Pycnidial Conidiomata Research Articles

Additions to Lentitheciaceae (Pleosporales, Dothideomycetes): Pseudosetoseptoria jiangxiensis sp. nov. from decaying culms of bamboo in China

In terms of morphology, ecology, and phylogeny, Lentitheciaceae species exhibit an enormous diversity. They have a cosmopolitan distribution worldwide, and lead diverse lifestyles. A novel species in Lentitheciaceae, Pseudosetoseptoria jiangxiensis occurring on decaying culms of bamboo in Jiangxi province, China is described and illustrated. Morphologically, Pseudosetoseptoria jiangxiensis has dark brown to black, semi-immersed, carbonaceous, pycnidial conidiomata and hyaline, obovoid, elliptic, or nearly spherical, aseptate conidia with minute guttules. Maximum likelihood and Bayesian inference analyses of combined ITS, LSU, SSU and tef1-α sequence data were used to clarify the phylogenetic affinities of the species.

First report of Diaporthe tulliensis causing leaf spot of betel vine

The betel vine (Piper betle) is grown in protected areas under the humid and shaded conditions required for the plant's growth. This damp, shady environment favours the spread of numerous diseases, particularly leaf spot disease (Chattopadhayay & Maity, 1990). During 2019–2022, diseased betel leaves were collected from the East Khasi Hills and Ri Bhoi districts of Meghalaya, India. The leaves had spots that were irregular in shape and size, light to dark brown and surrounded by a chlorotic yellow halo, which gradually spread to the leaf centre followed by yellowing, drying and drooping of the whole leaf. An area of approximately of 8 m2 was affected and 5–20% of the plants had symptoms, depending on the year. The number of leaf spots increased with temperature and humidity. To identify the causal agent, lesions (5-10 mm in diameter) were cut from ten infected leaves (Figure 1), surface disinfected with 70% ethanol, rinsed three times in sterile distilled water and then plated on potato dextrose agar (PDA) amended with 30mg/ml of streptomycin sulphate. The same colony type was obtained from all leaves which was white producing yellowish-green pigments and the reverse olive green to brown after seven days (Figure 2A-B) and after 30 days of incubation at 30°C, produced semi-immersed black pycnidial conidiomata which exuded pale yellow to black conidial droplets (Figure 2C). The α-conidia were one to two-celled, hyaline, oval to fusiform, guttulate and 5.3 × 2.5 μm (Figure 2D), conidiophores hyaline but no β- and γ-conidia were observed (Li et al., 2022), and conidiogenous cells were hyaline and septate (Figure 2E-F). The identification of the fungus was confirmed by sequence analysis using ITS1 and ITS4 primers (White et al., 1990) (GenBank Accession No. OQ402579). BLAST analysis showed 100% identity with sequences of Diaporthe tulliensis (e.g. MN911384). In phylogenetic analysis, the isolate in the current study clustered with D. tulliensis using the neighbour-joining method, showing 100% identity (Figure 3). To confirm pathogenicity, the isolate was grown in potato dextrose broth at 28°C for seven days, then inoculated onto five healthy betel leaves using pin-prick inoculation with 10 μl spore suspension (1×105 spores/ml). The untreated control did not show any symptoms, however, the inoculated plants had brown necrotic spots followed by yellowing after four days (Figure 4). On PDA, cultures isolated from infected plant leaf lesions exhibited the same morphology as D. tulliensis and similar conidia were discovered during microscopic analysis subsequently satisfying Koch's postulates. To the best of our knowledge, this is the first report of D. tulliensis as a fungal pathogen of betel leaves. Diaporthe tulliensis has been reported to cause leaf spots on coffee in China (Gorg et al., 2020) and on Parthenocissus tricuspidata in Taiwan (Huang et al., 2021), cacao pod rot in Puerto Rico (Serrato-Diaz et al., 2022), and stem canker of kiwifruit in China (Bai et al., 2017) and of jasmine in Taiwan (Hsu et al., 2023). The authors thank the Head of the Department of Botany, North-Eastern Hill University for providing laboratory facilities. The first author is also grateful to the Non-Net Fellowship, New Delhi, for financial support in the form of a research fellowship.

Neocamarosporium aquaticum (Neocamarosporiaceae, Dothideomycetes), a novel fungus from salt marsh habitats

Abstract Neocamarosporium aquaticum, collected from a drift dead stem of a halophyte at the Kench Nature Reserve, Hayling Island, UK, is described as a new species in the family Neocamarosporiaceae, Dothideomycetes. Multi-locus analyses based on LSU, SSU, ITS rDNA, and EF1-α placed the new fungus as a distinct species in the genus Neocamarosporium, a sister to Neocamarosporium aesturinum isolated from seawater. The new species is characterized by the asexual morph with pycnidial conidiomata, doliiform, enteroblastic, annellidic conidiogenous cells, and conidia that are initially hyaline, aseptate, with longitudinal and transverse septa when matured, muriform, and turning brown to dark brown on maturity. Neocamarosporium aquaticum and Ne. aesturinum differ in the colour of the conidia, degree of septation, and conidial measurements.

Dwiroopa punicae Causing Leaf Spot Disease on Pomegranate in Guizhou, China.

