Cylindrical Conidia Research Articles

First report of Colletotrichum nymphaeae causing anthracnose on Hass avocados in Brazil.

The Hass cultivar is one of the most cultivated and distributed avocado varieties throughout the world, having high productivity, nutritional quality, market acceptance and adaptation to different climatic zones (Dreher and Davenport 2013). Anthracnose affects avocado production in tropical and subtropical regions, causing economic losses, especially post-harvest (Fuentes-Aragón et al. 2020). Correct identification of Colletotrichum species causing anthracnose is essential, as different species vary in important phenotypes such as virulence and sensitivity to fungicides (Chen et al. 2016). Twelve samples from batches of avocados with anthracnose were collected in Minas Gerais State, Brazil, in September 2023. The observed symptoms were brown to black depressed circular spots, ranging from a few millimeters to 3 cm in diameter on the epicarp of the fruits, covered in center by mucilaginous layers of pathogen sporulation. Isolation was performed directly from the spore masses and monoconidial isolates were cultured in PDA at 25°C for 7 days for morphological characterization and preserved in sterile water at 4°C. One of the morphotypes commonly recovered from lesions, represented by isolate UCBV 362 (Culture Collection COAD 3843), formed fast-growing colonies having white aerial mycelium and intense salmon-colored sporulation. The cylindrical conidia were 13 to 17.5 μm long and 4.5 to 7 μm wide (average 14.5 x 5.7 μm, N=100), produced on conidiophores dispersed in the aerial mycelium or aggregated on melanized conidiomata formed on the agar. The partial sequence of the second largest subunit of the RNA polymerase II gene (RPB2) from isolate UCBV 362 (GenBank: PQ034617, 1116 nt) showed 99% of coverage and 99.37% of nucleotide identity with the RPB2 sequence of the ex-epitype strain of Colletotrichum nymphaeae ICMP 17918 (=CBS 515.78) (GenBank: JN985506). In a Maximum Likelihood phylogenetic tree composed with RBP2 sequences from reference strains of the Colletotrichum acutatum species complex, the isolate UCBV 362 formed a highly supported clade with the ex-epitype and other reference strains of Colletotrichum nymphaeae, occupying the Clade 2 of the species complex together with C. scovillae and C. simmondsii (Damm et al. 2012). This result shows the reliability of RPB2 for phylogeny and species delimitation within Colletotrichum. To confirm pathogenicity, 10-mm discs from a 7-day-old colony were inoculated at 3 different points on healthy-looking avocado fruits and incubated at 28°C. Uninoculated fruits served as controls. The first symptoms appeared 5 days after inoculation and were similar to those observed in the original samples, while the fruits in the control group remained asymptomatic. The pathogen was reisolated from the lesions and identified morphologically, fulfilling Koch's postulates. Colletotrichum nymphaeae has been associated with avocado anthracnose in Mexico (Fuentes-Aragón et al. 2020). In Brazil, a study based on molecular phylogeny identified Colletotrichum siamense and C. karsti as etiological agents of this disease (Soares et al. 2021). This report expands the range of species that cause avocado anthracnose in Brazil and provides etiological information for the implementation and monitoring of control methods, especially chemical control.

First report of anthracnose caused by Colletotrichum kahawae on Hypericum chinensis in China.

Hypericum chinensis is growing in popularity amongst consumers in cut-flower and pop-flower market as an ornamental woody plant for its florid berry and colorful flower. In August 2019, anewleafspot diseasewas observed onH. chinensis in three commercial nurseries in Kunming (25°05'N, 102°72'E), Yunnian province, China. Disease symptoms were observed on approximately 40% of the plants one year after planting and 30% of the leaves were infected. Leaf symptoms began as small, water-soaked lesions on young leaves which later became larger, dark brown and necrotic. The lesion size ranged from 0.2 to 2.8 cm in diameter.For pathogen isolation, three samples of symptomatic leaves were collected from four different nurseries. The leaves were cut into 0.5 mm pieces, surface sterilized using 70% ethanol for 30 s, and 3% NaOCl for 5 min, rinsed three times in sterilized distilled water and plated on potato dextrose agar (PDA) (Zhou et al. 2023). The plates were incubated at 26°C in the dark for 3 days. Eight isolates with comparable morphological characteristics were obtained. Initially, colonies produced pale gray to white aerial mycelia, turning dark gray after 5 days. The isolates produced hyaline, single celled, straight and cylindrical conidia, with mean size 9.7 to 14.8 μm long × 3.7 to 5.6 μm wide (n = 100). Morphological characteristics were consistent with Colletotrichum sp. (Bailey and Jeger 1992). For molecular analysis, genomic DNA was extracted from three representative isolates (XSD1, XSD3 and XSD5), amplified using the primers ITS1/ITS4 (Yin et al. 2012) and T1/Bt2b (Glass and Donaldson 1995) and submitted to sequencing (Weir et al. 2012). DNA sequences of the isolates XSD2, XSD3 and XSD8 were identical. DNA sequences of a representative isolate XSD2 were deposited in GenBank (accession no. MW202334 for ITS, and OR347007 for TUB 2). MegaBLAST analysis of the ITS and TUB2 sequences showed 99.5% and 99.3% similarity withC. kahawae strain ICMP 18539 (accession no. NR_120138.1 for ITS) and strain IMI319418 (JX145227.1 for TUB 2). Pathogenicity tests were conducted by inoculating the pathogen on healthy mature leaves of H. chinensis in the field. Ten leaves (two leaves/plant) were inoculated byspraying conidial suspension (106spores/ml) of isolates XSD1, XSD3 and XSD5, and covered with plastic bags to maintain high humidity for 48 hours, respectively. Leaves treated with sterile distilled water served as a control. All inoculated leaves showed symptoms similar to those observed in the field at 23±5°C 10 days after inoculation. No symptoms developed on non-inoculated leaves. The pathogen was re-isolated from inoculated diseased leaves and identified as C. kahawae based on morphological and molecular characters. C. kahawae has been reported to cause leaf spot on cultivated rocket in Italy (Garibaldi et al. 2016), and anthracnose disease on tree tomato in Colombia (Rojas et al. 2018), to our knowledge, this is the first report of C. kahawae causing anthracnose on H. chinensis worldwide. Due to important ornamental and economic value of H. chinensis, thedistribution of C. kahawae needs to be investigated and monitored for effective disease management strategies to be developed.

