Conidiogenous Loci Research Articles

Cercospora polygonatum, a New Species Causing Gray Leaf Spot Disease in Polygonatum cyrtonema.

This study identified a new species (Cercospora polygonatum) that causes gray leaf spot (GLS) disease in cultivated Polygonatum cyrtonema. This fungal species was isolated from the affected region of GLS on P. cyrtonema leaves. Pathogenicity bioassays were conducted based on Koch's postulates. Morphology was examined based on the features of conidiomata, conidiogenous loci, conidia/conidiophores, and conidiogenous cells. The rDNA internal transcribed spacer region, calmodulin, translation elongation factor 1-alpha, and histone genes were subjected to phylogenetic analysis using the MrBayes tool in Phylosuite. Bootstrap support analysis for phylogenetic placement confirmed the new species, which was significantly different from the closely related species C. senecionis-walkeri and C. zeae-maydis. The morphological characteristics also supported this finding, with the conidiogenous cells of C. polygonatum being considerably shorter than those of C. senecionis-walkeri or C. zeae-maydis. In addition, C. polygonatum was distinguished by its cultural characteristics. As this fungus was isolated from P. cyrtonema, it was named C. polygonatum F.Q. Yin, M. Liu & W.L. Ma, sp. nov. The type specimen (H8-2) was preserved at the China General Microbiological Culture Collection Center. This is the first report of GLS caused by C. polygonatum on P. cyrtonema leaves in China. The current study enriches the knowledge regarding Cercospora sp., contributes to the identification of a species causing GLS in P. cyrtonema, and provides useful information for the effective management of this disease.

Morphological and phylogenetic analyses reveal two new Alternaria species (Pleosporales, Pleosporaceae) in Alternaria section from Cucurbitaceae plants in China.

Alternaria species are commonly found as saprophytes, endophytes and plant pathogens. During a survey of small-spored Alternaria in China, two new species were discovered from Cucurbitaceae plants collected in Hubei and Sichuan provinces. This study identified two new species of Alternaria using seven genes (ITS, GAPDH, TEF1, RPB2, Alt a 1, EndoPG, and OPA10-2) for phylogenetic analyses and morphological characteristics. The two new species A.jingzhouensis and A.momordicae were described and illustrated. Alternariajingzhouensis sp. nov., associated with Citrulluslanatus, is characterized by producing muriform, ellipsoidal, flask-shaped, rostrate, and beaked conidia. It differs from A.koreana, A.ovoidea, and A.baoshanensis by bearing conidia in a simple conidiogenous locus with occasionally longer beaks in a chain, and from A.momordicae sp. nov. by having shorter beaks. Alternariamomordicae sp. nov. from Momordicacharantia was distinct from A.koreana, A.ovoidea, and A.baoshanensis by producing muriform, long ellipsoid or ovoid to obclavate, sometimes inverted club-shaped conidia on a single conidiogenous locus with a wider body and longer beak in a chain, and distinct from A.jingzhouensis sp. nov. by a longer beak conidia. These two species were clearly distinguished from other species in the section Alternaria based on DNA based phylogeny and morphological characteristics. The morphological features were discussed and compared to relevant species in the present paper.

First report of cobweb disease in Auricularia cornea caused by Hypomyces mycophilus in Guizhou, China.

Auricularia cornea is a widely cultivated mushroom in China, which has high medicinal values such as hemostaticity, analgesia, antioxidation and anti-tumor (Wu et al., 2019). In 2022, an investigation on edible mushroom diseases in Guizhou Province observed a suspected cobweb disease in an A. cornea growing factory, with up to 30% incidence. The pathogen first produced flocculent hyphae on the surface of the fruiting body of A. cornea, and then developed spider web-like aerial hyphae, covering the entire fruiting bodies. It hinders the normal growth of A. cornea, resulting in deformity and rot of the fruiting bodies. These symptoms seriously affect the quantity and quality of mushroom yields and cause huge economic losses. Three fungal isolates (GUCCX001, GUCCX002 and GUCCX003) were recovered from the diseased mushroom fruiting bodies and purified through single spore isolation. The colonies of three isolates spread rapidly on PDA, reaching 79-82 mm in seven days. The flocculent mycelium was whitish, and its reverse turned from yellowish to amber after 14 days. The branched conidiophores arising from aerial mycelia were septate and each cell contained several denticulate conidiogenous loci. Each denticle contained a single conidium. Conidia were observed at the tip of conidiophore branches and were 0-1-septate, oval or spherical, transparent, 5.2-11.3 × 11.7-18.7 μm (n = 35). Chlamydospores were visible as 3-4 thick-walled cells at the tip of lateral hyphal branches. Three isolates were tentatively identified as H. mycophilus based on their morphological characteristics similar to those described by Rogerson and Samuels (1993). The sequence of internal transcribed spacer (ITS) region (primers ITS5/ITS4) (Rehner and Samuels, 1994) and nuclear ribosomal large subunit (LSU) region (primers LR0R/LR5) (Vilgalys and Heste, 1990) of GUCCX001 (ITS: OP777905; LSU: OQ152071), GUCCX002 (ITS: OP862872; LSU: OQ152072) and GUCCX003 (ITS: OP862873; LSU: OP862873) were 99%-100% similar to H. mycophilus CBS 175.56 (ITS: MH857567; LSU: MH869110). Fifteen healthy fruiting bodies of A. cornea were inoculated by spraying spore suspension (106 conidia/mL) of the three isolates and five healthy fruiting bodies were sprayed with sterile water as control. All inoculated fruiting bodies were kept at 25 ℃. After three days, fruiting bodies of A. cornea treated with the spore suspension exhibited the same symptoms of cobweb as in the factory, while no symptom appeared in the control. Pathogens re-isolated from diseased fruiting bodies were confirmed to be H. mycophilus based on morphological characteristics, which fulfills the Koch's postulate. Zeng et al. (2017) reported H. mycophilus on the fruiting bodies of Auricularia sp. as a new record in Guangdong, China. H. mycophilus caused cobweb disease on A. auricula (Liu et al., 2020), A. cornea var. Li. (Cao et al., 2023) and A. heimuer (Zhang et al., 2023). To our knowledge, this is the first report of cobweb disease in A. cornea caused by H. mycophilus in Guizhou, China. Our findings will provide a basis for correct diagnosis and management of cobweb diseases on A. cornea.

