First Report of Green Mould on Leaf of Phaseolus vulgaris Caused by Cladosporium tenuissimum in Liaoning, China.

Phaseolus vulgaris Linn. is a widely cultivated vegetable throughout the world. From spring 2019 to 2022, green mould symptoms were observed on leaves of P. vulgaris in the greenhouse in Liaoning, China, with disease incidence of 8-75% (plants) and 6-23% (leaves). Symptoms appeared as chlorotic lesions covered with dark green mould. The infections started at the apex or margin of the leaves and then spread inward with a characteristic "V" shape. Lesions exhibited curly morphology. 15 leaf samples with typical symptoms were collected from 5 different greenhouses. A total of 75 (5 replicates of each sample) leaf tissues (0.5 cm × 0.5 cm) were selected from the boundary between diseased and healthy parts. These samples were surface sterilized in 0.5% NaClO formin, rinsed 3 times in sterile distilled water and subsequently incubated at 28℃ on potato dextrose agar (PDA) supplemented with streptomycin (50 µg/ml). Numerous morphologically uniform colonies had been purified, with no other fungi observed. Afterwards, the strains were subcultured on malt extract agar (MEA). Colonies on MEA reached 70 to 80 mm diam after 14 days, smoke-grey to pale olivaceous-grey, woolly, sometimes radially wrinkled. The mycelia were pale olivaceous-grey, with hyphae measuring 1-5 µm wide (n = 20). The conidiophores were solitary or in groups of 2 to 5, and measured 50-280(-350) × 2.5-4 µm (n = 20), with 2-7 septa. The conidiogenous cells exhibited a cylindrical-oblong morphology and measured 10-44 × 5 µm (n = 20), with 0-2 septa, and the loci frequently thickened. The conidia were catenate in densely branched chains, ellipsoid to obovoid, smooth, and measured 2.5-5 × 2-3 µm (n = 50), with 0-4 septa. The morphological characteristics were similar to Cladosporium tenuissimum (Zhang 2003). The representative isolate KZ-19 was selected for molecular identification. The rDNA-ITS, translation elongation factor 1-α and actin genes were amplified, sequenced, and the resulting sequence data were submitted to GenBank (ITS: OQ931048; EF-1α: OQ954495; ACT: OQ954496). The BLAST results exhibited a 99 to 100% similarity with the sequences of C. tenuissimum type strain CBS 125995(ITS: HM148197; EF-1α: HM148442; ACT: HM148687). Furthermore, a multi-locus phylogenetic tree was constructed using the PhyloSuite (v 1. 2. 2) software, which revealed that the strains were most closely related to C. tenuissimum (Zhang et al. 2020). Based on both morphological and molecular characteristics, KZ-19 was finally identified as C. tenuissimum (Bensch 2012). Pathogenicity testing was performed on healthy 1-month-old P. vulgaris plants by inoculating the spore suspension (1×106 conidia/ml) of KZ-19 onto leaf surfaces, while control plants were simulated inoculated with sterile water, and five pots were used for each treatment. The test was performed under field conditions of 16-28°C (temperature) and 24-56% (relative humidity). Chlorotic lesions became evident within 2 days of inoculation, followed by the appearance of green mold on leaves after 7 days. No symptoms were observed in the control group. To fulfill Koch's postulates, the pathogen was re-isolated from three inoculated leaves. The morphological identification of re-isolated pathogens was similar to that of originally isolated pathogens. No infection was observed in non-inoculated control. To the best of our knowledge, this is the first report of C. tenuissimum causing green mould on P. vulgaris. As a ubiquitous saprobic hyphomycete, C. tenuissimum has been implicated in leaf mold in Punica granatum and Trifolium repens, larch bud blight, and strawberry blossom blight in previous years (He et al. 1987; Zhang et al. 2003; Zheng et al. 2010; Nam et al. 2015), presenting a potential threat to numerous crops. Therefore, an investigation of its distribution and pathogenic potential is essential in addition to the development of effective disease management strategies.

First Reports of Cladosporium tenuissimum Causing Leaf spots on Hydrangea macrophylla in Anhui province in China.