Punica granatum 'Tunisia' is widely planted in several areas of Kaiyang County, Guizhou Province, China. Symptoms of leaf spot and fruit rot were observed in April 2020. Infected pomegranate leaves exhibited scattered, oval, yellow-to-brown spots (2-5 mm), and numerous melanized pycnidia were observed in the center of the lesions. All 7-year-old pomegranate trees in a private orchard (Shangyi orchard, E27°09'08", N107°10'58"), exhibited the same symptoms, and 25% of the diseased pomegranate trees had blighted leaves in the late stage. Eighty diseased P. granatum leaves were collected in the orchard. Abundant fruiting bodies were observed on the surface of diseased spots examined using a dissecting microscope. Portions of the symptomatic leaves were surface disinfected with 75% ethanol for 30 s and then in 2% NaClO for 1 min, rinsed three times with sterile distilled water, and transferred onto potato dextrose agar (PDA) at 28°C for 3 weeks. The morphological characteristics of potential pathogens were observed and photographed under a compound light microscope (Zeiss Scope 5) equipped with an AxioCam 208 color camera. Pycnidial conidiomata were erumpent, globose, black, with a central ostiole, and oozed a slimy black conidial mass. Conidiophores were reduced to conidiogenous cells, ampulliform to subcylindrical, hyaline to pale brown, and proliferated percurrently at the apex. Macroconidia were solitary, one-celled, obovoid, truncate at the base, rounded at the apex, dark brown, aseptate, granular, and thick-walled, (11.0-)12.8-18.2(-19.0) × (9.2-)10.0-15.1(-16.0) μm (av: 14.8 × 12.0 μm, n = 30). No meso- or microconidia were observed. In morphology, our fungi were very similar to Dwiroopa punicae K.V. Xavier, A.N. Kc, J.Z. Groenew, Vallad & Crous (Xavier et al., 2019). Primers ITS4 and ITS5 were used to amplify the ITS, LROR and LR5 were for the LSU (White et al. 1900), and fRPB2-5F and fRPB2-7cR for rpb2 (Liu et al. 1999). Phylogenetic analysis based on these three loci also suggested that our strain was D. punicae, since there were 100% match for ITS and LSU and only two base differences in rpb2 gene between our strain and the D. punicae (CBS 143163). The qualified sequences were submitted to GenBank (ITS: MZ816695, MZ816696, MZ816697; LSU: MZ816692, MZ816693, MZ816694; rpb2: MZ802953). To fulfill Koch's postulates, mycelial plugs (5 mm diameter) were used for an inoculation experiment, only because the fungal isolates failed to produce conidia on PDA, and conidia from diseased tissues were also fewer to make the required spore suspension. A total of 20 attached leaves were used from three pots of pomegranate seedlings in the artificial climate chamber (25°C). Each mycelial plugs cut from 1-week-old PDA cultures were placed on one healthy leaf wrapped with a plastic fresh-keeping film. As control, leaves were inoculated with sterile PDA plugs. After 24h, the plugs were removed. The inoculation experiment was performed three times. Symptoms similar to the original were observed at the 7th day after inoculation on all inoculated plant leaves and necrosis occurred in 30-50% of leaf tissue, but control plants remained healthy. Dwiroopa punicae was successfully re-isolated from the inoculated diseased tissues after morphological and phylogenetic analyses. To the best of our knowledge, this is the first report of D. punicae causing leaf spot disease on pomegranate trees in China. This first report of D. punicae in China provides a basis for the diagnosticians and researchers to identify the disease and formulate disease management strategies.

First Report of Stem Canker Caused by Diaporthe tulliensis on jasmine in Taiwan.

Diaporthe species can infect forest trees, ornamentals, and crops, causing root and fruit rots, stem cankers, leaf spots, etc. (Yang et al. 2018). In February 2021, about 10-20% of jasmine plants showing stem canker, foot rot, and wilting were observed in Changhua (24°01'57.7"N 120°34'54.7"E), Taiwan. The diseased plants initially showed chlorosis, leaf drop, and dieback. Sunken lesions were observed on the infected stem and kept expanding gradually. Eventually, plants wilted and black spots formed on the lesions. The margin of healthy and infected tissues of six samples were cut into 4 pieces, disinfected with 10% NaOCl for 30 seconds, rinsed twice in sterilized distilled water for 1 minute, and cultured on water agar at 28℃ under 12 h light / 12 h dark cycle. Hyphae grown out from isolated tissues were sub-cultured on potato dextrose agar (PDA). All tissues grown out of fungi showed similar colony morphology. Two hyphal tips from different tissues were isolated as representatives and deposited in Bioresource Collection and Research Center, Hsinchu, Taiwan, under BCRC numbers FU31566 and FU31567. The colonies on PDA were white to pale gray and produced black pycnidial conidiomata. The two-week-old conidiomata scattered or aggregated in small groups, exuded cream to pale yellow conidial droplets, 0.3-1.1 mm (n=50). The α-conidia were one-celled, hyaline, ovoid to cylindrical with one or two droplets, 3.8-6.3 × 2.5-3.8 μm (n=50). β-conidia were absent. The internal transcribed spacer (ITS), translational elongation factor subunit 1-α (EF1α), and β-tubulin of the two isolates were amplified using primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson, 1995), respectively. The ITS (MZ389113, MZ389114), EF1α (MZ419338, MZ419339), and β-tubulin (MZ408893, MZ408894) sequences of two isolates showed 98.55-98.56% (KR936130), 98.82% (KR936133), and 99.11-99.33% (KR936132) match to those of Diaporthe tulliensis R.G. Shivas, Vawdery & Y.P. Tan ex-type isolate BRIP 62248a (Dissanayake et al. 2017), respectively. Based on the morphological and molecular characters, this fungus is identified as D. tulliensis. To confirm the pathogenicity, the needle-wounded stem bases of eight-month-old cutting jasmine seedlings were inoculated with BCRC FU31566 by two PDA disks with actively grown fungal edges or conidial suspension at the concentration of approximately 2 × 105 conidia/ml. Each method inoculated five seedlings, performed in the greenhouse at 25 ± 2°C. Non-inoculated plants served as control. Two weeks after inoculation, three plants inoculated with PDA disks of the fungal culture showed wilting, and conidiomata formed on the stem base. The same symptoms were observed in one plant inoculated with the conidial suspension 3 weeks after inoculation. By contrast, the controls remained symptomless. Koch's postulates were completed by re-isolating the fungus from the inoculated plant. The re-isolated pathogen showed similar morphology and molecular characteristics to the original. D. tulliensis has been reported to cause cocoa rotted stem in Australia, kiwifruit stem canker in China, and Boston ivy leaf spot in Taiwan (Crous et al. 2015; Bai et al. 2017; Huang et al. 2021; Farr and Rossman 2021). To our knowledge, this is the first report of stem canker on jasmine associated with D. tulliensis in Taiwan. Furthermore, this is the first record of jasmine as a host of D. tulliensis worldwide.