First report of Alternaria burnsii causing leaf spot on Bletilla striata in China.

Bletilla striata (named "Bai Ji" in Chinese) is a plant from the Orchidaceae family that has been employed in traditional Chinese medicine for thousands of years in China. Polysaccharides extracted from B. striata have been shown to have an effect on Alzheimer's disease (Lin et al. 2021). Since 2021, leaf spots have been observed in the B. striata plantation in Chongqing, China. Out of 200 plants, the disease incidence was estimated at 56%, and the disease index was estimated at 32%. The symptoms were necrotic lesions with brown edges and yellow halos; severe infection caused the infected leaves to become blighted, dry and fall off. To identify the causal agent, eighteen leaves with typical symptoms were collected from the B. striata plantation (30.60°N, 108.64°E). The margins of infected tissue areas were cut into small pieces (5×5 mm), surface sterilized with 70% ethanol for 1 min, and rinsed twice with sterile distilled water. The tissue was then surface sterilized in 3% sodium hypochlorite for 2 min, followed by three rinses with sterile water. The tissue was then placed onto potato dextrose agar (PDA) plates and incubated at 25°C for 3 days, pure cultures of fungal isolates were obtained by single-spore isolation, stored on PDA slants and maintained at 4°C. Colonies of the fungal isolates showed three color types, ranging from grayish white to green above with olive green on the reverse, but conidial characteristics were more similar and indicated this was a single fungus. Conidiophores were single, lateral from hyphae or terminal; straight or curved; smooth-walled with 1 to 8 septa; pale brown; usually with only one pigmented terminal conidiogenous site, sometimes with one additional lateral conidiogenous locus; sometimes slightly swollen at the apex; and 15 to 170 μm long, 2.5 to 4.5 μm wide. Conidia were in short or moderately long chains of 2-8 conidia normally, sometimes with more; rarely branched; normally 14.07 to 50 × 5.24 to 10 μm in size; ellipsoid, fusiform, long ellipsoid, obclavate or ovoid with 1 to 11 transverse septa and 2 to 4 longitudinal septa; beakless or with subcylindric or cylindric secondary conidiophores, analogous to the beak 4.25 to 58.6 μm long, 3.2 to 4.8 μm wide. The fungal isolates were tentatively identified as Alternaria sp. The representative isolate BJ8 was selected for the pathogenicity test. The leaves of six healthy plants of B. striata (two years old) grown in pots were washed with sterile water. Ten mL of conidial suspension (1×106 conidia mL-1) contained in 0.05% Tween 80 buffer was brushed onto upper and lower surfaces of all the leaves on three plants, while other plants were brushed with 10 mL 0.05% Tween 80 buffer to serve as controls. Plants were placed in a greenhouse at 25°C and 95±1% relative humidity after inoculation and observed for symptoms. The symptoms initially developed as irregular brown necrotic lesions on the inoculated leaves after 7 days, with a yellow halo around the lesions, consistent with the symptoms in the field. Leaves on the control plants did not produce any symptoms. For molecular identification, the genomic DNAs of representative isolates BJ5, BJ6, and BJ8 were extracted. The internal transcribed spacer (ITS) region and RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-alpha (TEF1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were used for polymerase chain reaction (PCR), using primers ITS5/ITS4, GPD1/GPD2, EF-1F/EF-1B and RPB27cR/RPB25F2, respectively (White et al. 1990; Berbee and Pirseyedi et al. 1999; Carbone and Kohn 1999; Liu et al. 1999). The neighbor-joining tree revealed that these isolates are clustered together with the reference strain of A. burnsii. The sequences were deposited in NCBI GenBank BJ5 [ITS: OP897263; GAPDH: OQ544937; TEF1: OQ544941; RPB2: OQ544939], BJ6 [ITS: OP897262; GAPDH: OQ544938; TEF1: OQ544942; RPB2: OQ544940], and BJ8 [ITS: OK285209; GAPDH: OK340046; TEF1: OK340047; RPB2: OQ544936]. All three isolates showed 100% similarity with A. burnsii CBS 107.38 [ITS: KP124420; GAPDH: JQ646305; TEF1: KP125198; RPB2: JQ646457] ex-type sequence, thus the pathogen causing the leaf spot on B. striata was identified as A. burnsii. A. burnsii is an important pathogenic fungus causing blight of cumin (Shekhawat et al. 2013). Furthermore, Al-Nadabi et al. (2018) found that A. burnsii can cause leaf spots on wheat and date palms, and Sunapao et al. (2022) reported that A. burnsii can infect coconuts (Cocos nucifera), causing dirty panicle disease. This is the first report of A. burnsii causing leaf spot on B. striata in China. The new discovery shows that since A. burnsii can readily adapt to a variety of climatic conditions, controlling the fungus is crucial for the healthy growth of B. striata in the future. This study will provide a basis for further elucidating the pathogenic mechanism and development of effective control measures for this disease.

First Report of Cobweb Disease in Auricularia cornea var. Li. Caused by Hypomyces mycophilus in China.