Forever Summer Hydrangea (Hydrangea macrophylla) is a common flowering plant in the Yangtze River Valley area of China, and it is widely cultivated globally (Chen et al. 2015). In July 2023, H. macrophylla leaves exhibiting visible diseased lesions were reported in a nursery in Wuhu, Anhui Province, China. The incidence reached 40% in a 0.2 ha area. The primary disease symptom was multiple irregular necrotic spots (0.5 to 1 mm in diameter) appearing on the leaves. These spots on the leaves were faded yellow around the perimeter and grayish brown in the center.). 15 leaf samples were sterilized with 75% alcohol and rinsed three times in sterile distilled water, then transferred to antibiotic-added potato dextrose agar (PDA) for incubation at 27°C. The colonies were fluffy, flocculent, or hairy, dark green, gray-green to gray-brown in color, and spreading or protruding punctate with a colorless halo on PDA. The conidiophores were brown to dark brown, smooth or rough surface, mostly unbranched, clearly differentiated, erect or curved. The conidia displayed a light brown to brown hue, lemon shape, fusiform, elongated ellipsoid or others with obvious spore markings and spore umbilicus. Genomic DNA was extracted from fungal colonies on infected leaves of three collections separately (Braun et al. 2003) and the internal transcribed spacer regions (ITS), actin (ACT) genes and partial translation elongation factor-l-alpha (EF) were amplified and sequenced using the primers ITS1/4 (Yin et al. 2012), ACT-512F/ACT-783R and EF 1-728F/986R (Carbone and Kohn 1999), respectively. DNA sequences of isolates were identical and deposited in GenBank (accession no. OR362754 for ITS, OR611929 for ACT and PP209106 for EF). The consensus sequences from ITS, EF and ACT showed 100%, 98.98% and 100% identical to Cladosporium strains (accession no. OQ186140.1, MT154169.1 and OL322092.1), respectively. To confirm the pathogenicity of the isolates, hydrangeas were planted in 15-cm pots containing commercial potting mix (one plant/pot). Three healthy plants were inoculated at the five to eight leaf stage by spraying 50 µL of the isolate conidial suspension (4 × 106 spores/mL) on healthy leaves. Three plants treated with sterile distilled water were used as controls. After inoculation, all plants were placed in a humidity chamber (>95% relative humidity, 26°C) for 48 h and then transferred to a greenhouse at 22/27°C. All inoculated leaves exhibited symptoms similar to those observed in the nursery 10 days after inoculation, while no symptoms were observed for control leaves. The fungus was re-isolated and confirmed to be C. tenuissimum. Based on the above morphological characterization and molecular identification, the causal agent for this leaf spot disease was identified as C. tenuissimum. Although C. tenuissimum has been reported to cause disease on H. paniculata in northern China (Li et al.2021), this is the first time that C. tenuissimum has been found on H. macrophylla in southern China. This new disease of H. macrophylla caused by C. tenuissimum is a threat to urban greening and is worth further investigation.

First Report of Cladosporium tenuissimum causing Postharvest rot of Elaeagnus conferta Fruit in Kunming, China.