Pyrenochaeta fraxinina as colonizer of ash and sycamore petioles, its morphology, ecology, and phylogenetic connections

Pyrenochaeta fraxinina was first described in 1913 from the state of New York (USA) on petioles of Fraxinus sp. Since then, the species has not been reported from North America and reports from the other regions of the world are very sparse. The results of this study on P. fraxinina are based on the material collected in various regions of Poland from 2012 to 2019. The material comprised 2700 previous year’s leaf petioles of Fraxinus excelsior and 1970 petioles or leaf residues of eight other deciduous tree species. As a result, the occurrence of pycnidial conidiomata of P. fraxinina was confirmed on F. excelsior (3.4% of petioles), F. mandshurica (1.5%), F. pennsylvanica (3.2%), and Acer pseudoplatanus (2.0%). The morphology of the microstructures was described based on the fresh material and compared with the holotype of P. fraxinina. The optimal temperature for the growth of the fungus in vitro was estimated as 20 °C. The analyses based on ITS-LSU rDNA sequences and a protein coding sequence of TUB2 and RPB2 genes showed that P. fraxinina isolates form a well-supported clade in the phylogenetic trees. The species proved to be closely related to Nematostoma parasiticum (asexual morph Pyrenochaeta parasitica), a species occurring on Abies alba in connection with needle browning disease. Interactions between P. fraxinina and the ash dieback pathogen, Hymenoscyphus fraxineus, were analyzed in vivo on ash petioles and in vitro in dual cultures. Among 93 petioles of F. excelsior, for which P. fraxinina conidiomata were detected, 26 were also colonized by H. fraxineus. Mostly, these two fungi occurred separately, colonizing different sections of a petiole. For all dual cultures, both fungi, P. fraxinina and H. fraxineus, showed growth inhibition toward the counterpartner. The role of P. fraxinina as a saprotrophic competitor toward H. fraxineus in ash petioles is discussed.

Occurrence of Leaf Spot Caused by Neodeightonia phoenicum on Pygmy Date Palm (Phoenix roebelenii) in China.

The pygmy date palm (Phoenix roebelenii) is a popular ornamental plant widely cultivated in tropical regions as well as in China. In June 2018, a new leaf spot symptoms were observed on P. roebelenii in several different parks in Zhanjiang City of China. The early symptoms of infected leaves were presented with small, round, pale brown spots. As the size of these spots increased, they coalesced to form larger irregular necrotic lesions surrounded by dark brown edges, which eventually led to leaf wilted and defoliation. A filamentous fungus was consistently isolated from infected leaf samples. Colonies on PDA at 25°C (12 h light/dark) were initially white with abundant aerial mycelium, which turned fluffy and dark olivaceous after one-week culture. Pycnidial conidiomata were black and globose and formed on pine needles in water agar at 25°C (12 h light/dark) after 21 days. Conidiogenous cells were hyaline, cylindrical, holoblastic. The conidia was ovoid to ellipsoid, thick-walled, which was initially hyaline and aseptate, later turned into dark brown and 1-septate with a striate appearance to conidia, 11.6~25.0 μm×9.6~12.0 μm (av. 20.4 μm×10.1 μm). For molecular identification, the partial sequences of internal transcribed spacer (ITS) regions, translation elongation factor (EF-1α) and β-tubulin (TUB) genes of two representative isolates RYCK-1, RYCK-2 were amplified and sequenced using primer pairs ITS/ITS4 (White et al. 1990), EF-688F/EF-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The sequences of the above three loci of the two isolates (accession nos. ITS, OK329968 and OK329969; EF-1α, OK338067 and OK338068; TUB, OK338069 and OK338070) showed 98.4-100.0 % identity with the existing sequences of ex-type culture CBS 122528 of N. phoenicum. A multilocus phylogenetic analysis of the three loci concatenated sequences using the maximum likelihood method showed the isolates that belongs to N. phoenicum. Based on the morphological characteristics and molecular analysis of the isolates, the fungus was identified as N. phoenicum (Phillips et al. 2008). To confirm pathogenicity, five one-year-old potted plants were used for each isolate (RYCK-1 and RYCK-2) and the plants were inoculated by pricking the epidermis of the leaf with a needle. Five leaves of each plant were sprayed with 100 µl of a conidial suspension (1 × 106 conidia/ml) to the wounded surface for each plant. Sterilized distilled water was used as the control and the experiment was repeated. All the plants were incubated at 26 ± 2°C (12 h light/dark) and covered with plastic bags to maintain constant high humidity. After 14 days, all the inoculated leaves showed the same symptoms as those observed in the original diseased plants, but the control plants remained health. The reisolated fungus was identified as N. phoenicum by morphological and molecular characteristics. N. phoenicum is an important pathogen of Phoenix species plants worldwide, which have been reported to cause shoot blights and stalk rots on P. dactylifera and P. canariensis in Greece (Ligoxigakis et al. 2013) and root rot on P. dactylifera in Qatar (Nishad and Ahmed 2020). To our knowledge, this is first report N. phoenicum causing leaf spot on P. roebelenii in China.

