Centraalbureau Voor Schimmelcultures Research Articles

First report of Colletotrichum incanum causing leaf spots on common bean in Europe (Slovenia).

A Phaseolus vulgaris L. leaf showing necrotic spots was collected in an experimental bean field in central Slovenia in August 2021. The field contained diverse common bean lines sourced from genebank collections, with each line represented by 10 plants. While symptomatic leaves were seen across various lines, the reported species derived exclusively from a Huasca Huallaga Colorado plant (single-seed descent, USDA accession PI153714, doi: 10.18730/H7P9N), a Peruvian landrace. After incubating the leaf for 2 d at ambient temperature in a moist chamber, setose acervuli developed producing curved, distally tapering and proximately truncated conidia. Single-spore cultures developed equally-shaped conidia measuring 14.5-21.5 (avg. 18.5) × 3-4 (avg. 3.5) μm (n=60) on corn meal agar when mounted in lactic acid. Obtained morphological characters and sequences of the partial actin (GenBank accession, OR208162), beta-tubulin (OR208164), and histone 3 (OR208165) gene identified the isolate as Colletotrichum incanum H.-C. Yang, J.S. Haudenshield & G.L. Hartman. Sequences were identical to those from CBS 133485 (= NRRL 62592, IL6A), ex-type strain of C. incanum (KC110823, KC110814, and KC110796). Partial sequences of the chitin synthase (CHS) gene (OR208163), not available for the ex-type strain, was identical to sequences of other C. incanum strains reported from China (KP145539, ON189040, and OQ613679-OQ613686) or differed in two nucleotide positions (OL471268 and OL471269). The strain from Slovenia was deposited in the CBS biobanks of the Westerdijk Fungal Biodiversity Institute (Utrecht, The Netherlands) as CBS 150848. Pathogenicity of the strain was tested by spraying ca. 3×105 conidia as a watery spore suspension onto each leaf of 6 greenhouse-grown and 3 wk-old common bean plantlets (cv. KIS Amand). Nonsterile commercial substrate (Potgrond H, AGRO-FertiCrop) was used and natural light conditions at ambient temperatures (18-23°C) applied. Sterile water was sprayed on 6, equally grown negative control plants. Treated plants showed small brownish spots after 3 wks similar to those described by Yang et al. (2014) on soybean. Setose acervuli formed within 5 days after detached leaves were incubated in moist chambers. No acervuli formed on negative control plants. Conidia re-isolated from these acervuli and obtained cultures were morphologically identical to originally obtained conidia and cultures and those used for performing the pathogenicity test. Anthracnose is an important disease of common bean attributed to various races of C. lindemuthianum (Sacc. & Magnus) Briosi & Cavara (Nunes et al. 2021). Reporting an additional agent potentially able to cause diseases in common bean and so far not known to occur in Europe is of high relevance as the various genetic bean lines used in Europe may show alternative susceptibility levels to it. However, symptoms caused by C. incanum seem to be less severe as those caused by C. lindemuthianum and the species belongs to the C. spaethianum species complex, whose members have so far not been considered as pathogens of economic importance (Talhinhas & Baroncelli 2021). Yang et al. (2014) based C. incanum on isolates from soybean petioles (USA) and associated it with common bean by re-identifying strain ATCC 64682 obtained by Tu (1990) in Canada. Database queries revealed that it was encountered also on sugar beet (USA; Hanson et al. 2023) and on various crop hosts in China (e.g., chili; Diao et al. 2017), but not in Europe. The work was funded by the Ministry of Agriculture, Forestry and Food and conducted as part of research programs P4-0072 and P4-0431, financed by the Slovenian Research and Innovation Agency ARIS, and the Horizon 2020 project INCREASE funded by the European Union.

First Report of Globisporangium (Pythium) mastophorum causing Damping-off / Root Rot on Parsley in Slovenia.

Pythium-like species cause damping-off symptoms of various hosts, including umbelliferous crops. In April 2023, parsley plantlets (Petroselinum crispum), showing stunted growth, yellowing, decayed roots and damping-off, were obtained from a nursery in central Slovenia, where parsley was grown in polystyrene trays in a greenhouse. Nearly 30% of plants were symptomatic. Sampled roots of ten plants contained ornamented oogonia (avg. 33.3 ± 1.4 µm in diam) with conical projections (5.2 ± 0.5 µm long) (Figure S1 A, B) in microscopically analyzed squash mounts. The pathogen was isolated from root pieces treated for surface disinfection with 0.5% sodium hypochlorite for 30 s, and washed with sterile water. Four 1-2 mm root pieces were taken from each of 10 plants, plated on the selective medium P5ARP, and incubated at 21 °C. Mycelia emerging from root pieces were transferred to carrot piece agar (CPA). Twenty-two equally looking oomycetous colonies were obtained; all sampled plants were infested. Oogonia formed by all colonies were similar to those observed on decayed roots and suggested that Globisporangium (Pythium) mastophorum is the causal disease agent. Analyses of partial β-tubulin (Kroon et al. 2004) and mitochondrial cytochrome c oxidase I (COI) gene sequences (Robideau et al. 2011) confirmed the identification. Obtained COI (Genbank accession number OR725417) sequence was 100% identical to that from G. mastophorum strain CBS 375.72 (EU350523), whereas the β-tubulin sequence (OR725416) corresponded to 99.6 % pairwise identity (KJ595502). Further, pathogenicity of an obtained isolate was tested on 4 wk-old curly leaf (cv. Petra F1) parsley. Half of a 7 d-old CPA culture, consisting of mycelium and oogonia, was finely cut and mixed with ca 50 ml of nonsterile commercial substrate (Potgrond H, AGRO-FertiCrop) in each of six 400 ml pots. Pots were filled with ca 300 ml additional substrate, into which 5 parsley seedlings were planted. Control plants were treated equally but with sterile CPA. Plantlets were watered with sterile tap water and held at ambient light conditions and temperature (night 18 °C - day 23 °C). After 14 d, inoculated plants started wilting and yellowing and showed stunted growth. After 21 d, roots were severely decayed and the seedlings damped-off (Figure S1 C). Four pieces each from 10 decayed roots were plated. Thirty-one pieces revealed pythium-like colonies. Obtained isolates were morphologically identical to the strain used for inoculation and identified as G. mastophorum. Control plants developed no foliar or root symptoms and no pythium-like species was obtained. Agricultural advisors observed occurrence of parsley damping-off also in other nurseries in Slovenia what may lead to spreading the pathogen to parsley in production fields and private gardens. The case emphasizes the need for implementing phytosanitary measures in order to eliminate primary inoculum. Reports from field-infected plants showed that G. mastophorum is a pathogen of parsley in Australia (Petkowski et al. 2013) and the USA (Tsuchida et al. 2018), and celery in the Czech Republic (Šafránková and Holková 2017). Others isolated G. mastophorum from parsley in The Netherlands (online database of the Westerdijk Fungal Biodiversity Institute, strain CBS 243.86). However, the here described case is, to the best of our knowledge, one of the rare documentations of damping-off due to G. mastophorum in Europe (Šafránková and Holková 2017) and the first in Slovenia. Funding: The work was funded by the Ministry of Agriculture, Forestry and Food of Slovenia, and Slovenian Research and Innovation Agency (ARIS Programs P4-0431 and P4-0072).