Elaeagnus conferta Roxb. is a perennial evergreen climbing shrub and is mainly native to India, Vietnam, Malaysia, and South China (Gupta & Singh, 2021). Various parts of this plant are used to treat multiple diseases(Gupta et al., 2021). Between during the months of March and April of 2021, in Kunming city of grower fields, Yunnan Province (N 25°02'; E 102°42'), southwest China. Some postharvest E. conferta fruits showed brown spots of decay with a greyish mycelium, which symptom only appears on fruit, and did not find it on this plant. The incidence of this disease in postharvest E. conferta fruits ranges from 45 % to 65 % in natural conditions. This pathogen is harmful and causes many plant diseases. Such as rice, oriental persimmon, pear, panicles of mango, and so on (Cho & Shin, 2004; Guillén-Sánchez et al., 2007; Lee et al., 2009). The infected fruit samples surface was disinfected with 75 % ethanol and 0.3 % NaClO for 30 s and 2 min respectively, then aseptic water washing three times. The fruit tissue is rich in carbohydrates and water content, which aid the growth of fungal species. Putting these diseased tissues on a potato dextrose agar (PDA) medium, cultured at 25 ± 1 ℃ for 7 days. The colonies grow on the PDA medium, then separated and puried again. Three pure cultures (YNGH01, YNGH03, YNGH05) were obtained, which were stored in 15 % glycerol at -80 ℃ refrigerator in the State Key Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Agricultural University. After 7 days of cultivation, the colonies were round and the diameter attained up to 38 mm, the surface of the colony showed tiled, fluffy, with a velvet-like texture, greyish-green to smoke-gray, slightly raised in the middle, the edges were radial hollow and wrinkle (Fig. 1A). Conidiophores were solitary, erect, unbranched or rarely branched, slightly flexuous at the apex, septate, dark brown, 254 to 680 µm long, 3.6 to 4.5 µm wide, top of the conidiophores or the rostral were slightly swollen (Fig. 1B). Conidia were light gray or grey, solitary or bispora, irregular in shape and size (Fig. 1C), nearly circular (3.21 × 3.31 µm), oval to lemon-shaped (6.59 × 3.21 µm) or elliptical (8.35 × 4.65 µm). The CTAB method extracts 3 isolates (YNGH01, YNGH03, YNGH05) genomic DNA (Aboul-Maaty & Oraby, 2019). To confirm identity with molecular identification, performed by three different genomic DNA regions, fragments of internal transcribed spacer (ITS), partial translation elongation factor-1 alpha (TEF-1α), and actin (ACT) genomic regions. These genomic DNA were amplified with primers ITS1/4, EF1-728F/986R, and ACT-512F/783R, respectively (Carbone & Kohn, 1999). The sequences of these isolates were uploaded to GenBank (YNGH01: ON753810, ON868696, ON912090 YNGH03: ON753812, ON868698, ON912092, and YNGH05: ON753814, ON868700, ON912094). NCBI's BLASTn search of those ITS sequences showed 99.81% similar to C. tenuissimum (MG873077.1), and sequences TEF-1α and ACT were 100% identical to several isolates of C. tenuissimum (OM256526.1 and MT154171.1). Combined the ITS region, TEF-1α, and actin (ACT) genomic regions of isolates YNGH01, YNGH03 and YNGH05 to construct a phylogenetic tree with MEGA11. Maximum likelihood phylogenetic analyses further confirmed the results (Fig. 2)(Santos et al., 2020). Healthy and mature E. conferta fruits were used for pathogenicity test. Pathogens were washed with sterilized water at a final concentration of 2× 106 spores/mL (Jo et al., 2018). The test was divided into A and B groups (A: The surface of fruits was pierced with a sterilized needle that carried pathogenic fungus of final concentration at 2×106 spores/mL B: Sprayed at the concentration of 2×106 spores/mL on fruits). The control fruits were treated with sterilized water and stored at 25 ± 1 ℃ with a relative humidity of 80 %, average group with 10 fruits in this test, which was repeated three times. After 7 days, the fruits of group A were initially sesame seed size of the disease spots, nearly round, irregular, with grayish-brown spots, and slightly depressed. Later, the lesion gradually turns dark brown (Fig. 1D). And group B began with small patches of brown fungal growth on the pericarp, with the development of the disease, the necrotic spots enlarged and developed irregular and coalesced, the color of spots became gray or black gradually (Fig. 1E). The symptoms were similar to previously observed and the pathogen was reisolated and identified as C. tenuissimum. Control fruits were healthy (Fig. 1F). The pathogens test fulfilled Koch's postulates. According to morphology (Bensch et al., 2012), rDNA-ITS, TEF-1α, and ACT sequence analysis, phylogenetic analysis, and pathogenicity test, the pathogen was identified as C. tenuissimum. To our knowledge, this is the first report of C. tenuissimum occurring on E. conferta fruits in China.

First Report of Cladosporium tenuissimum causing spot diseases on leaves and fruits of cucurbits in Brazil.