Auricularia cornea var. Li. is a natural mutation strain of A. cornea which has been preferred by consumers for its white colour, good taste and pharmacological properties (Rebecca et al. 2020). In October 2021, a disease with symptoms similar to that of cobweb disease (Carrasco et al. 2017) was observed in A. cornea var. Li. in four mushroom farms in Fangshan District (115.83°E, 39.55°N), Beijing, China, infecting 20% of the fruiting bodies (Fig. 1A-D). White cottony mycelia formed typically on the casing soil and they gradually spread to the stipes and pileus, covering the whole fruiting body, which eventually died and lost commodity value. Cultures were obtained by aseptically transferring the diseased fruiting bodies onto potato dextrose agar (PDA); they were deposited in the culture collection (ID: JZBQA3) of the Beijing Academy of Agriculture and Forestry Sciences, China. The colonies were floccose with aerial mycelium white. Purplish grey diffusing pigments occasionally formed on the reverse side of the plate at 25 °C (Fig. 2A-B). Conidiophores arising in aerial mycelium, indefinite in length, branches septate, each cell producing denticulate conidiogenous loci, each denticle bearing a single conidium. Conidia mostly oblong to ellipsoidal, smooth, (9.0-)9.9-17.0(-18.0)×(6.0-)6.9-10.2 μm (n = 60), 0~1 septate (Fig. 2C-E). Chlamydospores forming as lateral branches of hyphae were commonly observed, globose, ellipsoid or oblong, 14.8-22×14.7-19.6 μm, l/w = 1.0-1.3 (Fig. 2F-G). The morphological characteristics were consistent with that of Hypomyces mycophilus, whose anamorph was Cladobotryum polypori (Rogerson et al. 1993, Zeng et al. 2017). For further species identification, genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, USA). The internal transcribed spacer (ITS) regions and large subunit ribosomal RNA (LSU) genes were amplified using the primer pairs ITS1/ITS4 (White et al. 1990) and LROR/LR5 (Bhattacharya et al. 2000), respectively. The sequences were deposited in GenBank with accession numbers OP430530 and OP430531. BLAST nucleotide searches showed more than 99% homology with corresponding sequences of Hypomyces mycophilus HMAS 275554 and CBS 175.56. Phylogenetic trees based on ITS and LSU revealed that the strain JZBQA3 was grouped with H. mycophilus with high support value (Fig. 3). A in vivo pathogenicity test was performed using eight mushroom sticks with healthy fruiting bodies in triplicate. Each four sticks were sprayed with conidial suspension (108 spores/mL) of strain JZBQA3 or sterile distilled water, respectively, and maintained in an artificial climate chamber at 25-26°C. Cobweb-like characteristics were observed on the fruiting bodies treated with the JZBQA3 conidial suspension 2-3 days after inoculation, while those treated with sterile distilled water remained symptomless (Fig. 4 A-B). The same pathogen was re-isolated and confirmed from the infected fruiting bodies by integrated analysis of morphological characteristics and gene sequencing data, fulfilling Koch's postulates. Hypomyces mycophilus was first reported on Trametes versicolor in North Carolina (Rogerson et al. 1993), and is the causal agent of cobweb diseases on Auricularia heimuer (Zhang et al. 2023). To our knowledge, this is the first report of cobweb disease caused by H. mycophilus in A. cornea var. Li. This finding is a valuable contribution to the knowledge of cobweb disease development in edible fungi.

Leaf spot disease on Juglans regia caused by Ragnhildiana diffusa in China.

Juglans regia L. is commercially important for its edible nuts, which is a major species of walnut trees in Sichuan Province (Luo et al. 2020). In September 2021, brown leaf spot symptoms were observed on roughly 75% of 60 J. regia trees surveyed in an orchard of Chongzhou city (30°40'6''N, 103°40'18''E). Initially, the lesions measuring 2-10 mm were reddish to brown with a yellowish halo, then increased in size and coalesced to cover the whole leaf, eventually resulting in severe defoliation. Six symptomatic leaves from different trees were collected, and a single fungal isolate was obtained from each of the sampled leaves using single-spore isolation (Chomnunti et al. 2014). The isolates were incubated on potato dextrose agar (PDA) with a 12h photoperiod at 25 ℃, and deposited at the Culture Collection of Sichuan Agricultural University. Colonies were identical with black center and reddish-brown periphery, and the diameter reached 2 cm after 7 days. On the host, conidiophores were mostly reduced to conidiogenous cells, with prominent and thickened conidiogenous loci. Conidia were light green to light brown, and curved with a thickened and darked hilum at the base, 0-17 septate, tapering toward the distal end, and measuring 20-120 × 3-5 μm ((x ) ̅= 56 × 4, n = 30). Morphological characteristics fit the description of Ragnhildiana diffusa (Heald & F.A. Wolf) Videira & Crous (Synonym: Sirosporium diffusum (Heald & F. A. Wolf) Deighton) (Poletto et al. 2017). The internal transcribed spacer (ITS) region, the large subunit of the nrDNA (LSU), and RNA polymerase II second largest subunit (rpb2) were amplified by polymerase chain reaction and sequenced with primers ITS5/ITS4 (White et al. 1990), LR0R/LR5 (Vilgalys & Hester 1990), fRPB2-5F/Rpb2-R3 (Liu et al. 1999, Videira et al. 2017), respectively. The nucleotide blast of the two isolates (SICAUCC 22-0077, SICAUCC 22-0078) showed 99.7% and 99.5% (ITS, 472/473 bp, 471/473 bp), 100% (LSU, 725/725 bp, 725/725 bp), 99.8% (rpb2, 866/867 bp, 866/867 bp) identities with the ex-type strain of Ragnhildiana diffusa (CBS 106.14). The phylogenetic tree combined with ITS, LSU, and rpb2 genes and morphological characteristics confirmed the identification as R. diffusa. These sequences of the three gene regions of two isolates were deposited in GenBank with accession numbers ON409525 and ON409526 (ITS), ON409559 and ON409560 (LSU), ON417473 and ON417474 (rpb2), respectively. The isolate SICAUCC 22-0077 was used for pathogenicity test to fulfill Koch's postulates. Three leaves of each walnut seedlings (2-year-old seedlings) were inoculated by placing a mycelium plug onto fresh wounds on the upper leaf surface punctured via a fine needle (0.7 mm in diameter), and three replicate seedlings were inoculated. For the control, a sterile PDA plug was placed on the same number of replicate leaves on the plants. The inoculated and control plants were placed in a growth chamber at 25°C with relative humidity >80% and a 12-h photoperiod. Irregular light to dark brown spots developed on inoculated leaves after twenty days, and no symptoms were observed on controls. The re-isolation and examination of the fungus showed it to be morphologically and phylogenetically identical to the originally isolated pathogen. R. diffusa has been described on J. regia in Mexico (Farr & Rossman 2022). To our knowledge, this is the first report of R. diffusa causing brown leaf spot on J. regia in China. The identification of the pathogen will provide a basis for disease management in walnut planting areas.