First Report of Colletotrichum fructicola Causing Anthracnosis on Glycine max in Brazil

Brazil is the largest producer of soybean [Glycine max (L.) Merrill], cultivated in diverse environments and systems. This scenario can contribute to emergence of new diseases or increase the severity of secondary diseases. In March 2023, elliptical to circular, brownish lesions, 5.2–6.1 cm length and 1.1–1.5 cm width, with salmon-colored masses of conidia in the center of the lesions, were observed on the stems of soybean cultivar ‘CZ 16B17 IPRO’, in the municipality of Campos Novos, Santa Catarina, Brazil (27º25'19''S and 51º14'14''05W). The presence of 210–355 μm length and 210–232 μm width acervuli was rare, with arrows larger than the mass of conidia (Figure S1). Fragments of the infected tissues were cut, disinfected and placed in Petri dishes containing Potato Dextrose Agar (PDA) or V8-agar medium and maintained at 23 ± 2ºC and a photoperiod of 12 h dark–light cycle. After 13 days, the development of grayish-white colonies was observed on both culture media, with the formation of a mass of septate hyaline, oblong, cylindrical conidia, 13.3–15.3 µm length and 2.9–3.5 µm width, with obtuse ends. One pure monosporic isolate was selected, isolate CF1. The presence of sexual structures was observed on PDA after 13 days and in V8 after 15–20 days. Perithecia were dark brown and globose, either immersed in the culture medium or on the surface between the mycelia. Inside of perithecia, unitunicate, clavate, and cymbiform asci, 39.1–61.0 μm length and 9.6–11.7 μm width were observed, containing eight spindle-shaped and slightly curved ascospores with rounded tips 13.8–18.3 μm length and 3.0–4.2 μm width (Figure S1). Pathogenicity tests were performed on young soybean plants at V1 phenological growth stage in four repetitions. PDA disks, 7mm in diameter, with growth mycelium were placed on stems while using uninfected PDA disks as a control. Plants were incubated in a chamber at 25 ± 2°C and 90% relative humidity. Anthracnose lesions were observed only on the stems of the inoculated plants. The same pattern of symptoms was observed on the stems, and the fungus were reisolated on PDA. The colony and morphological characteristics were identical to the previously isolated fungus. For molecular characterization, the growth mycelia were collected, macerated in liquid nitrogen, and DNA was extracted using the method Doyle and Doyle (1990) with CTAB. End-point PCR was performed using the GoTaq® Flexi DNA Polymerase (Promega, USA) and the primers, ITS-1F/ITS-4, T1/Bt2b, CL1C/CL2C, GDF/GDR, and SODglo2-F/SODglo2-R (Weir et al. 2012) for the amplification of internal transcribed spacer (ITS), β-tubulin (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and superoxide dismutase (SOD), respectively. Amplified fragments were sequenced and compared with the available sequences in the Genbank (www.ncbi.nlm.nih.gov/genbank/). The sequences of all five-genes (Accession numbers OR883777, OR891749, OR891750, OR891751 and OR891752, respectively) of the isolate CF1 characterized in this study showed 99% nucleotide identities whith the stand isolate ICMP 18581 of Colletotrichum fructicola. A phylogenetic tree was constructed in MEGA X (Kumar et al. 2021), containing the amplified and concatenated sequences and representative species from the Colletotrichum gloeosporioides complex. The isolate grouped only with C. fructicola clade, confirming the identity of the fungus (Figure S2). To our knowledge, this is the first study reporting the infection of C. fructicola in soybeans in Brazil, which has already been reported in China (Xu et al. 2023).

Two new species of Trichocomaceae (Eurotiales), accommodated in Rasamsonia and Talaromyces section Bacillispori, from the Czech Republic

During a previous study on microfungi associated with clematis roots, Penicillium-like fungi were isolated and identified based on morphology. In this study, we subjected those strains to a detailed examination which led to the proposal of two taxonomic novelties, named Rasamsonia chlamydospora and Talaromyces clematidis. The first taxon is characterized by rough-walled mycelium, acerose to flask shaped phialides, cylindrical conidia and by production of chlamydospore-like structures. The four-loci-based phylogeny analysis delineated the taxon as a taxonomic novelty in Rasamsonia. Talaromyces clematidis is characterized by restricted growth on Czapek yeast extract agar, dichloran 18% glycerol agar and yeast extract sucrose agar, and production of yellow ascomata on oatmeal agar. Phylogenetic analyses placed this taxon as a taxonomic novelty in Talaromyces sect. Bacillispori. Both taxa are introduced here with detailed descriptions, photoplates and information on their phylogenetic relationship with related species.

Root rot Caused by Setophoma terrestris on Illicium verum in Yunnan Province, China.

Star anise (Illicium verum Hook. f.), a genus of star anise in the family Magnoliaceae, is an important cash crop of "medicinal and food" origin, mainly from China. In August 2021, root rot of I. verum was first observed on more than 80% of the plants grown within a 500 hectares area in Wenshan city, Yunnan Province. At the early stage of the disease, the phloem of the root was dark yellow-brown, and the leaves turn yellow. With further disease development, the whole root became black (Fig. 1a, 1b), and the leaves gradually fall off, affecting the growth, yield and eventually caused death of the whole plant. A total of 20 root samples were collected from typical symptomatic plant roots with 20 years old in Wenshan City (23°18'12″N, 103°56'98″E) and were cut into 2 × 2 mm pieces at the junction of infected and healthy tissue. Each sample was surface-sterilized with 3% NaClO and 75% alcohol for 60 s before rinsing three times with distilled water. The sterile filter paper (5×5 cm) was used to dry the tissue, and samples were cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/ml). Plates were incubated at 25°C in the dark in the incubator. From 9 isolates obtained in culture, 7 exhibited the morphology described by Boerema et al. (Boerema et al. 2004) for Setophoma sp. The hyphae were hyaline and septate (Fig.1c). After 14 days of culture on V8 juice agar, white round colonies are formed, but there is no groove in the middle of the colonies (Fig.1d), and transparent, oval, or cylindrical conidia were produced, 6.0-8.0 x 2.5 to 4.0 um (Fig.1e). DNA was extracted from a representative isolate BJGF-04 for molecular identification using a fungal genomic DNA extraction kit (Solarbio, Beijing, China). Polymerase chain reactions (PCRs) were performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990) and primers T1/β-Sandy-R for the β-tubulin gene (TUB) region (Yang et al. 2017) and primers NL3/ LR5 for 28S large subunit rDNA (LSU) region (Hu et al. 2021) and NS1/ NS4 for 5.8S large subunit rDNA (SSU) region (Mahesha et al. 2021). Newly generated representative sequences were deposited in GenBank: ITS sequence (ON645256), TUB sequence (ON854484), and LSU sequence (ON644445), SSU sequence (ON644451). were sequenced and blasted, showing 99 to 100% sequence homology with known S. terrestris. Pathogenicity was performed using one-year asymptomatic plants of I. verum. A conidial suspension (1 x 106 conidia/ml) collected from V8 juice cultures with 0.05% Tween buffer was poured at a volume of 10 ml/plant. Three individual seedlings were used as replicates for each treatment, and sterile water was used as the negative control. All plants were placed in an artificial climate incubator at 25°C under 90% relative humidity. After 20 days, all inoculated plants showed symptoms identical to those described above, whereas controls remained healthy. Setophoma terrestris was reisolated from the infected roots, which was confirmed by morphological and molecular identification, which completed Koch's postulates. To our knowledge, this is the first report of S. terrestris as a causal agent of root rot on I. verum in China.