Taxonomic and Phylogenetic Insights into Novel Ascomycota from Forest Woody Litter.

Simple SummaryStudies suggest that fungi belonging to Ascomycota are sensitive to environmental changes which are disrupting ecosystems globally, with numerous extinction events, imbuing fungal diversity research with a sense of urgency. Thus, it is crucial we assess the diversity of Ascomycota across different habitats and substrates, and include programs to seek out novel taxa within Ascomycota. Our study provides some insights into the woody-based saprotrophic microfungi in Yunnan, China by introducing two novel Ascomycota species. Furthermore, these species were discovered by a 5-year-old boy, thus highlighting how young enthusiasts can be involved in field studies and make a significant impact.While surveying the mycobiomes of dead woody litter in Yunnan Province, China, numerous isolates with affinity to Pleosporales (Dothideomycetes, Ascomycota) were recovered. The present work characterizes two species associated with dead woody twigs found in terrestrial habitats in the Kunming area of Yunnan. The novel taxa were recognized based on a polyphasic approach, including morphological examination and multiple gene phylogenetic analyses (non-translated loci and protein-coding regions). Neokalmusia jonahhulmei sp. nov. is introduced in Didymosphaeriaceae (Pleosporales) as a woody-based saprobic ascomycete that possesses multiloculate ascostromata immersed under a black clypeus-like structure, and three-septate, brown, fusiform, guttulate ascospores. Thyridaria jonahhulmei (Thyridariaceae) is introduced with teleomorphic and anamorphic (coelomycetous) characteristics. The teleomorph has the following characteristics: globose to subglobose ascomata with an ostiolum, a pruinose layer of yellow to reddish- or orange-brown material appearing around the top of the ostiolar necks, and brown, ellipsoid to fusoid, two-to-three-septate, euseptate, rough-walled ascospores; the anamorph features pycnidial conidiomata, phialidic, ampulliform to doliiform, conidiogenous cells, and brown, guttulate, ellipsoidal, aseptate conidia.

Insight into the Taxonomic Resolution of the Pleosporalean Species Associated with Dead Woody Litter in Natural Forests from Yunnan, China.

In the course of investigating the systematics of woody litter micromycete associates in Yunnan Province, China, we found one new species in Phaeoseptaceae, one new genus and three new species in Sulcatisporaceae from 16 specimens collected (ten collections of ascomycetous teleomorphs, four collections of hyphomycetous and two collections of coelomycetes anamorphs) from Ailaoshan, Chuxiong, Diqing, Honghe, Kunming, Lancang, Mengla and Yuxi in Yunnan Province. These taxonomic novelties were recognized with the aid of morphological comparisons and phylogenetic analyses of multiple gene sequences (non-translated loci and protein-coding regions). Pleopunctum menglaense sp. nov. is accommodated in Phaeoseptaceae (Pleosporales) based on its hyphomycetous anamorph, which is characterized by superficial sporodochia on the host surface, macronematous, mononematous, cylindrical, unbranched, aseptate, hyaline and smooth-walled conidiophores, monoblastic, terminal, hyaline conidiogenous cells, hyaline, muriform α conidia, and brown, muriform β conidia with tri-lobed wing like basal cells. Kazuakitanaka gen. nov. (type: K. yuxiensis) is introduced in Sulcatisporaceae (Massarineae, Pleosporales) for a saprobic ascomycete with teleomorphic and anamorphic (coelomycetous) features. The teleomorph possesses globose to subglobose ascomata with acentric ostiole, a peridial wall of textura angularis to textura prismatica, cylindric-clavate, pedicellate asci with an ocular chamber, and 1–2-septate, hyaline, fusiform, guttulate ascospores with a distinct mucilaginous sheath. The anamorph features pycnidial conidiomata, phialidic, ampulliform to cylindrical, hyaline conidiogenous cells and ampulliform to cylindrical, one-to-three-septate, hyaline, guttulate conidia. Loculosulcatispora was known only from its anamorph of L. thailandica. We observed the teleomorph of Loculosulcatispora hongheensis sp. nov. and amended the generic description of Loculosulcatispora accordingly. Loculosulcatispora hongheensis is characterized by globose to subglobose ascomata with a central ostiole, a peridial wall of textura angularis to globosa, branched, septate, pseudoparaphyses, clavate asci with a short pedicel and a minute ocular chamber and hyaline, fusiform, 1-septate ascospores with a thick irregular mucilaginous sheath. This study provides some insights into the diversity of fungi on dead woody litter in terrestrial habitats.

