The phoma-like dilemma.
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Abstract
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The phoma-like dilemma
and Abstract
Species of Didymellaceae have a cosmopolitan distribution and are geographically widespread, occurring in diverse ecosystems. The family includes several important plant pathogenic fungi associated with fruit, leaf, stem and root diseases on a wide variety of hosts, as well as endophytic, saprobic and clinically relevant species. The Didymellaceae was recently revised based on morphological and phylogenetic analyses of ex-type strains subjected to DNA sequencing of partial gene data of the LSU, ITS, rpb2 and tub2 loci. Several poly- and paraphyletic genera, including Ascochyta, Didymella and Phoma were redefined, along with the introduction of new genera. In the present study, a global collection of 1 124 Didymellaceae strains from 92 countries, 121 plant families and 55 other substrates, including air, coral, human tissues, house dust, fungi, insects, soil, and water were examined via multi-locus phylogenetic analyses and detailed morphological comparisons, representing the broadest sampling of Didymellaceae to date. Among these, 97 isolates representing seven new genera, 40 new species and 21 new combinations were newly introduced in Didymellaceae. In addition, six epitypes and six neotypes were designated to stabilise the taxonomy and use of older names. A robust, multi-locus reference phylogenetic tree of Didymellaceae was generated. In addition, rpb2 was revealed as the most effective locus for the identification of Didymellaceae at species level, and is proposed as a secondary DNA marker for the family.
Introduction
The phoma-like circumscription is a pervasive and general concept, including species that produce pycnidia with aseptate, hyaline conidia occurring on herbaceous stems. The previously broad and ambiguous concept of the genus Phoma and the host-orientated nomenclature, together with the wide host range and occurrence, often resulted in incorrect taxonomic placements (Boerema et al., 2004, De Gruyter et al., 2009, Aveskamp et al., 2010, Chen et al., 2015). Species lacking good characteristics to be identified to genus level were often collectively deposited in collections as “phoma-like”, making this genus a “repository” to which more than 3 000 species epithets are associated in the MycoBank database (Crous et al. 2004). After more than 50 years of studying this group of fungi, Boerema and co-workers proposed that Phoma should be divided into nine sections (Boerema et al. 2004), although later studies again showed these sections to be poly- and paraphyletic (Aveskamp et al., 2008, Aveskamp et al., 2010). Recent progress in molecular phylogenetic analyses has greatly contributed to a substantial revision of phoma-like genera. This group of fungi has been elevated to family level and is now recognised as Didymellaceae based on the type genus Didymella (De Gruyter et al. 2009), which was also neotypified by de Gruyter in the same study.
Currently, Didymellaceae is one of the largest families in the fungal kingdom, which again belongs to the largest Dothideomycetes order, Pleosporales (Pleosporomycetidae, Dothideomycetes, Pezizomycotina, Ascomycota). This family is extremely species-rich. To date, more than 5 400 species belonging to at least 31 genera have been recorded, including recently established genera such as Neoascochyta and Paraboeremia and historical genera such as Ascochyta, Didymella and Phoma that remain highly problematic for the identification of new isolates (De Gruyter et al., 2009, Chen et al., 2015, Chen et al., 2017). Molecular characteristics have become increasingly essential in the identification of species in Didymellaceae. Based on sequence data obtained from 324 strains, Aveskamp et al. (2010) divided the family into 18 distinct clusters, and demonstrated that some important genera, such as Ascochyta, Didymella and Phoma, were highly polyphyletic. At the same time, phoma-like species that are located outside of this family were revealed to have affiliations to more than 25 families after phylogenetic analyses (De Gruyter et al., 2009, De Gruyter et al., 2010, De Gruyter et al., 2013). In a further study, Chen et al. (2015) provided an updated understanding of the taxonomy and evolution of Didymellaceae by including the rpb2 locus in a four-locus phylogenetic analysis. Several additional new genera, namely, Briansuttonomyces, Cumuliphoma, Ectophoma, Juxtiphoma, Neomicrosphaeropsis, Pseudoascochyta, Remotididymella, Similiphoma, and Vacuiphoma and a large number of new species have recently been introduced and accepted in the family (Ariyawansa et al., 2015a, Crous and Groenewald, 2016, Thambugala et al., 2016, Wijayawardene et al., 2016, Valenzuela-Lopez et al., 2018), revealing the great diversity and wide distribution of Didymellaceae.