The chalara-like anamorphs of Leotiomycetes

The chalara-like anamorphs of Leotiomycetes are phialidic hyphomycetes with cylindrical collarettes and deeply seated sporulating loci, and hyaline, aseptate or septate, cylindrical conidia. They are commonly found on plant litters in both terrestrial and submerged environments, and with broad geographical distribution. This paper reports our research result of diversity, taxonomy and phylogeny of these fungi in China, which is based on a systematic study by using an integrated approach of literature study, morphological observation and phylogenetic analyses of 153 chalara-like fungal species with diversified morphology in conidiomata, setae, conidiophores, phialides and conidia. The phylogenetic analyses employing different datasets of SSU, LSU and ITS sequences of 116 species showed that these chalara-like fungi were paraphyletic and scattered in 20 accepted genera belonging to five families of Leotiomycetes: Arachnopezizaceae, Hamatocanthoscyphaceae, Helotiaceae, Neolauriomycetaceae and Pezizellaceae. Additional six genera, Ascoconidium, Bioscypha, Chalarodendron, Didonia, Phaeoscypha and Tapesina, all reported with chalara-like anamorphs in literatures, are also accepted as members of Pezizellaceae or Leotiomycetes genera incertae sedis. Among of these 26 accepted genera of chalara-like fungi in Leotiomycetes, 17 genera are asexually typified genera (Ascoconidium, Bloxamia, Chalara, Chalarodendron, Constrictochalara, Cylindrochalara, Cylindrocephalum, Leochalara, Lareunionomyces, Minichalara, Neochalara, Neolauriomyces, Nagrajchalara, Parachalara, Stipitochalara, Xenochalara and Zymochalara), and 9 are sexually typified genera (Bioscypha, Bloxamiella, Calycellina, Calycina, Didonia, Hymenoscyphus, Mollisina, Phaeoscypha and Tapesina). The phylogenetic significance of conidial septation in generic delimitation was further confirmed; while other morphologies such as conidiomata, setae, conidiophores, phialides, conidial length, and conidial ornamentation have little phylogenetic significance, but could be used for species delimitation. The polyphyletic genus Chalara s. lat. is revised with monophyletic generic concepts by redelimitation of Chalara s. str. in a narrow concept, adaption of the emended Calycina to also include asexually typified chalara-like fungi, reinstatement of Cylindrocephalum, and introduction of six new genera: Constrictochalara W.P. Wu & Y.Z. Diao, Leochalara W.P. Wu & Y.Z. Diao, Minichalara W.P. Wu & Y.Z. Diao, Nagrajchalara W.P. Wu & Y.Z. Diao, Parachalara W.P. Wu & Y.Z. Diao and Stipitochalara W.P. Wu & Y.Z. Diao. Chaetochalara becomes a synonym of Chalara s. str., and the known species are disassembled into Chalara s. str. and Nagrajchalara. The polyphyletic genus Bloxamia is also redefined by introducing the new genus Bloxamiella W.P. Wu & Y.Z. Diao for B. cyatheicola. Five existing species of Chalara s. lat. were excluded from Leotiomycetes and reclassified: Chalara breviclavata as Chalarosphaeria breviclavata W.P. Wu & Y.Z. Diao gen. et sp. nov. in Chaetosphaeriaceae, C. vaccinii as Sordariochalara vaccinii W.P. Wu & Y.Z. Diao gen. et sp. nov. in Lasiosphaeriaceae, and three other Chalara species with hyaline phialides, C. hyalina, C. schoenoplecti and C. siamense as combinations of Pyxidiophora in Pyxidiophoraceae. For biodiversity of these fungi in China, a total of 80 species in 12 genera, including 60 new species, 17 new records and 1 new name, were discovered and documented in this paper. In addition, five species including three new species are reported from Japan. In connection to this revision, a total of 44 new combinations are made. The identification keys are provided for most of these genera. Future research area of these fungi should be the phylogenetic relationship of several sexually typified genera such as Bioscypha, Calycellina, Calycina, Didonia, Phaeoscypha, Rodwayella and Tapesina, and systematic revision of existing names under the genera Bloxamia, Chaetochalara and Chalara.

Introducing Querciphoma styphnolobii sp. nov., the first sexual morph of Querciphoma (Leptosphaeriaceae, Pleosporales)

Using combined morphological characteristics and molecular phylogeny, a new species Querciphoma styphnolobii sp. nov. (Dothideomycetes, Pleosporales) from China is described and illustrated. The new species is distinguished by dark green to black colony, eustromatic conidiomata, smooth, hyaline, cylindrical conidia with one middle constricted, septate and muriform ascospores. Phylogenetic analyses using multilocus sequence data of internal transcribed spacers (ITS), large subunit 28S rDNA (LSU), small subunit 18S rDNA (SSU), and translation elongation factor 1-alpha (TEF1-α) regions revealed that Q. styphnolobii is closely related to Q. minuta in Leptosphaeriaceae. The sexual stage of Querciphoma is characterized by superficial, rough, or hairy ascomata, cellular pseudoparaphyses, ellipsoidal and muriform ascospore with a multi-transverse vertical septum.

Molecular Variability of Colletotrichum spp. Associated with Anthracnose of Soybean

Background: Anthracnose is a major foliar disease of soybean associated with Colletotrichum species. In India, C. truncatum is the most widely associated species, whereas, other species are also being reported to cause anthracnose disease in soybean. Methods: Twenty-eight soybean infected samples were collected from different location of India, in order to obtain the pure cultures and were subjected to pathogenicity test. Microscopic observations were made for morphological characterization of all the isolates. Molecular detection was carried out using ITS primers and the sequences were deposited in the NCBI GenBank. Phylogenetic analysis was made using the MegaX bioinformatics tool. Result: The pure cultures were subjected to pathogenicity test, which had reproduced similar symptoms found under field conditions. Microscopic observations on spore morphology were made which revealed that truncate conidia were associated in twenty-seven isolates and cylindrical conidia in one isolate. Molecular detection made showed sequence similarity to Colletotrichum truncatum and the isolate with cylindrical spore was sharing homology with C. plurivorum. Phylogeny analysis clustered the two species into two groups. Hence, the study shows the association of two distinct species in causing anthracnose of soybean.

First Record of Ceratocystis fimbriata Causing Quick Wilt of Calotropis gigantea in Pakistan.