Cytospora: an important genus of canker pathogens

Cytospora species have commonly been reported as important plant pathogenic fungi with wide host ranges and geographic distributions. With the increase in the number of cryptic species being described, a comprehensive global taxonomic revision of the genus Cytospora is required. The present study includes 399 isolates from 32 countries. These isolates were subjected to DNA sequence analysis for five genomic loci (ITS, act1, rpb2, tef1-α and tub2). Based on these data, it could be confirmed that Cytospora, Leucostoma, Valsa, Valsella and Valseutypella are congeneric. Furthermore, 111 species of Cytospora could also be reassessed, 44 species and four combinations newly introduced, and new typifications proposed for a further three species. Three asexual morphological groups (including 13 asexual morphological types) and three sexual morphological groups (including eight sexual morphological types) were designated. The present study explored the species diversity of Cytospora and re-evaluated the identity of all cultures in the Westerdijk Fungal Biodiversity Institute (Utrecht, The Netherlands) that were deposited as either Cytospora or as one of its related genera. This is the most comprehensive phylogenetic analysis thus far conducted on Cytospora and the results contribute to an increased understanding of the taxonomy of these important fungi. It is also hoped that the findings will lead to improved management strategies for diseases associated Cytospora species.

First Report of Diplodia intermedia Causing Canker and Dieback Diseases on Apple Trees in Canada.

A dieback of apple trees (Malus domestica (Suckow) Borkh.) associated with cankers was observed in commercial orchards in southwestern Ontario, Canada, in 2019. Fifteen 2 to 10-year-old symptomatic trees were collected from three orchards exhibiting up to 37% disease incidence. Small sections of diseased wood (1 cm long) were surface sterilized with 70% ethanol for 30 sec and 1% NaClO for 20 min, rinsed thrice in sterile water, placed on 2% PDA (Difco) amended with kanamycin (50 mg liter-1), and incubated at 22°C for 5 days in the dark (Ilyukhin et al. 2023). Fungal colonies that were consistently isolated were hyphal-tipped, transferred to individual PDA plates and incubated at 22°C for 7 days in the dark. Purified isolates with same characteristics were classed into morphotypes. One morphotype was initially white and turned dark olivaceous with dense aerial mycelium. Pycnidia were produced on pine needles on PDA (Fig. S2) after incubation at 22°C for 17 days in the dark. Conidia were brown, aseptate, ovoid, and measured 27.9 to 31.3 μm x 12.1 to 14.2 μm (mean ± S.D. of 15 conidia = 29.9 ± 0.9 μm × 13.2 ± 0.6 μm), the typical morphology of a Diplodia sp. (Phillips et al. 2012). Genomic DNA was extracted from a 7-day-old culture of a representative isolate M45-28, using the Plant/Fungi DNA Isolation Kit (Norgen Biotech, Canada). The internal transcribed spacer (ITS), translation elongation factor 1-α (EF1-α) and β-tubulin gene regions were amplified and sequenced with primers ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b and deposited in GenBank with accession numbers MZ970605, MZ995430 and MZ995431, respectively. Based on the sequence, the fungus was identified as Diplodia intermedia A.J.L. Phillips et al. and matched isolates from different hosts and countries (ITS: 100%, MG220378; EF1-α: 100%, MG220385; β-tubulin: 99.24%, MT592502). The maximum likelihood-based phylogenetic analysis of ITS, EF1-α and β-tubulin concatenated sequences was performed using IQ-Tree 2.2.2.7 (Minh et al. 2020). M45-28 was clustered with high bootstrap support values with D. intermedia isolates from the Westerdijk Fungal Biodiversity Institute collection, including the ex-holotype (CBS 124462) (Fig. S1). A living culture of M45-28 was deposited in the Canadian Collection of Fungal Cultures (DAOMC 252253). Pathogenicity assay was conducted by inoculating mycelial plugs from a 7-day-old culture of M45-28 into wounds made on the trunk of 5 eight-month-old potted healthy 'Royal Gala' apple seedlings. Five control seedlings were inoculated with sterile plugs. Canker symptoms appeared 15 days after inoculation, spread around, up and down the main stem from the inoculation point, and by 7 weeks the upper portion of the seedling was dead (Fig. S2). Diplodia intermedia was re-isolated from all inoculated seedlings and species identity was confirmed by sequencing as described above, fulfilling Koch's postulates. Control seedlings remained symptomless and the fungus was not isolated from the wood. Diplodia intermedia was reported to cause cankers on apple in Uruguay (Delgado-Cerrone et al. 2016), wild apple (Malus sylvestris) in Portugal (Phillips et al. 2012), grapevines in France (Comont et al. 2016) and forest trees in Iran (Kazemzadeh Chakusary et al. 2019). To the best of our knowledge, this is the first report of D. intermedia causing canker and dieback diseases on apple trees in Canada. Further studies are required to better understand the epidemiology involved in the dynamic spread of the disease in order to recommend an adequate phytosanitary program for its control.

First report of Neofusicoccum parvum causing stem blight and dieback of highbush blueberry in the Czech Republic.

Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips is a cosmopolitan pathogen causing dieback of multiple diverse woody hosts including highbush blueberry (Vaccinium corymbosum L.). This fungus can survive inside colonized plants without causing any symptoms for several years. Once the endophytic lifestyle is switched to the parasitic one, the symptoms of dieback can rapidly occur (bronze leaves, necroses under the bark, apoplexy) and the plant usually declines within a few weeks (Slipper and Wingfield 2007). In August 2022, blueberry plants displaying symptoms described above were observed in a production orchard located in Hovorany, the Czech Republic. Around 3 % of 1000 observed plants were symptomatic. In order to identify the pathogen, leaves, stems and roots of three diseased plants were collected, sectioned into small pieces (5 × 5 mm), surface sterilized (60 s in 75% ethanol, followed by 60 s in 1% sodium hypochlorite and rinsed three times using sterile distilled water), plated on potato dextrose agar (PDA) supplemented with 0.5 g/liter of streptomycin sulfate (PDAS) (Biosynth, Staad, Switzerland) and incubated at 25°C for 2 weeks at dark. Newly developed mycelia were immediately transferred to fresh PDA plates and purified by single-spore or hyphal-tip isolation. In total 33 fungal isolates were obtained. All the 33 isolates shared similar morphology and resembled Botryosphaeriaceae spp. Colonies on PDA (7 d at 25°C) were felty, white to iron grey in the centre. Conidiomata were observed on sterile pine needles on 2 % water agar (WA) at 25°C under near-UV light after 2 wks (110-220 × 60-175 μm). Conidia (n=30) were cylindrical to ellipsoidal, hyaline, 0(-1)-septate, (3.8-8.1 × 2-3 μm). Two representative isolates were deposited at the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands (CBS 149846 and CBS 149847). The partial internal transcribed spacer (ITS) regions, beta-tubulin gene (tub2) and translation elongation factor 1-alpha (tef) gene were amplified from genomic DNA of both isolates following primers and protocols previously described (Eichmeier et al. 2020). Newly generated sequences were deposited in NCBI GenBank (acc. nos. ITS: OQ376566, OQ376567; tub2: OQ401701, OQ401702 and tef: OQ401699, OQ401700), being >99% identical (ITS 483/484 nt, tub2 426/430 nt and tef 230/232 nt) with the ex-type ITS (AY236943), tub2 (AY236888) and tef (AY236917) sequences of N. parvum strain CMW9081. Phylogenetically, newly obtained isolates grouped with ex-type and another three cultures of N. parvum in the three gene-based phylogenetic tree with strong 98/1.0 (BP/PP) support. To confirm pathogenicity, one-year-old canes of ten two-year-old V. corymbosum plants grown in pots were wounded by a 5 mm diam cork borer, and a 5-mm mycelial plug of a 7-day-old culture of both (CBS 149846 and CBS 149847) strains (five plants per strain) was inserted into the wound. Ten plants were inoculated with sterile PDA plugs and served as controls. Wounds were covered by sterile wet cotton, sealed with Parafilm® and inoculated plants were maintained in a growth chamber at 20 °C with 12/12 h light/dark period. Within two weeks, inoculated shoots changed colour from green to dark brown and exhibited dark necroses under the bark; after one month inoculated plants declined, while controls remained symptomless. The pathogen was reisolated from the inoculated plants with 100 % re-isolation rate, and its identity confirmed by sequencing ITS region. The experiment was repeated. Neofusicoccum parvum causing dieback of highbush blueberry was already reported from Australia, California, Chile, China, Italy, Mexico, Portugal and Uruguay (Rossman et al. 2023). Pecenka et al. (2021) reported a presence of another pathogen - Lasiodiplodia theobromae (Pat.) Griffon & Maubl. from the same plantation. This suggests that stem blight and dieback of highbush blueberry is caused by more than one Botryosphaeriaceae spp. as it was previously proved by Xu et al. (2015). To the best of our knowledge, this is the first report of stem blight and dieback of highbush blueberry caused by N. parvum in the Czech Republic.