Diversity of Cladosporium (Cladosporiales, Cladosporiaceae) species in marine environments and report on five new species.

Cladosporium species are cosmopolitan fungi, characterized by olivaceous or dark colonies with coronate conidiogenous loci and conidial hila with a central convex dome surrounded by a raised periclinal rim. Cladosporium species have also been discovered in marine environments. Although many studies have been performed on the application of marine originated Cladosporium species, taxonomic studies on these species are scarce. We isolated Cladosporium species from three under-studied habitats (sediment, seawater, and seaweed) in two districts including an intertidal zone in the Republic of Korea and the open sea in the Western Pacific Ocean. Based on multigenetic marker analyses (for the internal transcribed spacer, actin, and translation elongation factor 1), we identified fourteen species, of which five were found to represent new species. These five species were C.lagenariiformesp. nov., C.maltirimosumsp. nov., C.marinumsp. nov. in the C.cladosporioides species complex, C.snafimbriatumsp. nov. in the C.herbarum species complex, and C.marinisedimentumsp. nov. in the C.sphaerospermum species complex. Morphological characteristics of the new species and aspects of differences with the already known species are described herein together with molecular data.

Taxonomy and biology of <i>Cladosporium endophyticum</i> as the first record in Iraq

<italic>Cladosporium</italic> species are cosmopolitan organisms. Conidiogenous loci distinguish this genus. Their spores can be isolated from different sources. The morphological analysis and ITS sequence were used to identify <italic>Cladosporium endophyticum</italic> isolated from air. The findings demonstrate that <italic>C. endophyticum</italic> S1 and <italic>C. endophyticum</italic> Thailand strain were almost identical in this investigation. The <italic>C. endophyticum</italic> strain is isolated from Thailand, and it has a distinctive phylogenetic clade. This clade resided in the vicinity of <italic>Cladosporium sphaerospermum</italic> and <italic>Cladosporium halotolerans,</italic> respectively. In order to study the physiological factor effect on <italic>C. endophyticum</italic> on solid media, different temperatures were used (5, 10, 15, 20, 25, 30, 35, 40), pH (4, 5, 6, 7), media malt extract agar (MEA), potato dextrose agar (PDA), oatmeal agar (OA), synthetic nutrient-deficient agar (SNA), carbon (sucrose, glucose, sorbitol, xylose, manitol), nitrogen (ammonium nitrate, ammonium sulphat, urea, ammonium phosphate, potassium nitrate). The results showed all factors' highly significant (p ≤ 0.05) effect. The faster growth was found at 25 °C, pH suitable for growth 5 and 6. The maximum radial growth was found on PDA and MEA. The fungus <italic>C. endophyticum</italic> can utilize all carbon sources, and faster growth was recorded on sucrose, then glucose, xylose, sorbitol, and manitol. The fungus grows on all nitrogen sources, and mycelium growth was good on ammonium nitrate, ammonium phosphate, ammonium sulfate, urea, and potassium nitrate. <italic>Cladosporium endophyticum</italic> is newly recorded from Basrah, Iraq, and it has been deposited in GenBank with accession numbers for nucleotide sequence (MW298524).