Calotropis gigantea belongs to the family Asclepiadaceae, native to Asia and commonly known as crown flower. C. gigantea was identified as an important medicinal plant in Asia and also harvested to obtain the fibres from the stem (Ganeshan et al. 2018; Narayanasamy et al. 2020). In April 2021, a quick wilt epidemic of C. gigantea was observed in District Gujranwala (32°05'58.0"N 74°02'38.0"E) Punjab, Pakistan. The wilt symptoms were very severe on mature plants with 60% disease incidence. Affected plants exhibited yellowing and quick wilting of leaves on each branch that eventually spread to the whole plant (Figure 1). Black patches were observed on the stem bark and cross section of infected stem showed black streaks in the xylem. The samples (n=33) were collected from symptomatic plants. Infected tissues were excised into 4-5mm segments, surface disinfested with 1% NaClO and rinsed 3 times with sterilized distilled water. To induce the sporulation of the fungus, the described carrot baiting method of Moller et al., (1968) was used. Single ascoma from carrot baits were picked under a microscope and transferred to malt extract agar (MEA) medium and incubated at 25°C. Purified cultures were obtained by single spore culture method and all the isolates were preserved with 35% glycerol at -80°C. The fungal colony was greyish olive with overripe banana odour. Hyphae were smooth and segmented. The perithecia were globose, black, measuring 190.27 to 257.34 × 186.11 to 243.24 µm, and showed a long black neck (732 to 977.34 µm). Cylindrical conidia (9.46 to 22.97 µm × 2.7 to 5.41 µm), barrel-shaped conidia (6.34 to 7.95× 8.15-10.41 µm) and chlamydospores (7.65-11.38µm×10.17-16.81µm) were observed (Figure 2). The isolates were identified as Ceratocystis fimbriata based on morphology and similar results were reported by Engelbrecht et al. (2005) and Suwandi et al. (2021). Genomic DNA was extracted from all isolates and multi-locus sequence analysis approach was used for molecular identification. The Internal Transcribed Spacers (ITS) region, the Translation Elongation Factor 1-α gene (TEF) and β-tubulin gene (TUB) were amplified using ITS1/4 (White et al. 1990), EF1-728F/ EF1-986R (Carbone et at. 1999) and βt1a/βt1b (Glass and Donaldson, 1995). Based on the BLAST analysis, all isolates were identified as C. fimbriata. The sequences of the representative isolate AK-W17 were submitted to the GenBank, NCBI database with the accession numbers (ITS:MZ711226), (TEF: MZ714595) and (TUB:MZ714596) showing 100% similarity with AF395687(ITS), MG980731(TEF) and AY177227(TUB) accessions of C. fimbriata. Based on Sequences similarity, representative isolate AK-W17 grouped with the isolates which representing C. fimbriata. Pathogenicity test was conducted on healthy C. gigantea seedlings grown in pots. A fungal mycelium plug (4×4mm) from 15 days old culture of AK-W17 was inserted into a slit (5 × 8 mm and made by puncturing the bark surface) in the stem bark, and the artificial wound was covered with wetted tissue paper and secured with Parafilm to maintain humidity, while control treatment was inoculated with only MEA medium plug. The seedlings were incubated under 70% relative humidity (RH) and 28°C in greenhouse. After 24 days, the inoculated seedlings showed 100% wilting identical to the original plant from which they were isolated (Figure 3). The control treatment had no symptoms. The pathogenic fungus was reisolated and identifies as C. fimbriata based on morphological and molecular characterization. C. fimbriata has been also reported to cause wilt disease in Dalbergia sissoo (Harrington et al. 2015) and pomegranate (Alam et al. 2017) in Pakistan. To our knowledge, this is the first report of C. fimbriata to cause C. gigantea quick wilt in Pakistan. C. fimbriata is one of the most aggressive plant pathogens and rapidly spread worldwide, so it is critical to implement appropriate management practices to reduce the fungus attack on plants.

Curvicladiellapaphiopedili sp. nov. (Hypocreales, Nectriaceae), a new species of orchid (Paphiopedilum sp.) from Guizhou, China.

BackgroundAn asexual fungus, collected from diseased leaves of Paphiopedilum sp. from Guizhou Province, China, and based on the phylogenetic analyses and morphological characters, it was identified as a new species in Curvicladiella. The genus Curvicladiella are recorded for the first time for China.New informationThe morphology of Curvicladiellapaphiopedili sp. nov. is characterised by penicillate conidiophores with a stipe, dull, tapering towards the apex, the curved stipe extension and cylindrical conidia. In the phylogenetic analyses of combined cmdA, his3, ITS, LSU, tef1 and tub2 sequence data, this taxon was clustered as sister to Curvicladiellacignea within Nectriaceae.

First report of Colletotrichum henanense causing anthracnose on Chinese chestnut in the United States.

Culinary chestnut production in the United States (US) is a rapidly growing industry supporting fresh market and value-added industries. An estimated 200-400 new acres of chestnuts are planted every year in the US, with most growers east of the Rocky Mountains planting Chinese chestnut (Castanea mollissima) or Chinese chestnut hybrids. In 2018, Ohio producers of Chinese chestnut reported losses of up to 80% to blossom end rot. Symptoms were like those reported by Fowler and Berry (1958), including black spots on the chestnut shell, often at the stylar end, and blackening of the kernels. Spots covered 1-100% of the kernel, however, no signs of any pathogen were present on the shell or kernel. In 2020, cankers that were brown/black in color, sunken, and ~1 cm in length were observed on 1-yr twigs of chestnut seedlings from a nursery operation on the same farm from which the symptomatic kernels were observed. In some seedlings, distal portions of the twigs died, while in other seedlings only shoots and leaves within the cankered areas died. Black acervuli were observed erupting from the cankers. Colletotrichum spp. were isolated and cultured on potato dextrose agar from surface-sterilized tissue from kernel lesions (MLI246-21 to MLI249-21) and twig cankers (MLI250-21 and MLI251-21). All isolates produced grey aerial mycelia, pink sporodochia, and cylindrical conidia with rounded ends ranging in size from 12-20 um long by 5-8 um wide. Isolates were preliminarily identified as belonging to the Colletotrichum gloeosporioides species complex (CGSC). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, β-tubulin (TUB2) gene, and the intergenic spacer ApMat were amplified from genomic DNA and sequenced (Dowling et al. 2020). These genes are suitable for identifying species within the CGSC (Eaton et al. 2021). Sequences were submitted to the GenBank database (GAPDH OL687148 to OL687153, TUB2 OL741624 to OL741629 and ApMAT OL695914 to OL695919). BLASTn queries of NCBI GenBank showed that the GAPDH, TUB2, and ApMat sequences from all isolates had 98%, 98% and 99% identity with C. henanense isolates MT513015.1, MT513080.1, and MT512917.1 from apple (Martin et al. 2021). Representative isolates were used to demonstrate Koch's postulates and confirm pathogenicity on kernels (MLI246-21 and MLI249-21) and twigs (MLI250-21). Developing chestnuts in burs (n=4 per isolate) were gathered from the field and surface-sterilized with 70% ethanol. Nuts (n=12 per isolate) inside burs were inoculated by injecting 50uL of inoculum (~1.0 x 106 conidia/mL) directly into each kernel with a hypodermic needle and sterile syringe. Burs were incubated at room temperature in a moist chamber for 14 days. One-year-old seedlings (n=6) grown in containers were used for twig inoculations. Twig nodes (one/seedling) were surface sterilized with 70% ethanol and inoculated by wounding the stem with a sterile probe and dropping 100uL of inoculum into each wound. Seedlings were incubated in a glass house for 60 days and monitored daily for symptom development. Nuts (n=9) and twigs (n=3) were inoculated with sterile water using the same inoculation and incubation conditions to serve as negative controls. Inoculated kernels developed characteristic brown/black lesions at the wound site and inoculated twigs developed brown/black, slightly sunken cankers with acervuli. No symptoms developed on the control kernels or twigs. Fungi with the same colony, conidial, and molecular characteristics as C. henanense were re-isolated from inoculated kernels and twigs. Blossom end rot caused by Glomerella cingulata has been reported on chestnut kernels in Georgia (Fowler and Berry 1958), but this is the first report of C. henanense causing rot on kernels and twigs in the US. Since 2018, C. henanense has been isolated from infected nuts received from commercial orchards in Pennsylvania, Missouri, and Alabama. We propose to retire the name "blossom end rot" for the symptoms found in kernels and replace it with the name "chestnut anthracnose" for this disease that affects both twigs and kernels of Chinese chestnut.