Cucurbitaceae crops are widely cultivated in the Northeast region of Brazil, which is the biggest producer of melon and watermelon in the country (Oliveira, 2020). Between November and December 2020 leaves of pumpkins (Cucurbita maxima L.) and watermelon (Citrullus lanatus L.), and leaves and fruits of melon plants (Cucumis melo L.) were collected with moderate to severe necrotic, irregular, and brown lesions from farms in the state of Rio Grande do Norte, Brazil. Fragments of diseased tissues were cut into small pieces and surface disinfested in 70% ethanol for 30 seconds, then in 2% sodium hypochlorite for 1 minute, and washed in sterile distilled water. Disinfested pieces of tissue were plated on potato dextrose agar (PDA) and incubated for seven days in the dark at 28 ± 2 °C. A total of 12 fungal isolates (four from pumpkins, one from watermelon, and seven from melons) were isolated from leaves and symptomatic fruits. All isolates in this study shared similar morphological characteristics. The colonies were dark gray to olive green in color with a velvety texture and surrounded by gray-white hyphae. The conidiophores were erect, tall, dark, and irregularly branched at the apex containing dark conidia, with 0 to 3 septa, variable in shape and size, forming chains that were often branched, globose, or subglobose with 3 to 4.5 µm in diameter. DNA from each isolate was extracted using the SDS method (Smith et al., 2001) and submitted to PCR amplification of the ITS and TEF1α regions with the primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. The amplicons were sequenced and deposited in GenBank: ITS (OP493545-OP493556) and TEF1α (OP536836-OP536847). Blastn analysis of the ITS and TEF1α partial sequences revealed that all 12 isolates belong to the species Cladosporium tenuissimum, with 100% nucleotide similarity with sequences of many C. tenuissimum isolates deposited in GenBank. A phylogenetic tree was constructed using the Maximum Parsimony Analysis, with the concatenated sequences (ITS-TEF1α) on MEGAX software (version 11.0.8) (Tamura et al, 2018). All 12 isolates clustered in the same clade and were closely related to isolates A2PP5, A3I1, and XCHK2 with the respective accession numbers KU605789.1, KU605790.1, and MG873071.1 from GenBank, with 99% bootstrap support. The pathogenicity of the 12 isolates was evaluated in pumpkin and melon plants in a greenhouse. Spore suspensions (10 6 conidia/ml -1) were sprayed on the leaves of healthy seedlings until runoff, only water was sprayed on control plants as the mock, and five seedlings of each crop (melon and pumpkin) were inoculated in each treatment. All plants were covered with plastic bags for two days. Spots, similar to those observed on diseased plants in the field, developed on the inoculated leaves (after seven days from the inoculation day, no symptoms were observed on plants from the mock treatment) and the fungal morphology was identical to that observed on the originally diseased leaves, fulfilling Koch's postulate. The pathogenicity test was repeated and yielded the same results. The fact that all 12 isolates were pathogenic on pumpkin and melon leaves, indicates that many Cucurbits are susceptible to C. tenuissimum infection. Many growers in the region are reporting similar symptoms in their melon plantations and it appears that the disease incidence is getting more severe year after year, based on growers's reports. Therefore, more research needs to be conducted to determine the epidemiology and the extension of the economic impact caused by this pathogen to Cucurbits to develop strategies for disease control. To the best of our knowledge, this is the first report of C. tenuissimum causing disease in Cucurbits in Brazil.

First Report of Brown Spot of Dekopon Fruit Caused by Cladosporium tenuissimum in China.

Dekopon citrus (Citrus reticulata Shiranui) is a three-way hybrid (Citrus unshiu Marcov. × C. sinensis Osbeck × C. reticulata Blanco) developed in Japan in 1972. This citrus is popular in China due to its sweet and tender taste (Lim 2012). In November of 2021, a brown spot disease on Dekopon fruits with about 20% disease incidence was observed in an orchard of the Institute of Citrus Research in Ganzhou, Jiangxi Province, China. Initially, the symptoms appeared as slightly sunken deep red to purple spots on the fruit surface, with the disease progression, lesions became brown to brown-black large necrotic regions covered with a fluffy layer of gray spores. Infected fruits were surface sterilized with 70% ethanol for 30 sec and rinsed three times with sterile distilled water. Diseased tissues from the edge of lesions were cut into small segments, placed onto potato dextrose agar and incubated at 25℃ for 7 days. Ten single-spore isolates were obtained in total. Fungal colonies were olive green to dark green, velvet-like in texture and sporulated abundantly, surrounded by grayish-white hyphae. Conidiophores were subcylindrical, straight, septate, solitary or in clusters of two to three, and ranged in size from 65 to 550 × 3.8 to 6.3 µm (x ̅= 261.7 ± 60.5 × 5.2 ± 0.4 µm, n=50). Ramoconidia were cylindrical，aseptate, and 10 to 22 × 2.8 to 4.5 µm (x ̅= 15.5 ± 1.4 × 4.0 ± 0.9 µm, n=50). Conidia were lemon-shaped to oval-shaped, smooth-walled, and 1.8 to 5.0 × 1.4 to 2.5 µm (x ̅= 3.9 ± 0.4 × 2.2 ± 0.2 µm, n=50). The morphological characteristics of the pathogen were consistent with those of Cladosporium tenuissimum Cooke (Li et al. 2021). For further identification, DNA was extracted from two representative isolates. The internal transcribed spacer (ITS) region, translation elongation factor (EF1-α), and actin (ACT) were amplified by using primers ITS1/ITS4, EF1-728F/EF1-986R, and ACT-512F/ACT-783R (Bensch et al. 2012), respectively. ITS (OM232067, OM232068), EF1-α (OM256525, OM256526) and ACT (OM256529, OM256530) sequences were deposited in GenBank. Multi-gene (combined data set of ITS, EF1-α and ACT) phylogenetic analysis was conducted using the Maximum Likelihood method (Nguyen et al. 2015). Based on the morphological characteristics and the molecular data, two fungal isolates were identified as C. tenuissimum. To evaluate pathogenicity, fifteen fruits were surface sterilized with 1% NaClO solution for 30 sec, rinsed twice with sterile distilled water and dried. Dekopon fruits (n=10) were wounded with a sterile needle and inoculated with a 10 µL drop of conidial suspension (1 × 106 conidia/mL) of isolate GZCJ-1, followed by incubation at 25℃ and 80% relative humidity. The controls (n=5) were treated with sterile water and maintained under the same conditions. Five days after inoculation, small brown sunken spots were observed on the wounded and inoculated fruits. After 7 days, lesions were coated by a layer of brown conidia that were similar to those described above, whereas control remained symptomless. Pathogenicity test was repeated twice. Cladosporium tenuissimum was consistently re-isolated from inoculated fruits and confirmed by morphological and molecular data, fulfilling the Koch's postulates. To our knowledge, this is the first report of C. tenuissimum causing the brown spot of dekopon fruit in China and perhaps the world. The disease may become the potential risk for fruit production, making fruits unfit for marketing purposes, and the appropriate management actions will be necessary.

