 3 days. Pure cultures were obtained by hyphal tip transfer and five isolates were studied further. Colonies initially appeared white, turned gray from the center, then became taupe with cottony aerial mycelia, and finally black hard, round or irregular sclerotia (0.92 to 5.34 × 0.86 to 4.42 mm, n = 20) developed. The sealing film of several plates were removed after 5 days, and abundant conidia were produced 3 days later. The conidia are terminally arranged at the end of long, grayish branched conidiophores, conidia are unicellular, hyaline and round or elliptical, (9.2 to 11.4 × 6.7 to 9.2 μm, n = 50), and the conidiophores are (10.7 to 13.0 μm × 760 to 1080 μm, n=20) in size. The morphological characteristics were consistent with descriptions of Botrytis cinerea (Li et al., 2016). Genomic DNA of the five isolates was extracted, and the internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene, heat-shock protein 60 (HSP60) gene, and DNA-dependent RNA polymerase subunit II (RPB2) gene were amplified using the primers described by Aktaruzzaman et al. (2018). The sequences were deposited in GenBank (accession nos. ON533694 and ON566787-ON566790 for ITS; ON600613 to ON600617 for HSP60; ON600608 to ON600612 for G3PDH; ON600603 to ON600607 for RPB2). The BLASTn analysis of these sequences showed that the isolates had high similarity (99 to 100%) with other B. cinerea isolates. A phylogenetic tree was constructed by MEGA11, and our isolates clustered in the B. cinerea clade. In pathogenicity test, 2-week-old seedlings of pea cultivar 'Zhongqin1' were inoculated. Mycelial plugs (5 mm diameter) taken from a 3-day-old colony of each isolate were placed on the axil of a stipule at the 4th node of potted pea plants (n=5 per isolate), and PDA plugs were placed on the same location of control (n=3). Inoculated and control plants were kept in a humid plastic box at 23°C for 2 days, and then placed in a glasshouse. Symptoms with water-soaked lesions were observed on the inoculated plants after 2 days, stems showed soft rot and broke off after 3 to 5 days, disease symptoms similar to those in the field, while the controls remained healthy. The pathogen was re-isolated from the affected stems, fulfilling Koch's postulates. B. cinerea had been reported to cause foliar, pod, seed and stem rot of pea after flowering in many pea production regions in the world (Kraft and Pfleger, 2001). Pea was recorded as a host of B. cinerea in Zhejiang, Sichuan and Yunnan Provinces (Tai, F. L. 1979; Zhuang, W.-Y. 2005; Zhang, Z. 2006.), but there has been no detailed disease description and identification of pathogen. To our knowledge, this is the first report of B. cinerea causing stem rot on pea in vegetative stage in China. Since B. cinerea can infect pea at any developmental stage, it could have a high economic impact as green pea production increases in China.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Solanum carolinense in Korea.