First report of Sclerotinia sclerotiorum causing Sclerotinia wilt of hop (Humulus lupulus) in Poland.

The common hop (Humulus lupulus L., Cannabaceae) is a perennial plant cultivated in the temperate climate zone and used in the brewing and pharmaceutical industry. In June 2021, symptoms of wilting and subsequent drying of shoots were observed on hop plants (cv. Lubelski) in Lubelskie Province, in Poland (50°55'30.5"N, 22°10'35.4"E). Wilted shoots showed no symptoms of chlorosis. Usually both healthy and wilted shoots were present on the same plant. Approx. 20% of the plants in the 2 hectare hop garden showed these symptoms. An inspection of the underground parts of the infected shoots revealed a brown necrosis of the tissues partly covered by white mycelium with black sclerotia. These symptoms were characteristic for Sclerotinia sclerotiorum (Lib.) de Bary (Bolton et al. 2006), but this pathogen has never been detected in hop gardens in Poland before. In order to confirm the preliminary diagnosis, shoots from five different plants with symptoms of necrosis were collected, disinfected with 1% sodium hypochlorite for 1 min, rinsed with sterile water and dried. Twelve fragments from partially necrotized tissue were placed on potato dextrose agar (PDA) amended with chlortetracycline hydrochloride. After three days of incubation at room temperature and daylight, 75% of the explants had white or light grey mycelia present. Pure cultures were obtained by transplanting mycelium to fresh PDA. Within 4 days, the mycelium has reached the diameter of Petri dishes (90 mm). On PDA at room temperature and daylight, black spherical or cylindrical sclerotia formed after 6-14 days. Sclerotial size was 1.0 to 10.0 x 1.0 to 5.0 mm (average 2.8 x 2.0 mm; n = 57 from 3 plates). Five isolates were subjected to DNA extraction. Then ITS region was amplified and sequenced using primers ITS1/ITS4 (White et al. 1990). The sequences obtained from the five fungal isolates were identical with each other and BLAST analysis revealed also 100% identity with the GenBank records of S. sclerotiorum (e.g. KT224645, Baturo-Ciesniewska et al. 2017). One representative sequence of isolate Ss5HL was deposited to GenBank (OQ998981). Pathogenicity of the isolate Ss5HL was confirmed in inoculation tests on approx. 30 cm high, young shoots of hop plants (cv. Lubelski) grown from rootstock for 4 weeks in the greenhouse. Five plants were inoculated by placing mycelial discs from a 7-day-old culture on shoots and covering them with wet sterilized cotton pads and aluminum foil. Two control plants were treated in the same way but instead of mycelial discs pure agar discs were placed on the shoots. Then plants were kept in a climate chamber (80% relative humidity, 15-h light at 22°C, 9-h dark at 18°C). All fungal inoculated shoots wilted within 3-4 days, then necrosis developed and spread from the place of inoculation up the shoot. No tissue necrosis was observed below the place of inoculation. The control plants remained asymptomatic. The inoculation test was carried out twice with the same result. Fungal cultures reisolated from the inoculated shoots were morphologically identical with the culture used as inoculum. Isolate Ss5HL was deposited in the collection of the Westerdijk Fungal Biodiversity Institute (CBS 150077).To our knowledge, this is the first case of Sclerotinia wilt of hops in Poland and also in Europe, with the exception of one, nearly 100 years old report from the United Kingdom (Salmon and Ware 1936). S. sclerotiorum rarely occurs on hop but it can cause severe yield reduction if pathogen accumulates in the soil.

First report of Stemphylium leaf blight of onion (Allium cepa) caused by Stemphylium vesicarium in Italy.

In July 2019, severe leaf symptoms were observed on onion plants (Allium cepa L. cv. Dorata di Parma) in a commercial field located in the municipality of Medicina (Bologna, Emilia-Romagna region), in northern Italy. Diseased leaves showed yellowish-pale-brown and oval-shaped lesions, which later coalesced in larger necrotic areas, and black leaf tips. As the disease progressed, conidia developed on the necrotizing leaves, until premature desiccation of the whole plants. Disease incidence of approximately 70% was calculated in the affected field, together with yield losses that were estimated to be above 30%. Symptomatic tissue fragments excised from the leaf lesions were surface disinfested with NaOCl 1% for 2 minutes, rinsed with sterile water and transferred onto potato dextrose agar (PDA). Fungi were consistently isolated after 5 days of incubation at 27 ± 1°C in the dark. Single spore isolation was performed on PDA to obtain 7 pure cultures, whose morphological characteristics were consistent with the description of Stemphylium vesicarium (Ellis 1971). DNA from a representative single spore isolate was extracted and the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) was amplified using the universal primers P-ITS1 and P-ITS4 (White et al. 1990). The PCR product was sequenced and deposited in GenBank (Accession No. OP144057). A BLAST search in CBS-KNAW collection bank (Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands) showed 100% identity for the ITS gene with the strain of S. vesicarium under accession number CBS 124749. Moreover, the PCR assay using the primer pair KES 1999 and KES 2000 (Graf et al. 2016) for the cytochrome b gene displayed the specific fragments of 420 bp for S. vesicarium. The isolate was tested for pathogenicity on onion (potted plants cv. Texas Early Gran, fourth leaf stage) by spraying 4 ml of a conidial suspension (1 × 104 conidia/ ml) per plant. Inoculated and non-inoculated (sprayed with sterile distilled water) plants were kept at 24 ± 1°C and 90% relative humidity with a 16-h photoperiod. Seven days after inoculation, disease assessment was performed. Inoculated plants showed typical Stemphylium leaf blight (SLB) symptoms, similar to those observed in the field. No symptoms developed on the water-inoculated plants. S. vesicarium was consistently reisolated from the artificially inoculated onion plants and identified using a PCR assay, according to Graf et al. (2016). The assay was repeated twice with the same results. SLB is currently reported worldwide and it is considered a re-emerging threat and a truly challenging fungal disease, which can result in yield and quality losses of up to 90% in onion crops (Hay et al. 2021). In Italy, S. vesicarium has been reported several years ago on pear (Ponti et al. 1982) and, more recently, on radish sprouts (Belisario et al. 2008), chili pepper (Vitale et al. 2017) and spinach (Gilardi et al. 2022). To our knowledge, this is the first report of S.vesicarium on onion in Italy. Our results stress that development and implementation of innovative Integrated Pest Management (IPM) strategies are urgently needed to ensure an effective control of SLB, since only a few moderately resistant onion varieties are available (Hay et al. 2021) and no fungicides are currently registered to specifically control SLB in Italy. Further studies are underway to elucidate the pathogen geographic distribution and assess the impact of this disease on the onion crop in Italy.