First report of Cladosporium tenuissimum causing leaf spot of Arachis hypogaea in China.

Peanut (Arachis hypogaea L.), one of the most important oilseed crops in tropical and subtropical regions of the world (Kumar and Kirti 2011), is widely cultivated for its high protein and oil content in seeds. In August 2019, about 30% of A. hypogaea plants were found infected by leaf spot in the peanut-growing regions of Shandong Province, China. Disease symptoms appeared as the irregular and brown necrotic lesions on leaves that were 0.5 to 5.0 mm in diam. Twenty symptomatic plants were randomly sampled from peanut planting areas in Weihai and Yantai City. Small pieces (3 mm2) were cut from lesions, dipped in a 0.5% NaClO for 10 min, rinsed three times with sterilized distilled water, dried, placed onto potato-dextrose agar (PDA), and incubated in the dark at 25°C for 10 days. Three typical Cladosporium-like strains were isolated from diseased leaves of peanut. The colonies were grey to olivaceous green, reverse olivaceous black and woolly. The conidiophores were solitary, macronematous, unbranched or branched, straight or flexuous, cylindrical, slightly swollen at the apex, smooth. Conidiogenous cells were integrated, terminal and intercalary, with numerous loci on nodulose swelling. Ramoconidia were cylindrical, oblong, fusiform, 8.0 to 19.5×2.0 to 4.5 µm, aseptate or 1 septum, pale brown. Conidia were catenate, in densely branched chains, ellipsoid, ovoid, limoniform, aseptate, 4.0 to 11.5×2.5 to 5.5 µm, smooth, with conspicuous hila. The conidia easily break off from the chains. The morphological characteristics of these isolates matched the descriptions of Cladosporium tenuissimum (Bensch et al. 2010). For the molecular identification, the partial actin (act) and translation elongation factor 1-alpha (tef1) genes were amplified and sequenced using the respective primers ACT-512F/ACT-783R and EF1-728F/EF1-986R (Carbone and Kohn 1999). The representative sequences, deposited in GenBank (act: OL332701, OL332702 and OL332703; tef1: OL322090, OL322091 and OL322092), exhibited 99.6% and 100% identical to C. tenuissimum ex-type isolate CBS 125995 (HM148687 and HM148442). Phylogenetic analysis was done by Neighbor-Joining (NJ) analysis based on act+tef1 sequences. These three isolates were identified as C. tenuissimum by morphological and molecular characteristics. Pathogenicity of each C. tenuissimum isolate was tested on peanut in the greenhouse at 28°C with 75% relative humidity. Twenty plants of A. hypogaea were inoculated with the conidial suspension (1.0 × 105 conidia/ml) on the leaf surface. Ten plants were mock inoculated with sterile water as controls. Within 2 weeks, inoculated plants exhibited dark necrotic lesions on leaves which were similar to the symptoms observed in the field, while the mock inoculated plants remained symptomless. The fungal pathogen which was reisolated from inoculated rather than mock inoculated leaf tissues was identical to the original pathogen on the basis of morphological and molecular analysis, confirming Koch's postulates. To our knowledge, this is the first report of leaf spot caused by C. tenuissimum on peanut in China. The C. tenuissimum infection poses a serious threat by reducing the yield and quality of peanut in Shandong Province. This research is especially valuable to enhance epidemiological studies and implement effective control strategies.