Dothidea kunmingensis, a novel asexual species of Dothideaceae on Jasminum nudiflorum (winter jasmine) from Southwestern China

During a survey of saprobic microfungi in Southwest China, a coelomycetous fungus was found on dead twigs of Jasminum nudiflorum in Kunming, Yunnan Province. Based on a detailed morphological characterization coupled with multi-locus phylogenetic analyses, the fungus was identified as a new species in the genus Dothidea. Phylogenetic analyses using a combined matrix consisting of internal transcribed spacer (ITS), large subunit rRNA (LSU), small subunit rRNA (SSU), beta tubulin (tub2) and translation elongation factor-1 alpha (tef1-α) confirmed its placement in Dothideaceae and revealed a sister relationship to Dothidea eucalypti. The new species is characterized by pycnidial conidiomata, ampulliform or doliiform conidiogenous cells as well as aseptate, subglobose to ovoid, hyaline to pale-brown conidia. Comprehensive descriptions and illustrations are provided. Morphological characteristics of asexual morph taxa in Dothideaceae are also summarized and discussed.

First Report of Leaf Spot Caused by Diaporthe tulliensis on Boston Ivy (Parthenocissus tricuspidata) in Taiwan.

Starting from the May to August 2020 (average humidity 76.6% and temperature 25.2°C in Taipei), Boston ivy (Parthenocissus tricuspidata) plants on the campus of National Taiwan University (25°01'05.4"N 121°32'36.6"E) exhibited leaf rusts caused by Phakopsora ampelopsidis (Tzean et al., 2019) and leaf spots caused by an unknown pathogen. The leaf spots appeared reddish to brown color and mostly irregular to round shape on the simple and trifoliate leaflets (Supplemental Figure 1A-C). The leaf spots were surface-disinfected with 1% NaOCl for 30 seconds, and the margin of healthy and infected tissues was cut and placed onto water agar, which were incubated at room temperature. Hyphae grown out from leaf spots were sub-cultured on potato dextrose agar (PDA), and the majority of isolates exhibited white colony with black pycnidial conidiomata embedded in PDA. The pycnidial conidiomata of two-week-old has an average diameter of 463±193 μm (n=30) and the sizes of α-conidia were 5.71±0.49 μm in length and 2.42±0.32 μm in width (n=50) similar to the previous records (Crous et al. 2015). The α-conidium was one-celled, hyaline, and ovoid with two droplets (Supplemental Figure 1D-G). This putative pathogen was re-inoculated to confirm its pathogenicity on the leaves of Boston ivy plants. A PDA block with actively growing fungal edge was placed on the tiny needle-wounded leaves of detached branches (Supplemental Figure H-I) and the whole plants in pots (Supplemental Figure 1J-M) in a moist chamber at 28°C in dark. Reddish to brown leaf spots were observed by 2 days post-inoculation (dpi) and the leaf spots expanded by 5 dpi. To complete the Koch's postulates, the pathogen was re-isolated from inoculated leaves and the re-isolated pathogen exhibited identical morphology to the original isolate. The internal transcribed spacer (ITS), translational elongation factor subunit 1-α gene (EF1α), β-tubulin (BT), and calmodulin (CAL) was amplified using the primers ITS1/ITS4 (Martin and Rygiewicz. 2005), EF1-728F/EF1-986R, Bt2a/Bt2b, and CAL-228F/CAL-737R, respectively (Manawasinghe et al. 2019). Using BLAST in the NCBI database, the ITS (MT974186), EF1α (MT982963), and β-tubulin (MT982962) sequences showed 98.57% (NR_147574.1, 553 out of 561 bp), 98.04% (KR936133.1, 350 out of 357 bp), and 99.23% (KR936132.1, 518 out of 522 bp) identity to the Diaporthe tulliensis ex-type BRIP 62248a, respectively (Dissanayake et al. 2017). Phylogenetic analysis using concatenated sequences of ITS, EF1α, and β-tubulin grouped the D. tulliensis isolated from Boston ivy leaf spots with the D. tulliensis ex-type (Supplemental Figure 1N). In summary, the morphological and molecular characterizations supported the causal pathogen of Boston ivy leaf spot as D. tulliensis. While Diaporthe ampelopsidis was reported to infect Parthenocissus quinquefolia and P. tricuspidata (Anonymous, 1960; Wehmeyer, 1933), there is no record for D. tulliensis infecting Boston ivy according to the USDA National Fungus Collections (Farr and Rossman. 2020). Because pathogens of Boston ivy such as P. ampelopsidis may also infect close-related crops like grape (Vitis vinifera L.) and D. tulliensis has been known to infect kiwifruits (Actinidia chinensis) and cocoa (Theobroma cacao) (Bai et al. 2016; Yang et al. 2018), the emergence of D. tulliensis should be aware to avoid potential damage to economic crops.