First Report of Colletotrichum fructicola Causing Anthracnose on Camellia yuhsienensis Hu in China.

Camellia yuhsienensis Hu is an endemic species from China, where is the predominant oilseed crop due to its anthracnose resistance (Kuang 2015; J. Li et al. 2020; Nie et al. 2020). In April 2019, anthracnose symptoms were observed on C. yuhsienensis in a plantation in Youxian, Zhuzhou, Hunan Province, China (113.32°E, 26.79°N). It was detected approximately 10% anthracnose incidence in 500 two-year-old plants in a 5000 m2 cultivated area. Diseased leaves showed irregular grayish brown spots with dark brown edges and dark brown undersides. Symptomatic tissues (4 to 5 mm2) were surface-disinfected for 90 s in 75% ethanol, then rinsed twice with sterile water, and finally incubated on PDA (potato dextrose agar) at 28℃ (Jiang et al. 2018). Pure cultures were obtained by the single-spore isolation method. A total of 100 fungal isolates were obtained from 85 symptomatic leaves, from which 81 had similar colony morphology. Colonies on PDA were white, fluffy and cottony, and becoming dark gray after 5 days. The character of the reverse of the colony were similar to that of the upper of the colony, but the color was darker at the same time. The isolates produced a large number of single-celled, hyaline, straight and cylindrical conidia, with 10.35 to 17.58 length × 3.46 to 5.69 μm width (x=13.61 × 4.63 μm, n = 30). The isolates were preliminarily identified as Colletotrichum spp. according to morphological features (Weir et al. 2012). Representative isolate YX2-5-2 was used for molecular identification: internal transcribed spacer (ITS), partial actin (ACT), chitin synthase (CHS-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genomic DNA regions were amplified by PCR (Weir et al. 2012). Gene sequences were deposited in GenBank (GenBank accession no. MW398863 for ACT, MW886232 for CHS-1, MW398864 for GAPDH, MW398865 for ITS). BLAST analysis revealed that DNA sequences of YX2-5-2 at the ITS, GAPDH, ACT, and CHS-1 loci showed 100%, 99.25%, 100%, and 99.33% sequence identity, respectively to their corresponding loci in strains ZH6 (GenBank accession no. MT476840.1), ICKP18B4 (LC494274.1), YN17 (MN525804.1), and ICKG4 (LC469131.1) of C. fructicola. A Maximum Likelihood phylogenetic tree based on the combined ACT, CHS-1, ITS and GAPDH sequences revealed that the representative isolate YX2-5-2 clustered with C. fructicola. In addition, the morphological features of YX2-5-2 were similar to C. fructicola which has been reported (Weir et al. 2012). Pathogenicity was tested using isolate YX2-5-2 by inoculating leaves of 2-year-old C. yuhsienensis. Four leaves of each healthy C. yuhsienensis were sprayed with a conidial suspension (105 conidial/mL) of isolate YX2-5-2, and the above steps were repeated three times. Two additional mock-inoculated control plants were sprayed with sterilized liquid potato dextrose medium. The plants were incubated in a greenhouse at 28℃ and 90% humidity with a 12 h photoperiod. Anthracnose-like symptoms were observed 5 days post-inoculation. The control plant tissues remained healthy. C. fructicola was re-isolated on PDA from lesions, and the morphological features were consistent with YX2-5-2, confirming Koch's postulates. To our knowledge, this is the first report of anthracnose of C. yuhsienensis caused by C. fructicola in China. Anthracnose of Camellia. oleifera has been reported for a long time (H. Li et al. 2016). C. yuhsienensis, as a wild relative of C. oleifera (commonly known as tea-oil tree), has been concerned about its resistance to anthracnose. Therefore, the occurrence of C. yuhsienensis anthracnose hindered the control of anthracnose tea-oil tree. This finding will lay the foundation for studying the pathogenesis of anthracnose of tea-oil tree and developing effective prevention methods.

Detection of Bipolaris hawaiiensis (cutaneous phaeohyphomycosis) in Saudi Arabian Camels (Camillus dromederius)

Bipolaris hawaiiensis describes subcutaneous lesions caused by dematiaceous fungi, brown-pigmented mould. In a camel farm in Saudi Arabia, the owner complained of cases of skin infection among camels. Lesions persisted after sarcoptic mange outbreak, which was treated until the infection resolved. General examination revealed that four camels were affected showing alopecia, erythema, numerous small subcutaneous nodules and brownish blackish crusts. The affected areas were disinfected with 70% ethyl alcohol and deep scraping were prepared in 10% potassium hydroxide for microbiological examination. Cultures were done onto sabouraud dextrose agar with chloramphenicol 0.5mg/ml, incubated at 30⁰C. Another set cultured on the same media enriched with chloramphenicol 0.5mg/ml and cycloheximide 0.4 mg/l and incubated at 27⁰C and 37⁰C. Molecular mycology analysis was done by polymerase chain reaction on internal transcribed spacer (ITS) gene using ITS1 and ITS4 primers. Microscopic examination indicated an absence of dermatophytes. However, brownish to black, septate hyphae arranged as arthro-hyphae, and black yeast-like particles were showed microscopically. Cultures yielded multiple, velvety, gray colonies turning brownish black later. Lactophenol cotton-blue smears revealed septate, branched hyphae that are dark brown in colour1.5–5μ wide. Conidiophores are septate, unbranched with flexuose apexes, bearing brown, multi-septate, cylindrical conidia. ITS gene sequence analysis confirmed the isolate from camel skin scrapings to be bipolaris hawaiiensis. The case is interesting as this represents, to the best of our knowledge, the first authenticated report of bipolaris hawaiiensis in camels from a tropical country.

Phaeohyphomycosis Caused by Cochliobolus hawaiiensis in a Camel Farm in Saudi Arabia: An Emerging Disease

Phaeohyphomycosis describes subcutaneous lesions caused by dematiaceous fungi, brown-pigmented mould. In a camel farm in Saudi Arabia, the owner complained of cases of skin infection among camels. Lesions persisted after sarcoptic mange outbreak which was treated until the infection resolved. General examination revealed that four camels were affected showing alopecia, erythema, numerous small subcutaneous nodules and brownish blackish crusts. To collect specimens, affected areas were disinfected with 70% ethyl alcohol and deep were taken. Skin scrapings were prepared in 10% potassium hydroxide for microbiological examination. Cultures were done onto Sabouraud dextrose agar (SDA) with chloramphenicol 0.5 mg per ml, incubated at 30⁰C and another set with chloramphenicol 0.5 mg per ml and cycloheximide (Sigma), 0.4 mg/L, incubated at 27⁰C and 37⁰C. Molecular mycology analysis was done by Polymerase Chain Reaction (PCR) on internal transcribed spacer (ITS) gene using ITS1 and ITS4 primers.
 Microscopic examination indicated it was negative for dermatophytes. It showed brownish to black, septate hyphae arranged as arthro-hyphae, and black yeast-like particles. Cultures yielded multiple, velvety, gray colonies turning brownish black later. Lactophenol cotton-blue smears revealed septate, branched hyphae that are dark brown in colour1.5–5 μ wide. Conidiophores are septate, unbranched with flexuose apexes, bearing brown, multi-septate, cylindrical conidia. ITS gene sequence analysis confirmed the isolate from camel skin scrapings to be Cochliobolus hawaiiensis. The case is interesting as this represents, to the best of our knowledge, the first authenticated report of C. hawaiiensis in animals from a tropical country.