Endophytic fungus Cladosporium tenuissimum DF11, an efficient inducer of tanshinone biosynthesis in Salvia miltiorrhiza roots.

Salvia miltiorrhiza is a traditional medicinal plant mainly used for cardiovascular and cerebrovascular disease treatment. Tanshinones are the main bioactive constituents of S. miltiorrhiza, which mainly accumulate around its root periderm tissue. Endophytic fungi are important bioelicitors or probiotics that can promote the accumulation of secondary metabolites and sustainable cultivation of medicinal plants. Among them, endophytic Cladosporium spp., possessing a variety of biotransformation and metabolic abilities, is an ideal elicitor source. Here, we used a gnotobiotic system to investigate the effects of the endophytic fungus Cladosporium tenuissimum DF11 on tanshinone biosynthesis in S. miltiorrhiza roots. The results showed that C. tenuissimum DF11 mainly colonizes the intercellular space of the root tissues and promotes tanshinone biosynthesis and accumulation in S. miltiorrhiza roots by upregulating the expression of the genes encoding for key enzymes HMGR, DXS, DXR, GGPPS, CPS, KSL and CYP76AH1 of the tanshinone biosynthesis pathway. The expression levels of almost all genes encoding for key enzymes reached the response peak in the first or second week after DF11 colonization. Taken together, the endophytic fungus C. tenuissimum DF11 could promote secondary metabolite accumulation in S. miltiorrhiza roots. These results indicate that DF11 will be a potential biofertilizer fungus to regulate and stabilize the quality of cultivated S. miltiorrhiza medicinal materials.

First Report of Leaf Spot Caused by Cladosporium tenuissimum on Panicle Hydrangea (Hydrangea paniculate) in China.

Panicle Hydrangea (Hydrangea paniculate) is an ornamental flowering plant native to China and Japan. In August 2019, leaf spot symptoms with about 30% disease incidence were observed on panicle hydrangea in two grower fields (about 0.1 ha in total) of Northeast Agriculture University, China (126.72°E, 45.74°N). Symptoms initially appeared on the lower and older leaves and showed small subcircular brown spots with dark-brown edges on both sides. As the disease progressed, the necrotic spots enlarged, became irregular, coalesced, and the infected leaf blighted in approximately 2 weeks. Panicle hydrangea leaf samples (n=15) from different plants that showed spot symptoms were collected and surface sterilized with 70% ethanol for 10 s, followed by 0.5% NaClO treatment for 4 min, and rinsed in sterile water 3 times. Thereafter, leaf samples were placed on potato dextrose agar (PDA) and incubated at 25°C for 7 days. Fifteen hyphal-tipped pure cultures were obtained. Colonies growing on PDA for 7 days were olive green to dark green, exhibited a velvet-like texture and sometimes were radially furrowed and wrinkled. Margins varied from white gray to dark green without prominent exudates. The back of the plate showed dark green to black. Conidiophores were up to 180 to 600 µm long, 2.8 to 4.5 µm wide (n=50), subcylindrical-filiform, straight, septate, and unbranched or rarely branched. Ramoconidia were 0 to 1 septate, cylindrical to clavate, smooth-walled, 8 to 22 µm long (n=50). Conidia were single-celled, lemon-shaped, smooth-walled and 2.0 to 5.0 µm (diameter) (n=50). To confirm the identity, three genomic DNA regions, internal transcribed spacer (ITS), partial translation elongation factor-1 alpha (EF), and actin (ACT) of the representative isolate BAI-1 were amplified with primer pairs ITS1/4, EF1-728F/986R, and ACT-512F/783R, respectively (Bensch et al. 2012; Jo et al. 2018). DNA sequences of the isolate from ITS, EF, and ACT showed 99.81% (514/515 bp), 99.10% (219/221 bp), and 99.54% (216/217 bp) nucleotide identity with those of C. tenuissimum CBS 125995, respectively (GenBank accession nos. HM148197, HM148442, and HM148687). The sequences of isolate BAI-1 were deposited in GenBank (accession nos. MW045455, MW052465, and MW052466). To fulfill Koch's postulates, five healthy 2-year-old panicle hydrangea plants grown in pots were surface sterilized with 70% ethanol, washed twice with sterile distilled water, and sprayed with a conidial suspension of strain BAI-1 (adjusted to 1×106 conidia/ml using a hemocytometer), maintained in a greenhouse at 25°C and 85% relative humidity. Five plants sprayed with sterilized water served as controls. The inoculated plants showed leaf spot symptoms that were similar to those previously observed in the fields after 7 days, whereas control leaves remained healthy. The fungus was reisolated from symptomatic leaves and its identity was confirmed by morphological and molecular method. These experiments were repeated twice. So far, C. tenuissimum was reported to cause leaf spot of alfalfa (Han et al. 2019) and castor (Liu et al. 2019). To our knowledge, this is the first report of leaf spot disease in panicle hydrangea caused by C. tenuissimum in China. Leaf spot has a negative effect on the aesthetic value of panicle hydrangea, and this report will assist with monitoring distribution of the disease as well as developing management recommendations.