Alternaria malorum, a new causal agent of cowpea leaf spot disease in Iran

Cowpea (Vigna unguiculata, Fabaceae) is an annual herbaceous legume cultivated for seed and fodder. During a survey in May 2022, cowpea leaves with leaf spot symptoms were collected from a field in which c. 40% of the plants were diseased (Karaj City, Alborz province, Iran). Leaf spots began as small circular water-soaked lesions at the leaf centre and margins which expanded and on occasion, coalesced. The infected tissue became pale brown and necrotic. Isolation of the causal pathogen was performed using the high humidity blotter method. After five days, growing fungi were transferred with a fine sterile needle to 2% water agar. Pure cultures were obtained using the hyphal tip method on synthetic nutrient-poor agar (SNA) and potato carrot agar culture media. Fungal colonies were incubated at 25°C with an 8 hour light/16 hour dark photoperiod for seven days. A typical colony (ALT1) reached 58 mm in diameter after seven days incubation on SNA culture medium. The colony was olive-brown to dark brown at the centre, with a white margin. Aerial mycelia were sparse. Mycelia were septate, pale brown and smooth–walled. Sporulation was abundant on SNA, mostly from the aerial hyphae. Conidiophores arose singly from the aerial mycelia, and were brown, erect, simple, short, smooth, frequently reduced to conidiogenous cells with a slight swelling at the apices, 7–30 × 3–5 μm. Conidiogenous loci were 1–3 per conidiogenous cell, and brown. Ramoconidia were 0–3 septate, ellipsoid to cylindrical, smooth, brown, 15–32 × 3–5 μm, with a short beak (n = 50). Conidia were subcylindrical, ellipsoid or fusiform, brown, smooth, in long acropetal chains, simple or with lateral branches and 8–11 × 3–3.5 μm (n = 50) (Figure 1). Morphological and cultural characteristics of the recovered isolate (ALT1) were similar to the description of Alternaria malorum provided by Goetz & Dugan (2006) and Crous et al. (2009). Molecular characterisation of isolate ALT1 was done by PCR amplification and sequencing of the internal transcribed spacer (ITS) region using the ITS1 and ITS4 primers (White et al., 1990). The obtained sequence was deposited in GenBank (Accession No. OP006224) and a BLAST search showed it had 100% identity to isolates of A. malorum (MW335156, LR134074, FJ839611 and KY077556). Maximum likelihood analysis was performed by heuristic search with Mega X software. In the generated phylogenetic tree (Figure 2), isolate ALT1 was separated from closely related Alternaria species and other fungal genera, and clustered in the same group as A. malorum isolates. The molecular analysis confirmed the morphological identification of the pathogen. Pathogenicity testing was performed by spraying a conidial suspension (106 conidia/ml) of isolate ALT1 on healthy and surface-sterilised cowpea leaves without wounding, and was replicated three times. Sterile water was sprayed on the leaves of control plants. All plants were covered with plastic bags to maintain high humidity and placed on a glasshouse bench at 25°C with a 12 hour photoperiod until the development of disease symptoms. Leaf spots appeared twelve days after inoculation (Figure 3) and resembled those observed in the field. The pathogen was re-isolated from the newly generated leaf spots, fulfilling Koch's postulates. In Iran, Alternaria malorum was first reported by Asgari et al. (2004) as Cladosporium malorum from the leaves of the Hordeum vulgare in East Azarbaijan province. Alternaria malorum has also been reported as a fungus associated with declining Persian oak trees (Alidadi et al., 2018). Bagherabadi & Zafari (2022) also reported A. malorum as the causal agent of bark canker in walnut trees. Several diseases are known to infect cowpea, including anthracnose, Cercospora leaf spot, damping off, Fusarium wilt, Sclerotium stem, root and crown rot, Septoria leaf spot, and scab (Dugje et al., 2009). This is the first report of Alternaria malorum as a causal agent of cowpea leaf spot disease in Iran and globally. We are thankful to the University of Tehran for supporting this research.

First report of Colletotrichum fructicola causing anthracnose on water hyacinth in China.

Water hyacinth (Eichhornia crassipes), a worst invasive aquatic weed has been caused the widespread problems for the water bodies and water resources, particularly the case in China. Plant pathogens are a promising alternative as biocontrol agents (Dagno et al. 2011), but success in this strategy will require the selection of some highly virulent pathogen strains. In September 2020, irregular necrotic lesions on leaves, stems, as well as crown and petiole rots symptoms, occurred on water hyacinth, in Minjiang and Xiyuanjiang watershed, in Fuzhou, China. Fragments from symptomatic leaf tissue (5x5mm) were superficially disinfected in 0.1% MgCl2 solution for 30 s, followed by rinsing three times in sterile water, placed on potato dextrose agar (PDA), and then incubated in darkness at 28°C for 5 days. Two fungal isolates (F3 and F11) were recovered and obtained pure cultures from the affected leaves and deposited in the Institute of Oceanography, Minjiang University. The colonies were stale, with felted, dense, pale grey aerial mycelium, scattered dark based acervuli with orange conidial masses near centre; in reverse side pinkish orange with patches of grey pigment near centre. The hyphae were septate, branched, and 2 to 6 µm in width. Appressoria were not observed. Conidiogenous cells were 20-24 × 3.5-4.5 µm, cylindric to flask-shaped, towards margin the conidiophores with a much looser structure, conidiogenous loci at apex and often also at septa. Asci were 60-80 × 15-20 µm, cylindric to subfusoid, 8-spored. Ascospores were 17-23 × 4-6 µm, gently curved, tapering to quite narrow, rounded ends. Perithecia mature after about 15 days, and were dark brown, subglobose, and 50-150 μm in diameter, and with scattered, dark brown setae about 50-80 µm long. Conidia were 15-25 × 4.5-6 μm, unicellular, colorless, and cylindrical to fusiform. Genomic DNA from two isolates was extracted with a modified DNA Midi Kit (TIANGEN, Inc., Beijing, China), and amplified using ITS4/ITS1F, CL1/CL2A, CHS-79F/CHS-345R, T1/T2 and GDF/GDR primers by PCR (Weir et al. 2012; White et al. 1990). Sequences of F3 and F11 were submitted to GenBank (accession no. ITS, MW307302, MW307303; CAL, MW303427, MW303429; CHS-1, MW303428, MW303430; TUB, MW531006, MW531007; GADPH, MW531008, MW531009). A phylogenetic tree using the maximum likelihood methods and including ITS-CHS-CAL-TUB-GADPH concatenated sequences from Colletotrichum gloeosporioides complex was obtained (Cai et al. 2009; Damm et al. 2018; Weir et al. 2012). Phylogenetic analyses revealed that isolate F3 and F11 were grouped into the clade C. fructicola. To test Koch's postulates, conidial suspensions (107 CFU/ml) of the isolate F3 and F11 were micro-injected into 20 water hyacinth seedlings per isolate. Another 20 seedlings were injected with water without conidia as control. Inoculated plants were kept in 50-liter plastic tanks, and maintained in a greenhouse at room temperature (19-24ºC) for two weeks. The Koch's test was conducted twice. After 10 days, typical anthracnose symptoms similar to the field appeared on the inoculated leaves, while the control leaves remained asymptomatic. The C. fructicola was re-isolated and identified by microscopy, PCR and sequencing, but not on non-inoculated controls. Anthracnose disease caused by C. fructicola has been reported affecting numerous plants worldwide, including cotton, coffea, grape, citrus, ect (Guarnaccia et al. 2017). However, to our knowledge, this is the first report of C. fructicola causing anthracnose on water hyacinth in China. Further studies for the efficacy of C. fructicola and/or of the genus Colletotrichum as biocontrol agent for water hyacinth or another aquatic plant are required (Ding et al. 2007; Dagno et al. 2012).