Morpho-Phylo Taxonomy of Novel Dothideomycetous Fungi Associated With Dead Woody Twigs in Yunnan Province, China.

Within the field of mycology, macrofungi have been relatively well-studied when compared to microfungi. However, the diversity and distribution of microfungi inhabiting woody material have not received the same degree of research attention, especially in relatively unexplored regions, such as Yunnan Province, China. To help address this knowledge gap, we collected and examined fungal specimens from different plants at various locations across Yunnan Province. Our investigation led to the discovery of four species that are clearly distinct from extant ones. These taxonomic novelties were recognized based on morphological comparisons coupled with phylogenetic analyses of multiple gene sequences (non-translated loci and protein-coding regions). The monotypic genus Neoheleiosa gen. nov. (type: N. lincangensis) is introduced in Monoblastiaceae (Monoblastiales) for a woody-based saprobic ascomycete that possesses globose to subglobose or obpyriform ascomata with centric or eccentric, papillate ostioles, an ascomatal wall with thin-walled cells of textura globulosa, cylindric, pedicellate asci with an ocular chamber, and 1-septate, brown, guttulate, longitudinally striated, bicellular ascospores. Neoheleiosa has a close phylogenetic affinity to Heleiosa, nevertheless, it is morphologically dissimilar by its peridium cells and ornamented ascospores. Acrocalymma hongheense and A. yuxiense are described and illustrated as new species in Acrocalymmaceae. Acrocalymma hongheense is introduced with sexual and asexual (coelomycetous) features. The sexual morph is characterized by globose to subglobose, ostiolate ascomata, a peridium with textura angularis cells, cylindric-clavate asci with a furcate to truncate pedicel and an ocular chamber, hyaline, fusiform, 1-septate ascospores which are surrounded by a thick, distinct sheath, and the asexual morph is featured by pycnidial conidiomata, subcylindrical, hyaline, smooth, annelledic, conidiogenous cells, hyaline, guttulate, subcylindrical, aseptate conidia with mucoid ooze at the apex and with a rounded hilum at the base. Acrocalymma yuxiense is phylogenetically distinct from other extant species of Acrocalymma and differs from other taxa in Acrocalymma in having conidia with three vertical eusepta. Magnibotryascoma kunmingense sp. nov. is accommodated in Teichosporaceae based on its coelomycetous asexual morph which is characterized by pycnidial, globose to subglobose, papillate conidiomata, enteroblastic, annelledic, discrete, cylindrical to oblong, hyaline conidiogenous cells arising from the inner layer of pycnidium wall, subglobose, oval, guttulate, pale brown and unicelled conidia.

Ecology of Diaporthe eres, the causal agent of hazelnut defects.

Diaporthe eres has been recently reported as the causal agent of hazelnut defects, with characteristic brown spots on the kernels surface and internal fruit discoloration. Knowledge regarding the ecology of this fungus is poor but, is critical to support a rationale and effective hazelnut crop protection strategy. Therefore, a study was performed to describe and model the effect of different abiotic factors such as temperature (T, 5–35°C, step 5°C) and water activity (aw 0.83–0.99, step 0.03) regimes on D. eres mycelial growth, pycnidial conidiomata development and asexual spore production during a 60-day incubation period. Alpha conidia germination was tested in the same T range and at different relative humidities (RH = 94, 97 and 100%) over 48 h incubation period. Fungal growth was observed from the first visual observation; regarding pycnidia and cirrhi, their development started after 8 and 19 days of incubation, respectively and increased over time. The optimum T for growth was 20–25°C and for pycnidia and cirrhi development was 30°C; aw ≥ 0.98 was optimal for the tested steps of the fungal cycle. The best condition for conidial germination of D. eres was at 25°C with RH = 100%. Quantitative data obtained were fitted using non- linear regression functions (Bete, logistic and polynomial), which provided a very good fit of the biological process (R2 = 0.793–0.987). These functions could be the basis for the development of a predictive model for the infection of D. eres of hazelnuts.

<strong><em>Hyaloterminalis</em>, a novel genus of Coryneaceae in order Diaporthales </strong>

The dematiaceous coelomycete Hyaloterminalis alishanensis gen. et sp. nov. (Coryneaceae, Diaporthales, Sordariomycetes) is introduced in this paper. It was isolated from dead branches of Cerasus sp. from the Ali Mountain, Taiwan. Coryneum was previously the only genus in Coryneaceae, and morphological similarity of Hyaloterminalis with the type genus is limited to broadly fusiform, brown conidia. Hyaloterminalis differs from Coryneum in having pycnidial conidiomata, persistent paraphyses and conidiophore morphology, with a long narrow hyaline tip to the apical cell and hyaline basal cells. Multigene phylogenetic analyses, using a combined LSU, ITS and TEF1-α sequence data generated from maximum likelihood, maximum parsimony and MrBayes analyses, confirmed that Hyaloterminalis forms a distinct lineage within Coryneaceae and its establishment as a new genus is well-supported. The new taxon is described, illustrated and compared with Coryneum. The ITS (MH861215) and LSU (MH872982) sequences obtained from the ex-type strain of Talekpea foeticia, confirm its status as a member in Coryneaceae. Therefore, Talekpea is accepted as a distinct genus in Coryneaceae.