Solanum carolinense L. (Solanaceae) is a perennial herbaceous plant native to the southeastern United States. The plant has been invasive in Europe, Australia, and Asia. It was accidentally introduced to Korea in the 1960s (Oh et al. 2002) and is now widely naturalized mostly in the southern area of the Korean peninsula. Since 2002, it has been designated as one of 'harmful non-indigenous plants' by the Korean Ministry of Environment due to its adverse effects on native plants. In September 2014, several S. carolinense plants were found infested with a powdery mildew disease in Busan, which was the first for Korea. Later in 2020-2022, hundreds of plants were continuously observed with an approximately 50% disease incidence in Jeonju, Korea. Symptoms first appeared as circular to irregular white patches, which subsequently coalesced to develop into abundant hyphal growth on both sides of the leaves. Representative voucher specimens were deposited in the Korea University herbarium (KUS-F28240 and F32541). Hyphal appressoria were nipple-shaped. Conidiophores were cylindrical, 110 to 190 × 10 to 12 µm, and produced 2 to 5 immature conidia in chains with a sinuate outline. Foot-cells of conidiophores were straight, cylindrical, and 58 to 90 μm long, followed by 2 to 3 cells. Conidia were ellipsoid to barrel-shaped, 29 to 36 × 15 to 20 μm (l/w 1.6 to 2.0), and devoid of distinct fibrosin bodies. Germ tubes were at the perihilar position of the conidia. Sexual stage was not developed. These morphological characteristics are consistent with those of Golovinomyces ambrosiae (Schwein.) U. Braun & R.T.A. (Braun and Cook 2012). Sequences of the internal transcribed spacer (ITS) region and large subunit (LSU) gene of rDNA were determined using primer pairs ITS1/PM6 and PM3/TW14, respectively (Bradshaw and Tobin 2020). A comparison of the resulting sequences using the BLASTn algorithm showed 100% identity with reference sequences of G. ambrosiae (MT355556, AB769425) and G. spadiceus (MN365027) for ITS and LSU in NCBI. Obtained sequences were deposited in GenBank (Accession Nos: OP585651-OP585654). A Maximum parsimony tree was constructed based on ITS+LSU dataset consisting of 23 sequences. Our sequences were clustered with sequences of G. ambrosiae, G. latisporus and G. cichoracearum, and supported with 100% BS value. A pathogenicity test was performed by gently dusting conidia onto leaves of five healthy potted plants. Five non-inoculated plants served as controls. Powdery mildew colonies developed on all inoculated plants after 5 days, whereas the control plants remained symptomless. The fungus present on the inoculated leaves was morphologically identical to that observed on the originally diseased leaves, which supports Koch's postulate. Previously, Erysiphe cichoracearum (syn. of G. cichoracearum) was recorded on this plant in theUSA (Farr and Rossman 2022). Since G. cichoracearum has been divided into several distinct species (Takamatsu et al. 2013, Qiu et al. 2020), the current taxonomic position of the North American isolate is unknown. In Korea, G. ambrosiae was reported on several asteraceous hosts such as Bellis perennis, Brachyscome multifida, Helianthus annuus, H. salicifolius, and Verbena bonariensis (Farr and Rossman 2022). To our knowledge, this is the first report of powdery mildew on this plant outside the USA. According to our field observations, powdery mildew infestation had little effect on plant growth and vigor.

First Report of Fusarium citri Causing Postharvest Fruit Rot of Chayote in Mexico.

In November 2018, symptoms of brown rot were observed on chayote (Sechium edule) var. nigrum spinosum with a 20% disease incidence of 120 harvested fruits in the National Germplasm Bank of Sechium edule, located in the Centro Regional Universitario Oriente (CRUO) from the Chapingo Autonomous University (Huatusco, Veracruz, Mexico). For fungal isolation, pieces from symptomatic fruits were surface disinfected by immersion in a 1.5% NaClO solution for 2 min, rinsed in sterile distilled water, placed in Petri plates containing potato dextrose agar (PDA) amended with kanamycin sulfate, and incubated at 25ºC. Fusarium-like colonies were consistently isolated on PDA and five monoconidial isolates were obtained. A representative isolate was selected for morphological characterization, phylogenetic analysis, and pathogenicity tests. On PDA, colonies exhibited white and fluffy aerial mycelia, with diffused pale brown pigment in the center at 7 days of incubation at 25℃ in darkness. Macroconidia (n= 100) were hyaline, falcate, with 4 to 5 septa, measuring 23.9 to 31.9 × 2.9 to 4.2 μm, and foot-shaped basal cells. Microconidia and chlamydospores were absent. Morphological features were consistent with the description of the Fusarium incarnatum-equiseti species complex (Xia et al. 2019). The isolate was deposited as FUS2 in the Culture Collection of Phytopathogenic Fungi of the Laboratory of Plant Pathology at the Colegio de Postgraduados. For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region, partial sequences of translation elongation factor 1-alpha (EF1-α), and the second-largest subunit of RNA polymerase II (rpb2) genes were amplified, and sequenced with the primer sets ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and RBP2-5F/RPB2-7R (Liu et al. 1999), respectively. DNA sequences were edited in BioEdit 7.2 and compared with those in the NCBI nucleotide database. Alignments were implemented in MEGA X using reference sequences from Fusarium spp. A phylogenetic tree, including published ITS, EF1-α, and rpb2 sequence data, was constructed for the Fusarium incarnatum-equiseti species complex (FIESC) based on Maximum Likelihood. The sequences were deposited in GenBank (accession nos. ON878083, ON890421, and ON890420). The phylogenetic analysis grouped the isolate FUS2 within the F. citri clade. Pathogenicity of the fungus was verified on 10 healthy chayote fruits var. nigrum spinosum previously disinfested by immersion in a 1% NaClO solution for 3 min and washed in sterile water. A total of 5 mL of a conidial suspension (1 × 106 spores/ml) was sprayed on each whole fruit. Ten control fruit were sprayed with sterile distilled water. The fruits were kept in a moist plastic chamber at 25°C and 12 h light/dark for 30 days. All inoculated fruits developed water-soaked brown lesions (3 to 5 cm in diameter) covered with white mycelium at 15 days after inoculation, whereas no symptoms were observed on the control fruits. The fungus was consistently re-isolated only from the diseased fruits and found to be morphologically identical to the isolate used for inoculation, fulfilling Koch´s postulates. Fusarium citri has been associated with Capsicum sp. and mandarin orange in China, Triticum sp. in Iran, alfalfa in Denmark, and lettuce in the Czech Republic and Italy (Farr and Rossman 2022). To our knowledge, this is the first report of F. citri causing postharvest fruit rot of chayote in Mexico and worldwide.