Blodgettia chinensis and Nigrolentilocus saprophyticus spp. nov., and a first record of Anapleurothecium botulisporum from southern China

Two new anamorphic fungi, Blodgettia chinensis sp. nov. and Nigrolentilocus saprophyticus sp. nov., are described and illustrated from specimens collected on dead branches of unidentified plants in southern China. Blodgettia chinensis is characterized by monoblastic, integrated, terminal conidiogenous cells that produce broadly ellipsoidal, 5-euseptate, smooth conidia with darkened and thickened bands at the septa. Nigrolentilocus saprophyticus is distinguished by its polyblastic, sympodially extending, terminal and intercalary, integrated conidiogenous cells with conspicuous, lenticular conidiogenous loci and ellipsoidal to fusiform, 3-euseptate, versicolored conidia. A key to Nigrolentilocus species is provided. Anapleurothecium botulisporum is recorded for the first time from China.

Cladocillium musae, a new genus and species of cercosporoid fungi (Mycosphaerellaceae) on wild banana in Taiwan

A dematiaceous hyphomycete with erect unbranched conidiophore stipes and Cladosporium-like branched conidial chains arranged at nodes along the stipe was found on living leaves of wild banana (Musa itinerans) in Taiwan. Scanning electron microscopy indicated a type of conidiogenous loci differing from that of Cladosporium by lacking the cleft between raised center and margin. Molecular phylogenetic analyses of several loci (LSU, ITS, TEF, RPB2) indicated a relationship with cercosporoid fungi (Mycosphaerellaceae). Since there is no other known lineage with similar morphology or DNA sequences, the new genus and species Cladocillium musae is proposed.

First Report of Castor Leaf Spot Caused by Cladosporium tenuissimum in Zhanjiang, China

Ricinus communis L., common name castor, is an important oil crop (Liu et al. 2016). Leaf spots on castor with disease incidence of about 20.5% have been observed in about 1,800 plants in the field (0.66 ha) located in Zhanjiang (21.17 N, 110.18 E), China, since 2016. Early symptoms appeared on affected leaves as light brown or gray necrotic spots. Successively, a mold appeared on both sides of the spots. The spots were irregular in shape and increased in size and coalesced. Seven samples of symptomatic leaves were collected in the field, and they were washed and surface disinfested before isolation. Potato dextrose agar (PDA) was used to isolate the pathogen. Successively, pure cultures were obtained by transferring hyphal tips on PDA. Thirteen isolates were obtained from affected leaves. Two monospore isolates (RiK-1 and RiK-2) were obtained from the isolates, with each isolate being identical based on preliminary molecular analyses by BLASTn. Therefore, the representative isolate (RiK-1) with typical morphology and molecular characteristics was selected for further study. The isolate RiK-1 was grown on PDA and formed colonies of approximately 3 cm (diameter) in 7 days, at 25 ± 1°C. Colonies were olive green and appeared velvety. Conidiophores were up to 500 or 700 µm long, thick walled, septate, and unbranched or rarely branched. Ramoconidia were cylindrical to clavate, slightly inflated at the apex, and bearing two to three flat, thickened scars. Conidia were single celled, lemon shaped, and smooth walled, 2 to 5 µm (diameter), up to 9 µm long. They formed fragile chains of up to six conidia. Morphological characteristics of colonies of isolate RiK-1 were consistent with the description provided for Cladosporium tenuissimum (Bensch et al. 2010). Molecular identification was performed by sequencing of the nuclear ITS-rDNA region using primers ITS1 and ITS4. The sequence of isolate RiK-1 was deposited in GenBank (accession no. MH383051). A BLASTn search of the sequence showed 99 to 100% identity with C. tenuissimum (MF473305.1) and C. oxysporum (KX258798.1). Isolate RiK-1 was closer to C. tenuissimum, which is characterized by nodulose conidiophores with a head-like swollen apex, different from C. oxysporum, which is characterized by nodulose to nodose conidiophores with conidiogenous loci restricted to swellings. Therefore, isolate RiK-1 was identified as C. tenuissimum. A pathogenicity test was performed in vivo, in the greenhouse with a relative humidity of 80%, at temperatures ranging from 24 to 30°C. Two methods (wound inoculation and no wound inoculation) were used on leaflets. Sterile cotton ball was dipped in a spore suspension (1 × 10⁶ conidia/ml) or in sterilized water (as controls) for about 1 min. One leaflet was covered by a cotton ball and was kept for 48 h (four leaflets for each plant, three plants for each method). The first symptoms were observed after 1 month on treatments, whereas controls remained healthy. Pathogen reisolated from diseased leaflets was identical to the inoculated isolates by morphology and 18S rDNA analysis. It has been reported that C. tenuissimum caused leaf spot of banana (Pandey and Gupta 1983) and leaf blight in watermelon (Narain et al. 1985). The disease was not previously reported in castor. Thus, this is the first report of C. tenuissimum causing leaf spot in castor. This study will provide an important reference for the control of the disease. The epidemiology of C. tenuissimum on R. communis should be investigated further.