First Report of Alternaria alternata Causing White Leaf Spot on Allium tuberosum in China.

Puding County is the major Allium tuberosum growing area in Guizhou Province of China. In 2019, white leaf spots were observed on Allium tuberosum in Puding County (26.31°N, 105.64°E). The white spots, ranging from elliptic to irregular in shape, first appeared on leaf tips. With disease aggravation, spots gradually coalesced, forming necrotic patches with yellow margins causing leaf necrosis; sometimes there was gray mold on dead leaves. The incidence of the diseased leaf rate was estimated to be 27-48%. To identify the pathogenic agent, 150 leaf tissues (5 mm × 5 mm) were obtained from disease-healthy junctions of 50 diseased leaves. Leaf tissues were disinfected in 75% ethanol for 30 s, soaked in 0.5% sodium hypochlorite for 5 min, and flushed three times with sterile water, before being placed on potato dextrose agar (PDA) in the dark at 25 °C. When colonies appeared, the mycelial tips were picked and placed on new PDA. Purified fungus was obtained after repeating this last step several times. The colonies were grayish-green with white round margins. Conidiophores (2.7-4.5 μm × 27-81 μm) were brown, straight, or flexuous with branches and septa. Conidia (8-34 µm × 5-16 µm) were brown, with 0-5 transverse septa and 0-4 longitudinal septa. The 18S nuclear ribosomal DNA (nrDNA; SSU), 28S nrDNA (LSU), RNA polymerase II second largest subunit (RPB2), internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor 1-alpha (TEF-α) (Woudenberg et al. 2013) were amplified and sequenced. The sequences were deposited in GenBank (ITS: OP703616, LSU: OP860684, SSU: OP860685, GAPDH: OP902372, RPB2: OP902373, TEF1-α: OP902374). According to BLAST analysis, the ITS, LSU, GAPDH, RPB2, SSU, and TEF1-α of the straishowed 100% (689 of 731 base pairs; bp), 100% (916 of 938 bp), 100% (579 of 600 bp), 100% (946 of 985 bp), 100% (1093 of 1134 bp), and 100% (240 of 240 bp) sequence identity to those of Alternaria alternata (ITS: LC440581.1, LSU: KX609781.1, GAPDH: MT109295.1, RPB2: MK605900.1, SSU: ON055699.1 and TEF1-α: OM220081.1). A phylogenetic tree was constructed using PAUP4 and the maximum parsimony method with 1000 replicas of bootstrapping for all datasets. According to morphological characteristics and phylogenetic analysis, FJ-1 was identified as Alternaria alternata (Simmons 2007, Woudenberg et al. 2015). The strain was preserved in the Agricultural Culture Collection of China (preservation number: ACC39969). To determine the pathogenicity of Alternaria alternata against Allium tuberosum, wounded healthy leaves were inoculated with a conidial suspension (106 conidial/mL) and round mycelial plugs (4mm). Sterile agar PDA plugs with no mycelium or sterile water were inoculated as negative controls. Three days later, white spots appeared on the wounded leaves inoculated with mycelial plugs or conidial suspension. However, the symptoms caused by conidial suspensions were weaker than those caused by mycelial plugs. No symptoms were observed in the control group. The experimental symptoms were consistent with the phenomena observed in the field. The same fungus was reisolated from necrotic lesions and identified as Alternaria alternata using the method described above. To our knowledge, this is the first report of Alternaria alternata causing white leaf spots on Allium tuberosum in China, a disease seriously affected the yield and quality of Allium tuberosum and caused economic losses to farmers. Reference: Simmons EG (2007) Alternaria: an identification manual. CBS Fungal Biodiversity Centre, Utrecht, the Netherlands. Woudenberg JHC, Groenewald JZ, Binder M, Crous PW ( 2013) Alternaria redefined. Stud Mycol, 75: 171-212. https://doi.org/10.3114/sim0015. Woudenberg JHC, Seidl MF, Groenewald JZ, Vries M de, Stielow JB, Thomma BPHJ, Crous PW (2015) Alternaria section Alternaria: Species, formae speciales or pathotypes? Stud Mycol, 82:1-21. https://doi.org/10.1016/j.simyco.2015.07.001.

Revising Clonostachys and allied genera in Bionectriaceae.