Cladosporium cladosporioides and C. tenuissimum Cause Blossom Blight in Strawberry in Korea.

Blossom blight in strawberry was first observed in a green house in Nonsan, Damyang, and Geochang areas of Korea, between early January to April of 2012. Disease symptoms started as a grey fungus formed on the stigma, which led to the blossom blight and eventually to black rot and necrosis of the entire flower. We isolated the fungi purely from the infected pistils and maintained them on potato dextrose agar (PDA) slants. To test Koch's postulates, we inoculated the fungi and found that all of the isolates caused disease symptoms in the flower of strawberry cultivars (Seolhyang, Maehyang, and Kumhyang). The isolates on PDA had a velvet-like appearance, and their color ranged between olivaceous-brown and smoky-grey to olive and almost black. The intercalary conidia of the isolates were elliptical to limoniform, with sizes ranging from 5.0~10.5 × 2.5~3.0 µm to 4.0~7.5 × 2.0~3.0 µm, respectively. The secondary ramoconidia of these isolates were 0- or 1-septate, with sizes ranging betweem 10.0~15.0 × 2.5~3.7 µm and 8.7~11.2 × 2.5~3.2 µm, respectively. A combined sequence analysis of the internal transcribed spacer regions, partial actin (ACT), and translation elongation factor 1-alpha (TEF) genes revealed that the strawberry isolates belonged to two groups of authentic strains, Cladosporium cladosporioides and C. tenuissimum. Based on these results, we identified the pathogens causing blossom blight in strawberries in Korea as being C. cladosporioides and C. tenuissimum.

Cladosporium cladosporioides and C. tenuissimum Cause Blossom Blight in Strawberry in Korea

Blossom blight in strawberry was first observed in a green house in Nonsan, Damyang, and Geochang areas of Korea, between early January to April of 2012. Disease symptoms started as a grey fungus formed on the stigma, which led to the blossom blight and eventually to black rot and necrosis of the entire flower. We isolated the fungi purely from the infected pistils and maintained them on potato dextrose agar (PDA) slants. To test Koch's postulates, we inoculated the fungi and found that all of the isolates caused disease symptoms in the flower of strawberry cultivars (Seolhyang, Maehyang, and Kumhyang). The isolates on PDA had a velvet-like appearance, and their color ranged between olivaceous-brown and smoky-grey to olive and almost black. The intercalary conidia of the isolates were elliptical to limoniform, with sizes ranging from 5.0~10.5 x 2.5~3.0 microm to 4.0~7.5 x 2.0~3.0 microm, respectively. The secondary ramoconidia of these isolates were 0- or 1-septate, with sizes ranging betweem 10.0~15.0 x 2.5~3.7 microm and 8.7~11.2 x 2.5~3.2 microm, respectively. A combined sequence analysis of the internal transcribed spacer regions, partial actin (ACT), and translation elongation factor 1-alpha (TEF) genes revealed that the strawberry isolates belonged to two groups of authentic strains, Cladosporium cladosporioides and C. tenuissimum. Based on these results, we identified the pathogens causing blossom blight in strawberries in Korea as being C. cladosporioides and C. tenuissimum.

Mycoflora of soybeans used for meju fermentation.