First Report of Leaf Spot Caused by Pseudocercospora ligustri on Ligustrum japonicum ‘Howardii’ in China

‘Howardii’ privet (Ligustrum japonicum ‘Howardii’) is a cultivar of L. japonicum (L.) (Japanese privet) that is native to central and southern Japan and Korea. This cultivar is widely planted as an ornamental in parks and landscapes in China. From 2010 to 2013, a leaf spot disease on L. japonicum ‘Howardii’ was observed in parks in Luoyang, Henan Province, China. A survey revealed that the disease was significant, and more than 50% of leaves in some plantings were infected. Leaf spots initially appeared slightly chlorotic, enlarging slowly, becoming lemon-yellow to purple and finally brown. Fully matured lesions were 5 to 15 mm in diameter, circular to subcircular or irregular, with reddish-purple to black margins. The centers were tan with occasional brown flecks and faintly zonate. Lesions often coalesced to form extensive necrotic areas. A cercosporoid fungus was consistently isolated from diseased leaves. Fungal stromata were well-developed, globular, dark brown, predominantly hypophyllous, and evenly distributed over the leaf spot. Stromata (n = 50) were 22.6 to 58.3 μm in diameter. Conidiophores (n = 50) were densely fasciculate, olivaceous to pale brown, subcylindrical, geniculate-sinuous, 12.8 to 32.3 × 3.0 to 4.2 μm, and aseptate to one septa. The conidiogenous loci were inconspicuous, neither thickened nor darkened. Conidia (n = 50) were pale olivaceous, cylindrical to obclavate, usually slightly curved, sometimes straight, smooth, multiseptate, and 19.3 to 64.1 × 2.0 to 3.9 μm. Three representative isolates of the fungus (JS1201, JS1202, and JS1203) were obtained by single-conidium isolation from fascicles of conidiophores on lesions of diseased plants. Cultures on potato dextrose agar (PDA) were slow growing, 15 to 20 mm diameter after 20 days at 25°C. Colonies were raised, olive gray on topside, black on reverse, and without sporulation. Based upon these morphological and cultural characteristics, the newly collected fungus from China on L. japonicum ‘Howardii’ was similar to Pseudocercospora ligustri (Deighton 1976). For further confirmation, three nuclear gene regions (ITS, ACT, and EF-1α) were amplified and sequenced for multigene phylogenetic analysis. The DNA sequences generated were deposited in GenBank (accession nos. KU255727, KU255728, KU255729, KX429750, KX429751, KX429752, KX397350, KX397351, and KX397352). A BLAST search in GenBank revealed that the sequences showed 99 to 100% identity with several sequences of Pseudocercospora species, including P. lilacis, P. oenotherae, P. marginalis, and others. Phylogenetic analysis was further performed with the concatenated sequence alignment of three genes (ITS, ACT, and EF-1α) using MEGA 6.0 software (Tamura et al. 2013). The neighbor-joining phylogenetic tree showed that the three isolates of P. ligustri from L. japonicum ‘Howardii’ and an isolate of P. lilacis from L. japonicum clustered into one clade (bootstrap value of 100%). In this group, our isolates clustered in one separate subclade with 71% bootstrap support. Pathogenicity testing was conducted on young leaves of 3-year-old potted ‘Howardii’ privet plants with the JS1201 isolate cultured for 2 weeks on PDA plates. Because the cultures did not produce conidia, mycelial discs were used for inoculation (de Miranda et al. 2014). Twenty mycelial discs of P. ligustri, approximately 5 mm in diameter, were placed on the upper leaf surface of the test plants. Control plants were inoculated with agar discs without mycelium. Plants were covered with plastic bags for 48 h after inoculation. Necrotic spots appeared on the inoculated leaves 10 to 12 days after inoculation and were identical to the symptoms observed in the field. P. ligustri was reisolated from symptomatic leaf tissues, confirming Koch’s postulates. No symptoms were observed on control plants. To our knowledge, this is the first report of leaf spot caused by P. ligustri on L. japonicum ‘Howardii’ in China.

Classiculasinensis, a new species of basidiomycetous aquatic hyphomycetes from southwest China.

Classiculasinensis, isolated from decaying leaves from Mozigou, Chongqing Municipality, China, is described as a new species. The new species is a member of basidiomycetous aquatic hyphomycetes which represent a small proportion of all aquatic hyphomycetes. This species falls within the genus Classicula (Classiculaceae, Pucciniomycotina) and is closely related to C.fluitans, based on multiple gene sequence analyses. Morphologically, it is characterised by the apical, hyaline, obclavate or navicular conidia with several hair-like lateral appendages and by its holoblastic and monoblastic conidiogenesis, with a flat un-thickened conidiogenous locus. Clamp connections and haustorial branches were often observed in culture.

Disentangling Phialophora section Catenulatae: disposition of taxa with pigmented conidiophores and recognition of a new subclass, Sclerococcomycetidae (Eurotiomycetes)

A new genus Rhopalophora is described for Phialophora clavispora, a lignicolous species formerly placed in Phialophora section Catenulatae that possesses pigmented conidiophores, phialides with a single conidiogenous locus that occasionally appear as schizophialides, and clavate, aseptate conidia arranged in chains or sometimes in heads. Sexual morphs are not known for this taxon. Phylogenetic analysis of DNA sequences from five loci (nucSSU, ITS, nucLSU, mitSSU, rpb1 and rpb2) of this and related fungi supports the introduction of a new family, Sclerococcaceae, for which we establish the order Sclerococcales. This order belongs to the new subclass Sclerococcomycetidae, a strongly supported clade within the Eurotiomycetes that is basal to a lineage containing the Chaetothyriomycetidae, Coryneliomycetidae and Eurotiomycetidae. Rhopalophora clavispora fits in this new family and is closely related to an isolate of Fusichalara minuta. The Sclerococcales also encompass marine, lignicolous species of Dactylospora, two species of the lichenicolous genus Sclerococcum, and a lineage comprised of strains from the digestive tracts of Neotropical wood-inhabiting beetles. We confirm that Dactylospora is polyphyletic; the phylogenetic placement of D. parasitica, the generic type, remains unknown.