Clonostachys (Bionectriaceae, Hypocreales) species are common soil-borne fungi, endophytes, epiphytes, and saprotrophs. Sexual morphs of Clonostachys spp. were placed in the genus Bionectria, which was further segregated into the six subgenera Astromata, Bionectria, Epiphloea, Myronectria, Uniparietina, and Zebrinella. However, with the end of dual nomenclature, Clonostachys became the single depository for sexual and asexual morph-typified species. Species of Clonostachys are typically characterised by penicillate, sporodochial, and, in many cases, dimorphic conidiophores (primary and secondary conidiophores). Primary conidiophores are mononematous, either verticillium-like or narrowly penicillate. The secondary conidiophores generally form imbricate conidial chains that can collapse to slimy masses, particularly on sporodochia. In the present study, we investigated the species diversity within a collection of 420 strains of Clonostachys from the culture collection of, and personal collections at, the Westerdijk Fungal Biodiversity Institute in Utrecht, the Netherlands. Strains were analysed based on their morphological characters and molecular phylogeny. The latter used DNA sequence data of the nuclear ribosomal internal transcribed spacer regions and intervening 5.8S nrDNA (ITS) and partial 28S large subunit (LSU) nrDNA and partial protein encoding genes including the RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-alpha (TEF1) and β-tubulin (TUB2). Based on these results, the subgenera Astromata, Bionectria, Myronectria and Zebrinella are supported within Clonostachys. Furthermore, the genus Sesquicillium is resurrected to accommodate the former subgenera Epiphloea and Uniparietina. The close relationship of Clonostachys and Sesquicillium is strongly supported as both are inferred phylogenetically as sister-genera. New taxa include 24 new species and 10 new combinations. Recognition of Sesquicillium distinguishes species typically forming a reduced perithecial stroma superficially on plant tissue from species in Clonostachys often forming well-developed, through bark erumpent stromata. The patterns of observed perithecial wall anatomies, perithecial wall and stroma interfaces, and asexual morph diversifications described in a previously compiled monograph are used for interpreting ancestral state reconstructions. It is inferred that the common ancestor of Clonostachys and Sesquicillium may have formed perithecia superficially on leaves, possessed a perithecial wall consisting of a single region, and formed intercalary phialides in penicilli of conidiophores. Character interpretation may also allow hypothesising that diversification of morphs occurred then in the two genera independently and that the frequently stroma-linked Clonostachys morphs evolved together with the occupation of woody host niches and mycoparasitism. Taxonomic novelties: New species: Clonostachys aurantiaca L. Zhao & Crous, Clonostachys australiana L. Zhao & Crous, Clonostachys bambusae L. Zhao & Crous, Clonostachys buxicola L. Zhao & Crous, Clonostachys cylindrica L. Zhao & Crous, Clonostachys ellipsoidea L. Zhao & Crous, Clonostachys flava L. Zhao, Crous & Schroers, Clonostachys fujianensis L. Zhao & Crous, Clonostachys fusca L. Zhao, Crous & Schroers, Clonostachys garysamuelsii L. Zhao & Crous, Clonostachys hongkongensis L. Zhao & Crous, Clonostachys longiphialidica L. Zhao, Crous & Schroers, Clonostachys obovatispora, L. Zhao & Crous, Clonostachys palmae L. Zhao, Crous & Schroers, Clonostachys parasporodochialis L. Zhao & Crous, Clonostachys penicillata L. Zhao, Crous & Schroers, Clonostachys reniformis L. Zhao & Crous, Clonostachys vacuolata L. Zhao, Crous & Schroers, Clonostachys venezuelae L. Zhao, Crous & Schroers, Mycocitrus synnematus L. Zhao & Crous, Nectriopsis didymii L. Zhao & Crous, Sesquicillium intermediophialidicum L. Zhao & Crous, Sesquicillium neerlandicum L. Zhao & Crous, Sesquicillium symmetricum L. Zhao & Crous. New combinations: Mycocitrus coccicola (J.A. Stev.) L. Zhao & Crous, Mycocitrus coxeniae (Y.P. Tan et al.) L. Zhao & Crous, Sesquicillium essexcoheniae (Y.P. Tan et al.) L. Zhao & Crous, Sesquicillium lasiacidis (Samuels) L. Zhao, Crous & Schroers, Sesquicillium phyllophilum (Schroers) L. Zhao, Crous & Schroers, Sesquicillium rossmaniae (Schroers) L. Zhao, Crous & Schroers, Sesquicillium saulense (Lechat & J. Fourn.) L. Zhao & Crous, Sesquicillium sesquicillii (Samuels) L. Zhao, Crous & Schroers, Sesquicillium spinulosisporum (Lechat & J. Fourn.) L. Zhao & Crous, Sesquicillium tornatum (Höhn.) Schroers. New synonyms: Clonostachys aranearum W.H. Chen et al., Clonostachys chuyangsinensis H. Yu & Y. Wang, Clonostachys eriocamporesiana R.H. Perera & K.D. Hyde, Clonostachys granuligera (Starbäck) Forin & Vizzini, Clonostachys indica Prasher & R. Chauhan, Clonostachys spinulosa R.H. Perera et al., Clonostachys squamuligera (Sacc.) Forin & Vizzini, Clonostachys wenpingii (J. Luo & W.Y. Zhuang) Z.Q. Zeng & W.Y. Zhuang. Epitypes (basionyms): Fusidium buxi J.C. Schmidt ex Link, Verticillium candelabrum Bonord. Citation: Zhao L, Groenewald JZ, Hernández-Restrepo M, Schroers H-J, Crous PW (2023). Revising Clonostachys and allied genera in Bionectriaceae. Studies in Mycology 105: 205-266. doi: 10.3114/sim.2023.105.03.

(2952) Proposal to conserve the name Coccidioides posadasii against Posadasia esferiformis, Coccidium posadasi, Pseudococcidioides mazzae, Geotrichum louisianoideum, Glenospora meteuropea, Glenospora metamericana and Trichosporon proteolyticum (Ascomycota)

(2952) Proposal to conserve the name <i>Coccidioides posadasii</i> against <i>Posadasia esferiformis</i>, <i>Coccidium posadasi</i>, <i>Pseudococcidioides mazzae</i>, <i>Geotrichum louisianoideum</i>, <i>Glenospora meteuropea</i>, <i>Glenospora metamericana</i> and <i>Trichosporon proteolyticum</i> (<i>Ascomycota</i>)

First report of Fusarium verticillioides causing Pokkah boeng disease on maize in India.

Maize is a widely grown cereal crop in India and ranks third to wheat and rice in production (https://iimr.icar.gov.in). During a field survey in Kharif season in 2018, foliar chlorosis at the base and middle of leaves, and twisted top symptoms were observed in 40-50 days old maize plants in Belagavi district, Karnataka, India. Again during Kharif season in 2021, similar symptoms were observed on commercial maize hybrids and sugarcane at Agricultural Research Station, Sankeshwar Karnataka. The symptoms resembled Pokkah boeng disease of sugarcane (Vishwakarma et al. 2013). Symptomatic sugarcane and maize leaves were sampled, surface sterilized with 1.0% sodium hypochlorite, and 70% ethanol, and transferred on Potato dextrose agar, incubated for 10 days at 27±1°C. Fungal growth initiated with white mycelium later turned to pinkish-white with hyaline spores. The morphological features and sporulation patterns of maize and sugarcane samples were similar (e-Xtra 1). Microconidia were formed in long chains and clusters with oval to club-shaped, 0-septate, monophialide-borne microspores. DNA from representative pure culture isolates was extracted using the CTAB protocol (Doyle and Doyle, 1990). The ITS region of r-DNA was amplified with ITS1/ITS4 primers and sequenced. BLAST analyses of sequences of maize and sugarcane culture isolates at NCBI database revealed 100% homology with Fusarium verticillioides MK264336 (Lin et al., 2016). PCR amplification with Fusarium verticilliodes specific primers VER1/VER2 (Mule et al., 2004) confirmed the organism. CBS-KNAW Fungal Biodiversity Centre's Fusarium MLST database also revealed over 98.89% homology with Fusarium verticilliodes (NRRL 46612). The fungal isolates were named Fusarium verticilliodes maize isolate SNK 01 (ON110289) and Fusarium verticilliodes sugarcane isolate SNK 01 (ON564879), and their sequences were deposited in the GenBank. To test pathogenicity, artificial inoculation using maize isolate SNK 01 and cross-inoculation of sugarcane isolate SNK 01 were done on ten maize plants by spraying a conidial suspension (2×106 conidia ml-1) on nonwounded leaves. The plants sprayed with sterile water were used as control. After ten days, typical Pokkah boeng symptoms were observed in the plants inoculated with both maize and sugarcane isolates. Diseased leaves turned pale yellowish-green with small brown spots and a chlorotic appearance, further, these developed into stripes (e-Xtra 2). Wrinkling of leaves was noticed followed by splitting and rotting. No symptoms were noticed in the water-treated control. The pathogens re-isolated from diseased plants inoculated with maize and sugarcane isolates were similar morphologically and identical to the original isolates, fulfilling Koch's postulates. Hitherto, Fusarium verticilliodes was known to cause post-flowering stalk rot in maize. However, this is the first report of Pokkah boeng disease on maize in India caused by F. verticillioides. Considering the economic value of the maize crop, this identification can help develop appropriate disease management strategies to control the disease. References Lee, S. B., et al. 1988. A rapid, high yield mini-prep method for isolation of total genomic DNA from fungi. Fungal Genet. Newsl. 35:23-24. Lin, Z., et al. 2016. Deciphering transcriptomic response of Fusarium verticillioides in relation to nitrogen availability and the development of sugarcane Pokkah boeng disease. Sci. Rep. 6, 29692. Mule, G., et al. 2004. A Species-Specific PCR assay based on the Calmodulin partial gene for identification of Fusarium verticillioides, F. proliferatum and F. subglutinans. European J. Plant Path. 110:495-502 Vishwakarma, S.K., et al. 2013. Pokkah Boeng: an emerging disease of sugarcane. J. Plant Pathol. Microb. 4(3):170. https://iimr.icar.gov.in. Director's desk, ICAR-Indian Institute of Maize Research. (Accessed September 8, 2022).