First Report of Diaporthe fusicola Causing Postharvest Fruit Rot of Peach (Prunus persica) in China.

In May 2022, rot symptoms were observed on postharvest peach (Prunus persica [L.] Batsch) fruits in a market in Nanchang, Jiangxi province (28°44' N; 115°50' E), China. A total of 80 samples were collected from three different fruit stalls through the market survey. The incidence of this disease was 10 to 15%, and severity varies from approximately 30 to 50% of fruit surface coverage. The symptom of infected fruits was circular, pale brown to brown, rotten, necrotic lesions, covered with white hyphae and small spore masses. Eight symptomatic peach fruits were surface disinfected with 75% ethanol for 30 sec and incisions were made with a sterile scalpel. Small pieces from symptomatic tissues were placed on a potato dextrose agar (PDA) medium and incubated at 25℃ for 7 days. Six isolates were obtained in total. Colonies on PDA were initially white, aerial, fluffy at first, and darkened with age. Alpha conidia were fusoid, hyaline, aseptate, guttulate, tapering towards ends, and ranged in size from 9.8 to 5.1 µm × 3.2 to 2.1 µm (x ̅=7.1 ± 1.0 × 2.6 ± 0.3 µm, n=60). Beta conidia were not seen. For further confirmation, genomic DNA was extracted from three isolates (04-10, 04-11, and 04-12), the internal transcribed spacer (ITS) region, beta-tubulin (TUB), calmodulin (CAL), partial translation elongation factor 1-alpha (TEF1-α) and histone H3 (HIS) genes were amplified by using primers ITS1/ITS4, Bt2a/Bt2b, CAL228F/CAL737R, EF1-728F/EF1-986R, CYLH3F/H3-1b (Udayanga et al. 2015), respectively. Sequences were deposited in GenBank (Accession Nos. ON994257 to ON994259 for ITS, OP076824 to OP076826 for TUB, OP076827 to OP076829 for CAL, OP076821 to OP076823 for TEF1-α, OP076830 to OP076832 for HIS). BLAST results showed that ITS and TEF1-α have 99.8% pairwise identity to Diaporthe fusicola (MN816432, KF576256), and the TUB, CAL, and HIS sequences also have 100% pairwise identity to D. fusicola (KF576287, MT978147, MT978142). Phylogenetic analyses of concatenated sequences using Bayesian inference and the maximum likelihood confirmed the identity. To verify Koch's postulates, the pathogenicity of three isolates was tested on harvested healthy peach fruits. Five surface-sterilized fruits were wounded by a sterile scalpel and inoculated with 5-mm-diameter mycelial plugs from 10-day-old PDA plates. Another set of five fruits was inoculated with sterilized PDA plugs as controls. All fruits were incubated at 26℃ with 80% relative humidity. The experiment was repeated three times. After 5 days, the fruit inoculated with mycelial plugs showed pale brown lesions with whitish mycelium mass, similar to the previous rot symptoms, whereas the control fruit remained symptomless. The same pathogen was reisolated from the inoculated fruit with symptoms and identified as D. fusicola by molecular techniques, but never from the control. Diaporthe fusicola (Diaporthe amygdali complex) was first described on leaves of Lithocarpus glabra in China (Gao et al. 2015) and reported as an agent causing leaf blotch on Osmanthus fragrans (Si et al. 2020) and pear shoot canker (Guo et al. 2020). However, this is the first report of D. fusicola causing postharvest fruit rot on peach. The managers involved must consider the impact of this disease and develop an effective fruit storage strategy.