First Report of Ceratocystis changhui Causing Postharvest Fruit Rot of Peach in Kunming, China.

The peach (Prunus persica (L) Batsch) is a predominant commercially grown stone fruit in China (Lee et al. 1990). Ceratocystis changhui is an aggressive pathogen causing typical black rot symptoms on corms of taro (Colocasia esculenta) (Liu et al. 2018), it has not been reported on other hosts. During the summer and autumn of 2013, a postharvest fruit rot disease was observed on several peaches at a farmer's market (N 25°02'; E 102°42') in Kunming City, Yunnan Province, China. The incidence of the disease varied from 5 to 20%. Necrotic spots were first observed on the infected peach fruit (Prunus persica cv. shuimitao). The spots enlarged gradually and developed into a brown, water-soaked and rotted lesion. Eventually, the whole fruit became soft, rotted and covered with a gray-brown mycelium (Fig. 1 A, B). The isolates were obtained from the symptomatic tissues incubated on slices of fresh carrot root (Moller et al. 1968). After 5 to 10 days of incubation, perithecia and mycelium were observed growing on carrot slices. Spore masses were removed from the apices of perithecia, transferred to potato dextrose agar medium (PDA) and incubated at 25°C for 5 to 10 days, followed by single-spore isolation. All eight single-spore isolates from peach fruits obtained in this study were deposited in the State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, China. In culture, mycelium was initially white, gradually turned to greyish-green or brown (Fig. 1E, F). Measurements were made 7 days after the formation of perithecia. Perithecia (Fig. 1G) were black, globose, 185.71 to 305.56 μm × 142.86 to 264.29 µm and showed a long black neck, 600 to 957.14 µm (Fig. 1H). Ascospores (Fig. 1I) were helmet-hat shaped and 2.86 to 6.67 µm ×3.81 to 4.76 µm. Cylindrical conidia (Fig. 1J) 6.67 to 38. 95 µm × 2.86 to7.62 µm were observed. Chlamydospore (Fig. 1K), 8.57 to 13.33 μm × 5.71 to 9.52 μm, were ovoid or obpyriform, smooth. To further verify pathogen identity the internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS1F and ITS4 (Thorpe et al. 2005), and the total genomic DNA from the mycelia of five isolates was extracted using a CTAB method (Lee &Taylor 1990). The nucleotide sequences have been blasted and deposited in the GenBank database. Analysis of the ITS sequences from the isolates T1-1yp, T1-2yp, T2-1yp (GenBank accession no. KY580895-KY580897) showed 99% to 100% similarity with isolates C. changhui CMW43272 (KY643886), CMW43281 (KY643884), CMW46112 (KY643891) and CMW46113 (KY643892) from taro in China. Phylogenetic trees based on the maximum-likelihood (ML) method were constructed using MEGA 7. ITS sequences of other Ceratocystis spp. were attained from NCBI for comparative analysis (Liu et al. 2018), and Davidsoniella virescens (CMW11164) served as outgroup. The robustness of ML tree was evaluated with 1,000 bootstrap (BS) values. The pathogen was identified as C. changhui based on the phylogenetic analysis (Fig. 2). Three isolates (T1-1yp, T1-2yp, T2-1yp) were used for pathogenicity. Nine Prunus persica cv. yingzuitao fruits at early maturity (8 points out of 10) were wound inoculated with 200μL conidia suspension of the fungus (approximately 2.0 × 106 conidia / mL). Degreasing cotton dipped in sterile water was used to raise the humidity in preservation boxes. Boxes were incubated for 10 days at 25°C. Three peaches as controls were treated only with sterile distilled water in the same way. Symptoms of sunken lesions and fruit rot were observed two days after inoculation, and measured at 1.8 to 3.2 cm from the inoculation point within 5 days (Fig. 1C: right, D). The same pathogen was re-isolated from them confirming Koch's postulates. Control peaches remained symptomless. This fungus was morphologically and phylogenetically identified as C. changhui. To our knowledge, this is the first report of C. changhui on postharvest peach in Yunnan, China. The disease will affect quality and taste of peach, so it is critical to deploy appropriate management strategies to limit the fungus spread.

First report of quinoa anthracnose caused by Colletotrichum nigrum and C. truncatum in the United States.

Quinoa (Chenopodium quinoa Willd.) is increasingly produced outside its native Andean range. In September 2019, stem lesions were observed on all six plants of quinoa accessions PI 510547 (25% severity) and PI 596293 (75% severity) in a demonstration plot in Ames, IA. Lesions were bleached, silvery-white to dark gray, slightly sunken, oval to linear with slightly tapered tips and contained setose acervuli. Fungi were isolated from both accessions following disinfestation with 70% ethanol and plating onto ½ acidified potato dextrose agar (APDA) and V8 medium. Isolates were examined morphologically. On V8 medium, isolate CQ1 produced sparse, flat, gray mycelia with profuse sclerotia and hyaline, aseptate, cylindrical conidia (n= 50, mean: 21.0 (range: 19.2-24) by 4.3 (2.4-4.8) µm); isolate CQ2 produced fluffy, gray to dark gray mycelia with profuse sclerotia and acervuli and hyaline, aseptate, falcate conidia (n =50, 26.8 (24-31.2) by 2.4 µm). Direct hyphal PCR was used to amplify ITS (ITS1/ITS4), ACT (ACT-512F/ACT-783R), GAPDH (GDF1/GDR1), CHS-1 (CHS-79F/CHS-234R), and TUB2 (T1/Bt-2b, CQ1 only) (Fu et al. 2019, Liu et al. 2013), and products were sequenced bidirectionally (MT772082-3, MT786524-30). A maximum likelihood tree was generated in MEGA X (Kumar et al. 2018) from a multiple sequence alignment of vouchered CBS isolates (Liu et al. 2013) and CQ1 was identified as Colletotrichum nigrum. CQ2 sequences showed 99-100% similarity to Colletotrichum truncatum sequences in Genbank (MN581860, MK675238, MF682518, MK118057). Koch's postulates were completed once with two isolates of each species grown on V8 medium under 12 hours of near UV light for two weeks. Greenhouse conditions were a 12 hr day/night cycle and temperature range of 26-30° C. Approximately 5 mL of mycelium on agar medium was sterilely removed and macerated in 6 mL of sterile distilled water. Non-inoculated medium was macerated in sterile water as a control. Forty-day old quinoa PI 634920 were inoculated by making three, 2-3 mm incisions between the cotyledons and first true leaves with a sterile razor blade. Next, 500 µL of slurry was placed on 2.54 cm2 of sterile cheesecloth and placed against the wound and wrapped with Parafilm. Six plants were inoculated per isolate and control. After two weeks, sunken, bleached to tan areas extended past wound sites of inoculated plants. No discoloration or sunken tissue was observed on control plants. Plants were tented with plastic film for one week. Acervuli were observed on C. truncatum- and C. nigrum-inoculated stems, and sclerotia were observed on C. nigrum-inoculated stems. Stems were surface disinfested with 10% bleach and plated onto ½ APDA. Colony morphologies of isolated fungi matched those of original inoculum for inoculated plants. Colletotrichum spp. were never isolated from control plants. When stems were inoculated, approximately 100 µL of slurry was also placed on 3-5 detached quinoa leaves in Petri dishes with moistened blotter paper and incubated for 48 hours at 25° C. Brownish, circular lesions developed on leaves inoculated with either species, but no lesions developed on control-slurry leaves. Colletotrichum spp. cause disease in quinoa relatives including spinach (Kurt 2015), beets (Gourley 1966) and amaranth (Wu 2001). This is the first description of Colletotrichum spp. causing stem lesions on quinoa in the United States. This disease may emerge in new quinoa production regions and may cause yield losses due to lodging.