Calophoma clematidina causing leaf spot and wilt on Clematis plants in the Czech Republic.

Clematis L. is one of the largest genera of Ranunculaceae, accommodating over 300 plant species (Wang & Li 2005). They are mostly flowering creepers commonly grown as ornamentals. Clematis leaf spot and wilt is a fungal disease caused by Calophoma clematidina (Thüm.) Q. Chen & L. Cai. Infected plants initially show irregular brown to black leaf spots which later turn into large necroses, usually leading to wilt disease. In June 2021, Clematis plants displaying symptoms described above were observed in three independent nurseries located in three counties (Brno-venkov, Břeclav and Nymburk) in the Czech Republic. Around 60% of 120 inspected plants were symptomatic, including both mother plants and young plants. Leaves, stems and roots of 43 diseased plants originating from the three nurseries were collected, sectioned into small pieces (5 × 5 mm), surface sterilized (60 sec in 75% ethanol, followed by 60 sec in 1% sodium hypochlorite and rinsed three times using sterile distilled water), plated on potato dextrose agar (PDA) and incubated at 25°C for 5 weeks. Newly developed mycelia were immediately transferred to a fresh PDA plates and purified by single-spore isolation. A total of 21 strains morphologically resembled the genus Calophoma. Colonies on PDA (7 d at 25°C) were felty, white to olivaceous/iron grey in the centre. Conidiomata were dark brown, pycnidial, solitary or in groups, (117-220 × 65-170 μm). Conidia were cylindrical to ellipsoidal, hyaline, 0(-1)-septate, (4-8 × 2-3 μm). Two representative isolates were deposited at the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands (CBS 149230 and CBS 149231). The partial internal transcribed spacer (ITS) regions, large ribosomal subunit of the nrRNA gene (LSU), beta-tubulin gene (tub2) and RNA polymerase II second largest subunit gene (rpb2) were amplified from genomic DNA of both isolates following protocols previously described (Spetik et al. 2022). Sequences were deposited in NCBI GenBank (accession nos. ITS: ON107539, ON107540; LSU: ON108575, ON108576; tub2: ON314832, ON314833; rpb2: ON125007, ON125008), being 100% identical with that of the ex-type strain of C. clematidina (CBS 108.79), ITS (NR_135964), LSU (FJ515632), tub2 (FJ427100), and rpb2 (KT389588). Phylogenetically, the two representative isolates formed a fully supported clade with sequences of the ex-type and another culture of C. clematidina in the multigene phylogeny. To confirm Koch's postulates, leaves of ten two-month-old Clematis plants grown in pots were wounded by a needle and inoculated with a conidial suspension (1.0 × 106 conidia ml-1) of both strains (five plants per strain) following Golazar et al. (2011). Ten plants were mock-inoculated with sterile distilled water and served as controls. Within one month, inoculated plants exhibited dark necrotic leaf spots similar to the symptoms observed in the nurseries, while controls remained symptomless. Calophoma clematidina was reisolated from the inoculated plants, and its identity confirmed (ITS, GenBank OP363927). The experiment was repeated. Although known from Europe, this is the first report of Clematis leaf spot and wilt caused by C. clematidina in the Czech Republic. Clematis leaf spot and wilt represents a serious disease in Czech nurseries, with the pathogen present in leaves, stems and roots of Clematis spp.

Dnabarcoder: An open-source software package for analysing and predicting DNA sequence similarity cutoffs for fungal sequence identification.

The accuracy and precision of fungal molecular identification and classification are challenging, particularly in environmental metabarcoding approaches as these often trade accuracy for efficiency given the large data volumes at hand. In most ecological studies, only a single similarity cutoff value is used for sequence identification. This is not sufficient since the most commonly used DNA markers are known to vary widely in terms of inter‐ and intraspecific variability. We address this problem by presenting a new tool, dnabarcoder, to predict local similarity cutoffs and measure the resolving powers of a biomarker for sequence identification for different clades of fungi. It was shown that the predicted similarity cutoffs varied significantly between the clades of a recently released ITS DNA barcode data set from the CBS culture collection of the Westerdijk Fungal Biodiversity Institute. When classifying a large public fungal ITS data set—the UNITE database—against the barcode data set, the local similarity cutoffs assigned fewer sequences than the traditional cutoffs used in metabarcoding studies. However, the obtained accuracy and precision were significantly improved. Our study showed that it might be better to extract the ITS region from the ITS barcodes to optimize taxonomic assignment accuracy. Furthermore, 15.3, 25.6, and 26.3% of the fungal species of the barcode data set were indistinguishable by full‐length ITS, ITS1, and ITS2, respectively. Except for these indistinguishable species, the resolving powers of full‐length ITS, ITS1, and ITS2 sequences were similar at the species level. Nevertheless, the complete ITS region had a better resolving power at higher taxonomic levels.

First report of fruit blight caused by Alternaria alternata on sesame in Northeast China.