Diverse fungi are present in Korean traditional meju and they are known to play an important role in fermented soybean products. To determine the origin of the fungi in meju, we examined the mycoflora of soybeans from 10 traditional meju factories. The samples were untreated or treated with sodium hypochlorite, and placed on malt extract agar (MEA), dichloran 18% glycerol agar (DG18), and dichloran rose bengal chloramphenicol agar (DRBC) medium. A total of 794 fungal strains were isolated and they were identified as 41 genera and 86 species. From sodium hypochlorite untreated soybeans, the genera, Cladosporium (55%), Eurotium (51%), Fusarium (33%), Penicillium (22%), and Aspergillus (exclusion of Eurotium) (20%), were mainly isolated, and Eurotium herbariorum (22%), Eurotium repens (18%), Cladosporium tenuissimum (18%), F. fujikuroi (18%), Aspergillus oryzae/flavus (7%), and Penicillium steckii (6%) were the predominant species. In case of sodium hypochlorite-treated soybeans, Eurotium (31%) and Cladosporium (5%) were frequently isolated, but Aspergillus (excluding Eurotium), Penicillium and Fusarium which were frequently isolated from untreated soybeans, were rarely isolated. Eurotium herbariorum (21%), Eurotium repens (8%), and Cladosporium tenuissimum (3%) were the predominant species. Of the 41 genera and 86 species isolated from soybeans, 13 genera and 33 species were also found in meju. These results suggest that the fungi on soybeans may influence the mycoflora of meju.

Mycoflora of Soybeans Used for Meju Fermentation

Diverse fungi are present in Korean traditional meju and they are known to play an important role in fermented soybean products. To determine the origin of the fungi in meju, we examined the mycoflora of soybeans from 10 traditional meju factories. The samples were untreated or treated with sodium hypochlorite, and placed on malt extract agar (MEA), dichloran 18% glycerol agar (DG18), and dichloran rose bengal chloramphenicol agar (DRBC) medium. A total of 794 fungal strains were isolated and they were identified as 41 genera and 86 species. From sodium hypochlorite untreated soybeans, the genera, Cladosporium (55%), Eurotium (51%), Fusarium (33%), Penicillium (22%), and Aspergillus (exclusion of Eurotium) (20%), were mainly isolated, and Eurotium herbariorum (22%), Eurotium repens (18%), Cladosporium tenuissimum (18%), F. fujikuroi (18%), Aspergillus oryzae/flavus (7%), and Penicillium steckii (6%) were the predominant species. In case of sodium hypochlorite-treated soybeans, Eurotium (31%) and Cladosporium (5%) were frequently isolated, but Aspergillus (excluding Eurotium), Penicillium and Fusarium which were frequently isolated from untreated soybeans, were rarely isolated. Eurotium herbariorum (21%), Eurotium repens (8%), and Cladosporium tenuissimum (3%) were the predominant species. Of the 41 genera and 86 species isolated from soybeans, 13 genera and 33 species were also found in meju. These results suggest that the fungi on soybeans may influence the mycoflora of meju.

Species and ecological diversity within the Cladosporium cladosporioides complex (Davidiellaceae, Capnodiales).

The genus Cladosporium is one of the largest genera of dematiaceous hyphomycetes, and is characterised by a coronate scar structure, conidia in acropetal chains and Davidiella teleomorphs. Based on morphology and DNA phylogeny, the species complexes of C. herbarum and C. sphaerospermum have been resolved, resulting in the elucidation of numerous new taxa. In the present study, more than 200 isolates belonging to the C. cladosporioides complex were examined and phylogenetically analysed on the basis of DNA sequences of the nuclear ribosomal RNA gene operon, including the internal transcribed spacer regions ITS1 and ITS2, the 5.8S nrDNA, as well as partial actin and translation elongation factor 1-α gene sequences. For the saprobic, widely distributed species Cladosporium cladosporioides, both a neotype and epitype are designated in order to specify a well established circumscription and concept of this species. Cladosporium tenuissimum and C. oxysporum, two saprobes abundant in the tropics, are epitypified and shown to be allied to, but distinct from C. cladosporioides. Twenty-two species are newly described on the basis of phylogenetic characters and cryptic morphological differences. The most important phenotypic characters for distinguishing species within the C. cladosporioides complex, which represents a monophyletic subclade within the genus, are shape, width, length, septation and surface ornamentation of conidia and conidiophores; length and branching patterns of conidial chains and hyphal shape, width and arrangement. Many of the treated species, e.g., C. acalyphae, C. angustisporum, C. australiense, C. basiinflatum, C. chalastosporoides, C. colocasiae, C. cucumerinum, C. exasperatum, C. exile, C. flabelliforme, C. gamsianum, and C. globisporum are currently known only from specific hosts, or have a restricted geographical distribution. A key to all species recognised within the C. cladosporioides complex is provided.