First Report of Colletotrichum brevisporum Causing Soybean Anthracnose in China.

In March 2020, widespread anthracnose was observed on soybean (Glycine max) in southeastern Jiangsu (Nantong municipality; 120.53° E, 31.58° N) in China. Plants exhibited irregular brown necrotic lesions in stem and leaves, and pronounced wilting. The symptoms were detected in one soybean field, 0.42 ha, surrounded by healthy wheat fields. Approximately 65% of the soybean plants showed the disease symptoms, and crop yield was reduced by 28-35% with respect the yield achieved in previous years, when no symptoms were observed. The symptoms were consistent with those previously reported for anthracnose on soybean caused by Colletotrichum chlorophyti, C. cliviae and C. gloeosporioides (Barbieri et al. 2017; Mahmodi et al. 2013; Yang et al. 2012). Diseased, 3-week old plants were collected. Small pieces, approximately 1 cm2 in size, of symptomatic tissue were surface sterilized in 1.5% NaOCl for 1 min, and washed twice with sterile ddH2O. The pathogen was isolated and cultured on potato dextrose agar (Song et al. 2020), containing chloramphenicol (50 µg/mL), under darkness at 28 °C for 3 days. Sequence of internal transcribed spacer (ITS), actin (ACT), β-tubulin (TUB2) and glyceraldehyde 3-phosphate dehydrogenase (GAP/span>DH) genes was performed as reported by Yang et al. (2015). Sequences were submitted to GenBank under accession numbers MT361074 (ITS) and MT415548-MT415550 (ACT, TUB2 and GAPDH). Blast search revealed that the amplified sequences had 100% (ITS; C. brevisporum TCHD, MH883805), 97.66% (ACT; C. brevisporum S38, KY986905), 99.06% (TUB2; C. brevisporum PF-2, KY705061) and 100% (GAPDH; C. brevisporum LJTJ27, KP823797) matches to multiple C. brevisporum strains, whereas all reported C. chlorophyti, C. cliviae and C. gloeosporioides strains showed no similarity to at least 2 of the studied genes. Molecular phylogenetic tree constructed using MEGA7 confirmed the identity of the pathogen. ACT and ITS sequences were blasted separately in Muscle (https://www.ebi.ac.uk/Tools/msa/muscle/) and then combined together to make the phylogenetic tree. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model, and the tree with the highest log likelihood (-1749.2186) is shown in Figure 1. The Colletotrichum strains previously found causing anthracnoseon soybean, and other relevant strains used in taxonomic analyses were included in the phylogenetic tree. Microscope observations showed the presence of 15-µm-long cylindrical conidia and septate mycelium, and agree with those reported for the morphology of C. brevisporum by Damm et al. (2019). To confirm pathogenicity, the mycelia from a 2 day-old culture on PDA was collected and suspended in sterile ddH2O (≈ 106 cells/mL) to prepare the inoculum. The pathogen was sprayed-inoculated on stem and leaves of healthy soybean plants. In control plants, sterile ddH2O was used. Inoculated plants were maintained in growth chamber at 28 °C and 50% relative humidity. Typical anthracnose symptoms were obsered 20 days after inoculation (Figure 2). C. brevisporum was reported to produce anthracnose on pumpkin, papaya, mulberry, coffee, passion fruit and pepper in China (Liu et al. 2017; Liu et al. 2019; Xue et al. 2019). Here, we report for the first time C. brevisporum causing anthracnose on soybean, an economically-relevant crop in China.

First Report of Anthracnose Caused by Colletotrichum gloeosporioides on Akebia trifoliata in China.

Akebia trifoliata (Thunberg) Koidzumi (three-leaf akebia), a climbing deciduous woody plant, grows wild in mountains of China and Japan. It has long been prized for its delicious sweet taste and medicinal value (Lu et al., 2019). Few pests and diseases reportedly affect this plant (Ye et al., 2013), but with more commercial planting of A. trifoliata in China, symptoms of anthracnose on leaves and fruits have increased. Between December 2018 and May 2019, typical anthracnose symptoms were first observed on A. trifoliata grown in Wuhan, China, with an incidence up to 15%. Diseased leaves exhibited irregular gray-brown spots with dark brown edges, and dark brown undersides, substantially affecting photosynthesis and growth. As disease progressed, white mycelium appeared on stems causing stem rot and fruit drop. Several round or needle-shaped dark brown spots formed on fruit peel, coalescing into irregular, slightly sunken blotches. Under high humidity, the whole fruit turned brown and the spots were covered by white mycelia, greatly affecting the fruits' ornamental quality. To isolate the pathogen, 5-mm2 pieces of symptomatic tissue from 10 infected leaves and fruits were surface-disinfected for 90 s in 1% sodium hypochlorite then 30 s in 75% ethanol, rinsed twice with sterile water, then incubated on potato dextrose agar (PDA, Oxoid) at 25°C under 12 h light/dark photoperiod. Pure cultures were obtained from hyphal tips of each colony. Initially, colonies produced white mycelia, turning gray after 5 days. The isolates produced abundant hyaline, single celled, straight and cylindrical conidia, with mean size 10.35 to 15.58 × 3.46 to 5.69 μm. Morphological characteristics were generally consistent with those of Colletotrichum gloeosporioides (Cannon et al. 2012). Genomic DNA of three isolates was extracted for PCR amplification of the internal transcribed spacer (ITS) region, and β-tubulin (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes (Weir et al. 2012). BLAST search identified all sequences (GenBank accession nos. MT451846 to MT451848 for ITS, MT573957 to MT573959 for TUB2, and MT573960 to MT573962 for GAPDH) as 100% matches to C. gloeosporioides (Penz.) Penz. & Sacc. CBS 112999 strain (JQ005152 for ITS, JQ005587 for TUB2, JQ005239 for GAPDH) (Damm et al. 2009). Identification was confirmed by maximum likelihood phylogenetic analysis using MEGA7 . To evaluate pathogenicity, isolates were inoculated onto one side of 10 wounded healthy leaves of 1-year-old pot-grown A. trifoliata plants and 10 nearly mature fruits, with 10 μl of conidial suspension (106/ml) and colonized PDA pieces (5 mm diam.) from 7-day-old cultures of the fungus in Petri dishes; control sides received 10 μl sterile distilled water and sterile agar pieces. The test was performed twice. After incubation at 25°C, 70% humidity under 12 h fluorescent illumination/12 h dark for 5 days, similar spots were observed on all inoculated leaves and fruits. Controls remained asymptomatic. The re-isolated pathogen was identified as C. gloeosporioides by biological characteristics and sequencing analysis, indicating that C. gloeosporioides was a causal agent of anthracnose of A. trifoliata. Anthracnose caused by C. acutatum has been reported on A. trifoliata in Japan (Kobayshi et al. 2004). To our knowledge, this is the first report of C. gloeosporioides found on Akebia species. The new disease primarily reduces the quality and yield of A. trifoliata. Effective measures should be taken to manage this disease. Funding: This study was supported by the National Natural Science Foundation of China (31701974; 31901980), Science and technology program funded by Wuhan Science and Technology Bureau (2018020401011307).