Sesame (Sesamum indicum L.) is an important oilseed crop in China and it is also used in food and health products. In August of 2019, a blight sesame fruit was observed in a field of Liaoyang city, Liaoning province of China. Initial disease symptoms consisted of brown or dark brown spots on fruit. With time, lesions coalesced and the whole fruit turned dark brown or black. Most of the diseased fruit had thin and small, deformed, necrotic, hardened cracked epidermal lesions. Lesions were also produced on stem and petioles leading to leaf abscission. The disease results in premature fruit death, and in turn, considerable yield losses. To determine the causal agent, symptomatic fruit with developing lesions were collected, and surface sterilized in 2% NaClO for 3 min, rinsed three times in distilled water, and plated onto PDA medium. After incubation at 25°C for 5 days, a dark olivaceous fungus with abundant, branched, brown to black, and septate hyphae was consistently isolated. Twenty single spores were separated with an inoculation needle under stereomicroscope. The conidia were in chains, brown, obclavate, ovoid or ellipsoid, with 1-6 transverse septa and 0-4 longitudinal or oblique septa 12.5 to 45 × 6.5 to 14.5 μm in size. Conidiophores were septate, light brown to olive brown, measuring 22-60 μm × 2-4 μm. The morphological characteristics of the 20 isolates all matched the description of Alternaria alternata (Simmons, 2007). The internal transcribed spacer (ITS) region of rDNA of 15 isolates was amplified using primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone et al. 1999) and sequenced. Identical sequences were obtained and the sequence of the isolate ZMHG12 was submitted to GenBank (Accession no. MW418181 and MW700316). BLAST analysis of the sequences of the isolates of ZMHG12 showed 100% to A. alternata (KP739875 and LC132712). In pathogenicity tests, a conidial suspension (2.5 × 105 conidia per ml) was prepared from 7 days-old cultures of isolate ZMHG12 grown on PDA at 25°C. Fruit of 10 two-month-old potted sesame plants (Variety "Liaozhi 8") were sprayed with the conidia suspension until runoff. Another 10 plants sprayed with distilled water to served as non-inoculated controls. All plants were maintained for 48 h in a humid chamber with a temperature of 25°C to 26°C, and then moved to a greenhouse. Ten days after inoculation, all fruit of inoculated plants exhibited symptoms similar to those observed in the field and non-inoculated control plants remained symptomless. The experiment was repeated twice with similar results. A. alternata has been reported as a pathogen caused leaf blight disease of sesame in Pakistan (Nayyar et al. 2017). To our knowledge, this is the first report of A.alternata causing fruit blight of sesame in China. To date, we have observed the disease on sesames in fields of Fuxin, Chaoyang and Tieling city in Liaoning Province, and Tongliao city in Inner Mongolia of China, and it has become an important disease in sesame production of China. References : Simmons E. G. 2007. Alternaria: An identification manual. CBS Fungal Biodiversity Center, Utrecht, Netherlands. White T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego. Carbone I., et al. 1999. Mycologia, 91: 553-556. Nayyar, B. G., et al. 2017. Plant Pathology Journal, 33 (6): 543-553.

Intraspecific nucleotide divergence in Saccharomycodes ludwigii, and proposal of Saccharomycodes pseudoludwigii sp. nov, a new apiculate yeast isolated from China.

The six synonyms currently accepted under Saccharomycodes ludwigii were investigated for by phenotypic properties, however, the sequence diversity of the rRNA and protein coding genes have not yet been determined. Nine strains including the type strains of synonyms of S. ludwigii deposited in the CBS yeast collection, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands, were analyzed using a multi-locus sequence analysis (MLSA) approach that included sequences of 18S ribosomal RNA (rRNA), the D1/D2 domains of the 26S rRNA, the ITS region (including the 5.8S rRNA) and fragments of genes encoding the largest subunit of the RNA polymerase II (RPB1 and RPB2) and translation elongation factor 1-α (TEF1). Our results showed that the nine strains have identical D1/D2, 18S and RPB2 sequences and similar ITS, RPB1 and TEF1 sequences, which indicated that they are conspecific. In addition, a novel species of Saccharomycodes, S. pseudoludwigii sp. nov. (type CGMCC 2.4526T) that was isolated from fruit and tree bark in China, is proposed. The MycoBank number of this new species is MB 811,650.

Citizen science project reveals novel fusarioid fungi (Nectriaceae, Sordariomycetes) from urban soils.

Soil fungi play a crucial role in soil quality and fertility in being able to break down organic matter but are frequently also observed to play a role as important plant pathogens. As part of a Citizen Science Project initiated by the Westerdijk Fungal Biodiversity Institute and the Utrecht University Museum, which aimed to describe novel fungal species from Dutch garden soil, the diversity of fusarioid fungi (Fusarium and other fusarioid genera), which are members of Nectriaceae (Hypocreales) was investigated. Preliminary analyses of ITS and LSU sequences from more than 4 750 isolates obtained indicated that 109 strains belong to this generic complex. Based on multi-locus phylogenies of combinations of cmdA, tef1, rpb1, rpb2 and tub2 alignments, and morphological characteristics, 25 species were identified, namely 22 in Fusarium and three in Neocosmospora. Furthermore, two species were described as new namely F. vanleeuwenii from the Fusarium oxysporum species complex (FOSC), and F. wereldwijsianum from the Fusarium incarnatum-equiseti species complex (FIESC). Other species encountered in this study include in the FOSC: F. curvatum, F. nirenbergiae, F. oxysporum and three undescribed Fusarium spp.; in the FIESC: F. clavus, F. croceum, F. equiseti, F. flagelliforme and F. toxicum; Fusarium tricinctum species complex: F. flocciferum and F. torulosum; the Fusarium sambucinum species complex: F. culmorum and F. graminearum; the Fusarium redolens species complex: F. redolens; and the Fusarium fujikuroi species complex: F. verticillioides. Three species of Neocosmospora were encountered, namely N. solani, N. stercicola and N. tonkinensis. Although soil fungal diversity has been well studied in the Netherlands, this study revealed two new species, and eight new records: F. clavus, F. croceum, F. flagelliforme, F. odoratissimum, F. tardicrescens, F. toxicum, F. triseptatum and N. stercicola.

Characterization and Optimization of L-Malic Acid Production by Some Clinical Isolates of Aureobasidium pullulans

Background: Poly-L-malic acid (PLMA) comprises aliphatic polyester polymers with broad applications in pharmaceutical industries. The fungal microorganisms are among the best natural sources recruited to supply L-malic acid (MA) as a precursor of PLMA. In this study, we investigated MA production ability of 7 clinical isolated of the fungus Aureobasidium pullulans. Materials and Methods: Seven clinical isolates of A. pullulans acquired from Westerdijk Fungal Biodiversity Institute were studied, and the isolate with the highest total MA production was selected for the optimization process. We tried to optimize the output by applying different concentrations of CaCO3 in fungus medium (1.5%, 3%, and 6%) and various incubation temperatures (27°C, 32°C, and 37°C) during 3, 7, and 14 days. Results: Intra-strains variation was significantly strong (P&lt;0.0001), and the highest production of MA was carried out by the isolate A. pullulans var. melanigenum dH 21931, UTHSC 06-456. The amount of MA produced by this strain was significantly higher in medium with 3% CaCO3 compared with other concentrations of CaCO3 and after 7 days incubation than the other fermentation times (P&lt;0.05). Although MA production was higher at 27°C, the differences between the investigated various temperatures were not significant (P&gt;0.05). Conclusion: Overall, we obtained the highest MA production in Sabouraud dextrose agar (SDA)medium with 3% CaCO3 at 27°C after 7 days of incubation. Our study indicated that the fermentation period and CaCO3 concentration significantly alter MA production in A. pullulans var. melanigenum.

First report of Boeremia exigua var. exigua causing Black Spot-like symptoms on commercially grown soybean in Germany.