Additions to Phaeosphaeriaceae (Pleosporales): Elongaticollum gen. nov., Ophiosphaerella taiwanensis sp. nov., Phaeosphaeriopsis beaucarneae sp. nov. and a new host record of Neosetophoma poaceicola from Musaceae.

A novel ascomycetous genus, Elongaticollum, occurring on leaf litter of Hedychium coronarium (Zingiberaceae) in Taiwan, is described and illustrated. Elongaticollum is characterized by dark brown to black, superficial, obpyriform, pycnidial conidiomata with a distinct elongate neck, and oval to oblong, hyaline, aseptate conidia. Phylogenetic analyses (maximum likelihood, maximum parsimony and Bayesian) of combined ITS, LSU, SSU and tef1-α sequence data revealed Elongaticollum as a distinct genus within the family Phaeosphaeriaceae with high statistical support. In addition, Ophiosphaerella taiwanensis and Phaeosphaeriopsis beaucarneae are described as new species from dead leaves of Agave tequilana and Beaucarnea recurvata (Asparagaceae), respectively. Neosetophoma poaceicola is reported as a new host record from dead leaves of Musa acuminata (Musaceae). Newly described taxa are compared with other similar species and comprehensive descriptions and micrographs are provided.

Taxonomic novelties in Magnolia-associated pleosporalean fungi in the Kunming Botanical Gardens (Yunnan, China)

This paper represents the first article in a series on Yunnanese microfungi. We herein provide insights into Magnolia species associated with microfungi. All presented data are reported from the Kunming Botanical Gardens. Final conclusions were derived from the morphological examination of specimens coupled with phylogenetic sequence data to better integrate taxa into appropriate taxonomic ranks and infer their relationships. Shearia formosa, the type species of Shearia, lacks type material, and its phylogenetic position accordingly remains unresolved. A fresh collection of Shearia formosa, obtained from Magnolia denudata and M. soulangeana in China, therefore, designated a neotype for stabilizing the application of the species and/or genus name. Phylogenetic analyses of a combined DNA data matrix containing SSU, LSU, RPB2 and TEF loci of representative Pleosporales revealed that the genera Crassiperidium, Longiostiolum and Shearia are a well-defined monophylum. It is recognized as the family Longiostiolaceae and strongly supported by Bayesian and Maximum Likelihood methods. Its members are characterized by immersed to semi-immersed, globose to subglobose ascomata with a central, periphysate ostiole, a peridium composed of rectangular to polygonal cells, cylindrical to clavate asci, broadly fusiform, hyaline to pale brown ascospores, a coelomycetous asexual morph with pycnidial conidiomata, enteroblastic, annellidic, ampulliform, doliiform or cylindrical conidiogenous cells and cylindrical to fusiform, transverse and sometimes laterally distoseptate conidia without a sheath or with a basal lateral sheath. Nigrograna magnoliae sp. nov. is introduced from Magnolia denudata with both asexual and sexual morphs. We observed the asexual morph of Brunneofusispora sinensis from the culture and therefore amended the generic and species descriptions of Brunneofusispora.

Pseudoplagiostoma myracrodruonis (Pseudoplagiostomataceae, Diaporthales): a new endophytic species from Brazil

Two initially unidentifiable isolates were found when endophytic fungi from healthy leaves of Myracrodruon urundeuva (Anacardiaceae) were being investigated in Brazil. Based on phylogenetic analyses using internal transcribed spacer (ITS) and partial large subunit (LSU) of ribosomal DNA, RNA polymerase II (RPB2), translation elongation factor EF-1 alpha (TEF1), and tubulin (TUB2), the isolates were identified as members of the family Pseudoplagiostomataceae (Diaporthales). In this paper, we introduce a new species, Pseudoplagiostoma myracrodruonis, thus accepting eight species in Pseudoplagiostoma (Pseudoplagiostomataceae). The new species is proposed to accommodate endophytes characterized by a coelomycetous asexual morph, pycnidial conidiomata with lageniform to ampulliform and hyaline to pale brown phialidic conidiogenous cells that produce unicellular, ellipsoid to oblong-cylindrical, hyaline, and guttulate conidia. In the current phylogenetic analysis, P. myracrodruonis is placed in a well-supported clade in Pseudoplagiostoma (Pseudoplagiostomataceae). A discussion about the relationship of Pseudoplagiostoma species is included in the paper.

Cytospora elaeagnicola sp. nov. Associated with Narrow-leaved Oleaster Canker Disease in China

Cytospora is a genus including important phytopathogens causing severe dieback and canker diseases distributed worldwide with a wide host range. However, identification of Cytospora species is difficult since the currently available DNA sequence data are insufficient. Aside the limited availability of ex-type sequence data, most of the genetic work is only based on the ITS region DNA marker which lacks the resolution to delineate to the species level in Cytospora. In this study, three fresh strains were isolated from the symptomatic branches of Elaeagnus angustifolia in Xinjiang Uygur Autonomous Region, China. Morphological observation and multi-locus phylogenetic analyses (ITS, LSU, ACT and RPB2) support these specimens are best accommodated as a distinct novel species of Cytospora. Cytospora elaeagnicola sp. nov. is introduced, having discoid, nearly flat, pycnidial conidiomata with hyaline, allantoid conidia, and differs from its relatives genetically and by host association.