First Report of Colletotrichum karstii Causing Anthracnose on Cigar Tobacco in Hainan, China

Cigar tobacco (Nicotiana tabacum L.) is an important cash crop in China. Due to suitable ecological conditions, Hainan province is a major area for growing cigar tobacco. Cigar tobacco leaves are both an important source of fiscal taxation and also the main source of income for farmers in Hainan province. In 2018 and 2019, necrotic leaf spots were observed on 5 to 15% of cigar tobacco plants in Danzhou city (109.58°E, 19.53°N) and Wuzhishan city (109.52°E, 18.78°N) in Hainan province, China. Lesions on plants in the fields were small, nearly round spots (0.2 to 0.5 cm in diameter) with light gray centers and narrow dark brown margins, and sometimes there were tiny black dots composed of sporophores scattered in the lesion center. To determine the causal agent, more than 10 symptomatic leaves from Danzhou city and Wuzhishan city were used for isolation. Small pieces of tissue were cut from lesion borders, sterilized with 2% sodium hypochlorite (NaClO) for 4 min and 70% ethanol for 30 s, rinsed with sterilized water (SW), and dried on a sterile tissue paper. Clean and excised tissue pieces were placed on potato dextrose agar plates and incubated at 28°C for 5 days in the dark. Pure colonies of the fungus had grayish white aerial mycelia and orange conidial masses in the centers, and sometimes there were tiny black dots composed of clusters of conidiophores and conidia scattered in the centers. A pure culture was deposited in the China General Microbiological Culture Collection Center (CGMCC, no. 3.19502). Microscopic observations showed single, straight to slightly curved, hyaline, cylindrical conidia that were 12.1 to 14.7 μm long and 6.4 to 8.2 μm wide. To confirm the identities of the isolate, the internal transcribed spacer (ITS) rDNA of the fungus was amplified using PCR and sequenced, as described by Yang et al. (2011). The sequencing results were then compared with other DNA sequences in the GenBank database by BLAST. Analysis of the PCR product (GenBank accession no. MK050853) showed that the sequence had 99.5% identity with the nucleotide sequences of Colletotrichum karstii (HM585409). Glyceraldehyde-3-phosphate dehydrogenase (GPDH, GDF1/GDR1) (Yang et al. 2011) was also sequenced and BLAST searched. The PCR product sequence had 99.8% identity with the nucleotide sequences of C. karstii (HM585391). Based on the morphological and molecular characteristics, the isolate CGMCC 3.19502 was identified as C. karstii. Pathogenicity tests were conducted by artificially inoculating 20 cigar tobacco plants (40 days old, cv. H211). A spore suspension (10⁶ conidia/ml) was evenly sprayed on all leaves of the 20 plants. Ten control plants were treated similarly with SW. Twenty inoculated plants and 10 noninoculated plants were placed in a chamber maintained at 30°C and 90% relative humidity. After 7 days, the same symptoms observed from natural infections were on inoculated leaves, whereas the controls remained symptomless. This isolate also infected detached tobacco leaves. The pathogen was reisolated from inoculated leaves, and Koch’s postulates were completed by confirming the identity of the isolated fungus based on the morphology and ITS sequence. Anthracnose caused by C. karstii was previously detected on Schefflera octophylla in Sichuan province (Li et al. 2017). To our knowledge, this is the first report of C. karstii causing anthracnose on cigar tobacco in China. Damage caused by C. karstii could cause significant economic losses if not managed. More research is needed to better understand this disease and establish control strategies.

Morphological characteristics and distribution of colletotrichum isolates morphotypes infecting mango (Mangifera indica L.) in the north of Côte d’Ivoire

Anthracnose caused by Colletotrichum species is the most important field and postharvest disease infecting mango worldwide. A survey was conducted in the seven mango producing districts in the north of Cote d'Ivoire to identify and characterize Colletotrichum species isolates associated with mango leaves necrosis and fruit rots. A total of 70 isolates were identified based on morphological and cultural characters. Most of isolate colour were grey or grey whitish (36%), cottony (71%) with cylindrical conidia (97%) rounded on both ends (57%). Three texture types were observed in colonies of the seventy isolates. Most of the isolates were cottony (71%) followed by abundant aspect (23%) and closely appressed (6%). According to conidia shape, two species of Colletotrichum; Colletotrichum gloeosporioides (97%) and C. acutatum(3%) were identified in the seventy isolates. Conidia length and width varied respectively from 15.4to 19.7μm and 4.8 to 5.2 μm. The Average Linear Growth Rate (ALGR) 7 days after culture varied from 0.76 to 0.91 cm day-1. Multivariate analysis grouped the 70 isolates into four morphotypes containing, respectively: 5 (7%), 7 (10%), 17 (24%) and 41 (59%) isolates. Isolates of morphotype 1 were characterized by whitish obverse and reverse colour with an abundant mycelia aerial aspect. The second morphotypes were represented by isolates with obverse and reverse brownish grey colour and contained both conidia with two rounded ends, and one rounded and one sharped ends. The third morphotype consisted of isolates with obverse and reverse grey whitish colour. The fourth morphotype consisted of isolates with obverse and reverse respectively dark grey and grey or vice versa. Conidial length and width of morphotypes varied, respectively, from 14.5 to 17.2 μm and 4.6 to 5.2 μm. ALGR of morphotypes varied from 0.84 to 0.87 μm day-1. The present study highlighted that morphological variation of Colletotrichum species existed among the different isolates and the districts surveyed. However, to overcome the inadequacies of this traditional morphological identification, sequence analyses are needed to be carried out to confirm the identity of these Colletotrichum species isolates.