All that glitters is not Ramularia.

Ramularia is a species-rich genus that harbours plant pathogens responsible for yield losses to many important crops, including barley, sugar beet and strawberry. Species of Ramularia are hyphomycetes with hyaline conidiophores and conidia with distinct, thickened, darkened, refractive conidiogenous loci and conidial hila, and Mycosphaerella sexual morphs. Because of its simple morphology and general lack of DNA data in public databases, several allied genera are frequently confused with Ramularia. In order to improve the delimitation of Ramularia from allied genera and the circumscription of species within the genus Ramularia, a polyphasic approach based on multilocus DNA sequences, morphological and cultural data were used in this study. A total of 420 isolates belonging to Ramularia and allied genera were targeted for the amplification and sequencing of six partial genes. Although Ramularia and Ramulariopsis proved to be monophyletic, Cercosporella and Pseudocercosporella were polyphyletic. Phacellium isolates clustered within the Ramularia clade and the genus is thus tentatively reduced to synonymy under Ramularia. Cercosporella and Pseudocercosporella isolates that were not congeneric with the ex-type strains of the type species of those genera were assigned to existing genera or to the newly introduced genera Teratoramularia and Xenoramularia, respectively. Teratoramularia is a genus with ramularia-like morphology belonging to the Teratosphaeriaceae, and Xenoramularia was introduced to accommodate hyphomycetous species closely related to Zymoseptoria. The genera Apseudocercosporella, Epicoleosporium, Filiella, Fusidiella, Neopseudocercosporella, and Mycosphaerelloides were also newly introduced to accommodate species non-congeneric with their purported types. A total of nine new combinations and 24 new species were introduced in this study.

Occurrence of Leaf Blight on Cosmos Caused by Alternaria cosmosa in Korea.

In 2011, a leaf blight disease was observed on cosmos (Cosmos bipinnatus) leaves in Nonsan, Korea. The causal pathogen was isolated and identified based on morphological and molecular approaches. Morphological characteristics of the pathogen matched well with the Alternaria cosmosa and also easily distinguishable from Alternaria zinniae reported from cosmos seeds by producing branched beak. Phylogenetically, the pathogen could not be distinguished from A. passiflorae based on the sequence analysis of a combined data set of Alt a1 and gpd genes. However, A. passiflorae was distinguished from the present species by having conidiophores with 4 to 5 conidiogenous loci. The results indicate that the present Alternaria species is A. cosmosa. Pathogenicity tests revealed that the isolate was pathogenic to the leaves of Cosmos bipinnatus. This is the first report of Alternaria blight disease caused by A. cosmosa on cosmos in Korea.

Curviclavula, a new genus of anamorphic Helotiales (Leotiomycetes) isolated from air

A new genus Curviclavula, with C. anemophila as the type species, is established to accommodate a hyphomycetous anamorph isolated from a culturable air sample collected outdoors in New Mexico, USA. The fungus is morphologically distinct in having 1–5 times dichotomously branched conidiophores and integrated, terminal or intercalary, indeterminate, irregularly sympodial conidiogenous cells with 1–2 flattened, inconspicuous, non-protuberant, and neither thickened nor darkened conidiogenous loci. The conidia are variable in shape, occasionally didymosporous or phragmosporous, but usually minutely cheiroid at first and composed of a subcylindrical to obconical, often inflated, straight or curved basal cell that bears a subglobose lateral cell and a short row of 2–4 cells. The apical cell of the developing conidium curves and fuses with the lateral and adjacent cells once in contact to eventually forming a tightly appressed, subglobose to broadly clavate conidium. Conidial secession is schizolytic, but the conidial basal cells often show minute marginal frills and darkened hila. Phylogenetic analyses using DNA sequences from the nuclear ribosomal large subunit and internal transcribed spacer region also supported Curviclavula as distinct and placed it within the Helotiales (Leotiomycetes, Ascomycota) with strong affinity to the family Hyaloscyphaceae.

Reassessment of the Lichenicolous Hyphomycete GenusTalpapellis

Heuchert, B., Zhurbenko, M. P. & Braun, U. 2014. Reassessment of the lichenicolous hyphomycete genus Talpapellis. — Herzogia 27: 83–92.Based on a re-examination of type material of Talpapellis peltigerae, type species of the monotypic genus Talpapellis, conidiogenesis and structure of conidiophores and conidia have been reassessed, which led to a confirmation of Talpapellis as a separate, recognized lichenicolous genus with superficially Cladosporium-, Fusicladium- and Pseudocladosporium-like morphology. Owing to similar structures of conidiogenous loci and conidial hila as well as the characteristic structure of the walls of conidiophores and conidia, the lichenicolous genus Verrucocladosporium is considered to be morphologically similar to and confusable with Talpapellis, but differs with regard to proliferation of conidiophores and formation of conidiogenous cells. Therefore, Talpapellis and Verrucocladosporium are maintained as separate genera. The descriptions of Talpapellis and T. peltigerae are amended, and the new variety T. peltigerae var. rossica is introduced, illustrated and discussed, supplemented by a key to the recognized taxa of Talpapellis and Verrucocladosporium.