First Report of Twig Dieback Caused by Neopestalotiopsis clavispora on Blueberry in Korea

Blueberry (Vaccinium corymbosum) is an economically important fruit with increasing cultivation in Korea, expanded to about 4,000 ha in 2017. In January 2018, dieback symptoms were observed on young twigs and stems of blueberry plants at an orchard located in Goheung, Jeonnam Province, Korea. Initial red spots developed into larger lesions with brown margins, which gradually enlarged and developed into gray cankers. Affected twigs showed blight symptoms that gradually expanded along the stems, culminating in dieback. No acervuli and pycnidia were observed on diseased twigs and/or stems. Ten twigs displaying typical blight lesions were collected from each of 10 individual V. corymbosum ‘Duke’ plants. Small pieces of tissue were cut from the edge of each lesion, surface sterilized with 70% ethanol for 1 min, rinsed three times in sterilized water, and cultured on potato dextrose agar at 25°C. A total of 19 isolates were identified by morphological examination and molecular analysis. Colonies were white with cottony aerial mycelium, irregular margins, and light yellow on the reverse side of the plate, with black, globular acervuli (120 to 400 μm, average 230 μm) distributed in concentric circles. Pycnidial conidiomata were swollen and globose to oval. Conidia were straight to fusiform, measuring 19.5 to 30.1 × 5.0 to 11.0 μm (n = 50) and consisted of five cells with the three median cells being much darker than the end cells. The three median cells were doliform, rugose, and versicolored and had septa darker than the rest of the cell. The apical cell was hyaline, conic, and subcylindric, with two to three apical hyaline appendages (average 26.5 μm). The sexual stage was observed. These morphological characteristics are consistent with those of Neopestalotiopsis clavispora (Borrero et al. 2018). The ITS (MH423964-82), TUB2 (MH423945-63) and tef1 (MH423926-44) sequences matched with N. clavispora strains LB14 (99%; KY319134), FC-DB1 (99%; KY568918), and TOR-802-803-804 (99%; KU096881), respectively. Using MEGA X program (Kumar et al. 2018), maximum likelihood analysis based on the concatenated ITS, TUB2, and tef1 sequences placed the isolates within a clade comprising N. clavispora (G.F. Atk.) (Jayawardena et al. 2016; Song et al. 2014). To confirm pathogenicity of the isolates, three twigs of three 1-year-old V. corymbosum ‘Duke’ were inoculated by spraying conidial suspension (1 × 10⁵ conidia/ml) with and without wounding using a sterilized needle. Inoculated plants were maintained in a plastic bag for 16 h in a greenhouse with 16-h photoperiod, temperature between 18 and 30°C, and humidity maintained at 75%. Red-brown specks were observed on both the wounded and nonwounded twigs at 3 days postinoculation (dpi) and 2 months postinoculation, respectively. In the wound-inoculated twigs, the developed specks gradually grew larger at 7 to 14 dpi. After 14 dpi, the twigs showed gray canker symptoms typically observed on naturally infected plants. Control plants showed no symptoms on both the wounded and nonwounded plants. The pathogen was successfully reisolated using the methods described above and identified via analysis of the three genes and morphological characteristics. This pathogen has been previously reported to cause twig blight on blueberry in Chile, Uruguay, China, and Spain (Borrero et al. 2018; Chen et al. 2016; Espinoza et al. 2008; Gonzalez et al. 2012). To our knowledge, this is the first report of N. clavispora causing twig dieback on V. corymbosum ‘Duke’ in Korea, and this information will aid development of disease control strategies.

Tzeananiaceae, a new pleosporalean family associated with Ophiocordycepsmacroacicularis fruiting bodies in Taiwan.

The order Pleosporales comprises a miscellaneous group of fungi and is considered to be the largest order of the class Dothideomycetes. The circumscription of Pleosporales has undergone numerous changes in recent years due to the addition of large numbers of families reported from various habitats and with a large amount of morphological variation. Many asexual genera have been reported in Pleosporales and can be either hyphomycetes or coelomycetes. Phoma-like taxa are common and have been shown to be polyphyletic within the order and allied with several sexual genera. During the exploration of biodiversity of pleosporalean fungi in Taiwan, a fungal strain was isolated from mycelium growing on the fruiting body of an Ophiocordyceps species. Fruiting structures that developed on PDA were morphologically similar to Phoma and its relatives in having pycnidial conidiomata with hyaline conidia. The fungus is characterised by holoblastic, cylindrical, aseptate conidiogenous cells and cylindrical, hyaline, aseptate, guttulated, thin-walled conidia. Phylogenetic analysis based on six genes, ITS, LSU, rpb2, SSU, tef1 and tub2, produced a phylogenetic tree with the newly generated sequences grouping in a distinct clade separate from all of the known families. Therefore, a new pleosporalean family Tzeananiaceae is established to accommodate the monotypic genus Tzeanania and the species T.taiwanensis in Pleosporales, Dothideomycetes. The Ophiocordyceps species was identified as O.macroacicularis and this is a new record in Taiwan.