Soybean (Glycine max [L.] Merr.) is economically the most important protein crop grown worldwide. However, Europe largely depends on soybean imported from the Americas (European Commission 2019; Haupt and Schmid 2020). In Germany, soybean production was not formally recorded before 2016, but since then a steady increase along with an expansion of the growing area from the south of Germany to northern states occurred. In 2019 an area of 29,000 hectares was under soybean cultivation (Federal Ministry of Food and Agriculture (Germany) 2019). In the state of North Rhine-Westphalia (NRW, western part of Germany) farmers have started in recent years to cultivate soybean, making it increasingly important to monitor pathogens associated with this new crop. At the beginning of October 2019, shortly before harvest, rows of black spots on pods and stems of soybean plants cv. Viola throughout a field site near Jülich (NRW) were observed. Close observation identified them as pycnidia with similarity to symptoms reported from soybean in Austria in 2015 (Hissek and Bedlan 2016). The collected samples were thoroughly surface sterilized (two washes with 70 % EtOH, a rinse in 0.5 % sodium hypochlorite solution and a final wash in sterile double distilled water) and placed on plates containing potato dextrose agar (PDA) at 22 °C in the dark. Fungal colonies were transferred to malt extract agar plates (MEA) and examined by microscopy. Thus, 34 of 41 isolates looked morphologically similar, producing colonies that appeared dark grey with white aerial mycelium and round to irregular margins. A single spore isolate was generated and designated IPP1903. Spores derived from IPP1903 were unicellular and mostly oblong to cylindrical with a mean width of 2.6±0.3 µm and a mean length of 5.9±0.8 µm (N=50, mean value ± standard deviation). Colonies on MEA were 5.4 to 5.8 cm in diameter after growth for seven days at 20 to 25°C with a photoperiod of 12 h and 3.3 to 3.7 cm in diameter after growth for seven days in the dark at 22°C. These morphological observations led to the conclusion that the isolate may belong to the genus Phoma. To test this hypothesis, we performed a drop test with 5 M NaOH which is used routinely to check for the presence of a genus-specific metabolite. We observed a change in color, indicating a positive test result. The color change was even more pronounced on the plates incubated in the light, further confirming the presence of "metabolite E" (Boerema et al. 2004; Kövics et al. 2014). Next, DNA was extracted and PCR was performed with primers specific for ITS regions (GenBank MT397284), LSU (MT397285), rbb2 (MT414713) or tub2 (MT414712). Sequencing results of PCR products were used to create a combined phylogenetic tree, including sequences published previously (Chen et al. 2015). Our sequencing results together with the morphological observations clearly identified the fungal isolate to be Boeremia exigua var. exigua. The isolate is publicly available in the CBS collection of the Westerdijk Fungal Biodiversity Institute with the accession no. CBS 146730. Koch's postulates were fulfilled by inoculating a spore suspension of the isolate IPP1903 (5x105 ml-1 in 0.05% Tween 20 solution in distilled water) onto healthy primary leaves of twenty 14 days old soybean plants of the cultivar Abelina. While the mock-inoculated plants (inoculated with 0.05% Tween 20 solution in distilled water) stayed healthy, the inoculated plants developed lesions on the leaves after seven days. Six weeks after inoculation the fungus could be reisolated from cuttings of the infected leaves after surface-sterilization. Fungal colonies were confirmed to be B. exigua var. exigua by morphological examination and via NaOH drop test. To our knowledge, this is the first report of B. exigua var. exigua causing disease on commercially grown soybean in Germany.

First record of Septoria rudbeckiae on Rudbeckia in the United Kingdom

Species of Rudbeckia, often known as black-eyed Susan or coneflower, are used as bedding plants in UK gardens. The large, daisy-like flowerheads typically have bright yellow petals and a conspicuous black centre which may be raised into a cone shape. During the summer of 2016, Rudbeckia fulgida var. sullivantii cv. Goldsturm plants, purchased in the preceding May from a nursery in south-east England, were observed with black leaf spots in outdoor beds at the Royal Horticultural Society (RHS) Garden Wisley. Lower leaves exhibited small black lesions in July, which coalesced as the season progressed leading to complete necrosis of the bottom leaves and spotting on higher leaves. Spots showed no halos and became necrotic only later in the season. Flowering did not appear to be reduced. Pycnidia within the leaf spots were epiphyllous, 50-75 μm diameter, with a neck protruding slightly above the leaf surface. Conidia were filiform, 30-60 times 1.5-2 μm, with three septa. A single-spore isolate was obtained on water agar and cultured on potato dextrose agar. Living cultures were deposited in the RHS culture collection held at RHS Garden Wisley (Accession No. RHS454672) and at Westerdijk Fungal Biodiversity Institute, Netherlands (Accession No. CBS145765). The internal transcribed spacer (ITS) region of rDNA, the β-tubulin (Btub) gene and the translation elongation factor 1-alpha (EF1) gene were amplified using the primers ITS4:ITS5, T1:B-Sandy-R, and EF1-728F:EF2, respectively, according to the method by Verkley et al. (5). The DNA amplicons were sequenced (GenBank Accession Nos. MN093336 (ITS), MN105980 (Btub) and MN166626 (EF1)). The ITS sequence differed by one base pair from the only ITS sequence available for Septoria rudbeckiae (JQ677043). No previous sequences were available for comparison for Btub and EF1 and none of the available sequences had more than 90% identity. Pathogenicity was confirmed by spraying Rudbeckia fulgida sullivantii ‘Goldsturm’ plants with a conidial suspension (1 × 106 conidia/ml) prepared from spores from 21-day-old cultures on potato dextrose agar, incubated at 20°C with a 12 hr light/ 12 hr dark cycle. Plants were kept in high humidity for 72 hr after inoculation. After four weeks, black lesions were observed on leaves. Pycnidia and conidia consistent with S. rudbeckiae were found within each lesion. Control plants sprayed with sterilised water showed no symptoms. Septoria rudbeckiae was described from the USA (Ellis & Halsted, 1) where it is now widespread causing disfigurement of Rudbeckia in gardens. Although the fungus has been recorded from a number of different Rudbeckia species, Rudbeckia fulgida var. sullivanti cv. Goldstrum has been recognised in the USA as one of the most susceptible cultivars. Septoria rudbeckiae has also been reported from Canada, Bulgaria and Romania (Farr & Rossman, 2), Turkey (as Septoria sp.; Gumrukcu, 3) and Korea (Park, 4). To our knowledge, this is the first report of S. rudbeckiae in the United Kingdom. The authors would like to thank Jane Renshaw for aid in preparation of fungal cultures.

Multi-locus phylogeny of the genus Curvularia and description of ten new species

Curvularia is a cosmopolitan genus that includes species associated with plants, animals and humans, several of which are of clinical significance. Some of these species are important pathogens of grasses, causing devastating diseases on cereal crops in the family Poaceae. In the present multi-locus study, ex-type and reference strains of Curvularia, as well as several strains deposited in the CBS culture collection of the Westerdijk Fungal Biodiversity Institute, were included. Based on ITS, GAPDH and TEF1 sequences, as well as phenotypic data, ten new species are described and illustrated: C. arcana, C. austriaca, C. canadensis, C. ellisii, C. pseudoclavata, C. pseudoellisii, C. pseudointermedia, C. pseudoprotuberata, C. siddiquii and C. tribuli. Moreover, the new combinations C. cactivora and C. patereae are proposed, and an epitype for C. oryzae-sativae is designated. In addition, illustrations and descriptions are provided for C. cactivora, C. ellisii, C. crassiseptata, C. neergaardii, C. oryzae, C. oryzae-sativae, C. protuberata and C. verruciformis. The description of C. pseudobrachyspora is emended, and its host and distribution records are updated.