First Report of Colletotrichum tropicale Causing Anthracnose on Pitahaya Fruit in Mexico.

Pitahaya (Hylocereus spp.), also called dragon fruit, is a cultivated cactus that is native to Mexico as well as Central and South America. In October 2021, anthracnose symptoms were observed on fruit of pitahaya (Hylocereus costaricensis) in a commercial orchard located in Culiacán, Sinaloa, Mexico. Lesions on fruit were circular, sunken, dark brown and with halo. To fungal isolation, small pieces from adjacent tissue to lesions of symptomatic fruits were surface disinfested by immersion in a 2% sodium hypochlorite solution for 2 min, rinsed in sterile distilled water, and placed in Petri plates containing potato dextrose agar (PDA). The plates were incubated at 25 ºC for 5 days in darkness. Colletotrichum-like colonies were consistently observed on PDA and five monoconidial isolates were obtained. An isolate was selected as a representative for morphological identification, multilocus phylogenetic analysis, and pathogenicity tests. The isolate was deposited as CCLF186 in the Culture Collection of Phytopathogenic Fungi at the Research Center for Food and Development (Culiacán, Sinaloa). On PDA, initially white colonies turned grey with abundant orange conidia masses at 8 days after incubation at 25 ºC. Conidia were cylindrical, with ends rounded, aseptate, hyaline, and measuring 15.2 to 18.9 × 4.3 to 6.4 μm (n= 100). Appressoria were terminal, subglobose to clavate, of 7.4 to 11.6 × 5.9 to 8.2 µm (n= 30). Setae were not observed. These morphological characters were consistent with those reported for the Colletotrichum gloeosporioides species complex (Weir et al. 2012). To determine the phylogenetic identity of the isolate CCLF186, genomic DNA was extracted following the CTAB method (Doyle and Doyle 1990), and the internal transcribed spacer (ITS) region, the ApMat intergenic region, as well as partial sequences of actin (act) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes were amplified and sequenced using the primers pairs ITS5/ITS4 (White et al. 1990), AM-F/AM-R (Silva et al. 2012), GDF/GDR, and ACT-512F/ACT-783R (Weir et al. 2012), respectively. The sequences were deposited in GenBank under accession nos. OP269659 (ITS), OP302778 (gapdh), OP302777 (act), and OP302779 (ApMat). BLASTn searches revealed high identity with sequences of C. tropicale (CBS 124949) for ITS (100%), ApMat (100%), act (100%), and gapdh (100%). A phylogenetic tree based on Bayesian inference and Maximum Likelihood methods, including published ITS, ApMat, act, and gapdh sequence datasets for isolates in the Colletotrichum gloeosporioides species complex was generated. The phylogenetic analysis based on the concatenated sequences clustered the isolate CCLF186 with the C. tropicale reference isolates. Pathogenicity of the isolate CCLF186 was confirmed on 10 healthy pitahaya fruits without wounds. A drop of a conidial suspension (1 × 105 spores/ml) was placed on two locations on each fruit. Ten control fruits were treated with sterilized water. The fruits were kept in a moist plastic chamber at 25°C and 12 h light/dark for 8 days. The pathogenicity test was repeated twice. All inoculated pitahaya fruits exhibited sunken and necrotic lesions 6 days after inoculation, whereas no symptoms were observed on the control fruits. The fungus was consistently re-isolated only from the diseased fruits and found to be morphologically identical to the isolate used for inoculation. Recently, C. tropicale causing anthracnose in dragon fruit (Selenicereus monacanthus) was reported from Philippines (Evallo et al. 2022). Now, this is the first report of C. tropicale causing fruit anthracnose in H. costaricensis in Mexico and worldwide. These findings provide a basis for research about the distribution and effective disease-management strategies.