The Didymellaceae includes plant pathogens, opportunists, endophytes, and saprobes from a wide range of substrates, such as asbestos, cement, crockery (Aveskamp et al. 2008), soil, oceans and their fauna, glaciers, and even deep-sea sediments (Dorenbosch, 1970, Zucconi et al., 1996, Clipson et al., 2001, Yarden, 2014, Zhang et al., 2014, Zhang et al., 2016, Chen et al., 2017, Hou et al., 2020). As pathogens, species in Didymellaceae infect plants and also parasitise fungi, lichens, insects and vertebrates (Perrotta and Graniti, 1988, Costa et al., 1993, Hutchinson et al., 1994, Sullivan and White, 2000, Hawksworth and Cole, 2004, Aveskamp et al., 2008).
Some species of Didymellaceae are recognised as important quarantine species, posing a threat to a diverse range of crops; for example, Phoma foveata was considered to pose a high risk to the potato industry and was subsequently added to the quarantine organism list in Europe in 1975 (De Gruyter 2012). Although it was eventually removed from the European quarantine list in 1999, Phoma foveata is still a quarantine organism in many countries outside Europe, e.g. in South America (Mendes et al., 2007, EPPO Global Database, 2019). In addition, six Phoma-related species are listed as quarantine species in China (Announcement No. 862 of the Ministry of Agriculture of the People’s Republic of China 2007), as well as Phoma andigena (as Stagonosporopsis andigena) and Phoma tracheiphila (as Plenodomus tracheiphilus) in the European and Mediterranean regions (Smith et al., 1992, EPPO Global Database, 2019) and Phoma macdonaldii in Australia (Miric et al. 1999). As time for examination of potentially infected plants/animals is always limited, accurate and fast identification of quarantine organisms is of extreme importance. Detection and identification based on morphological characteristics are time-consuming and require a high degree of taxonomic experience, reducing the chance to successfully identify phoma-like species to species level in the laboratory (Aveskamp et al. 2008). Considering the importance of correct identification of Didymellaceae pathogens from intercepted material and the limitation of traditional morphological methods, authentic reference sequences with high discriminatory power are urgently needed.
As the most widely sequenced marker for fungi, the ITS locus has been proposed as a universal DNA barcode for fungi (Schoch et al. 2012). In Didymellaceae, however, the nuclear ribosomal RNA gene sequences (ITS, LSU and SSU) are insufficient to distinguish closely related species (Aveskamp et al., 2008, Chen et al., 2015). Therefore, identifying a different DNA locus (or secondary DNA marker) (Hebert et al., 2002, Summerbell et al., 2005, Stielow et al., 2015) has been a promising initiative for the rapid detection of potentially serious plant pathogens (Armstrong & Ball 2005).
The objectives of the present study were: 1) to elucidate the taxonomy of a global collection of 1 124 phoma-like strains of Didymellaceae, including all isolates used in the monograph of Boerema et al. (2004) deposited in the CBS culture collection of the Westerdijk Institute; 2) to revise the taxonomy of previously introduced genera, as well as designate epitypes/neotypes to fix the application of older names where possible; and 3) to evaluate rpb2 as potential secondary species-level DNA barcode across all genera included in Didymellaceae.
Materials and methods
Isolates
All isolates in this study were obtained from the CBS culture collection of the Westerdijk Fungal Biodiversity Institute (WI; Utrecht, the Netherlands, formerly CBS-KNAW). A total of 1 124 isolates identified as Didymellaceae based on morphological characters or preliminary DNA sequence data were included in this study, including 209 ex-type strains (Table S1 and Table S2). Freeze-dried strains from the CBS culture collection were revived in 2 mL of malt/peptone (50 %/50 %) liquid medium and subsequently transferred to oatmeal agar (OA) (Crous et al. 2019b). Strains stored in liquid nitrogen were transferred to OA directly from straws (Stalpers et al. 1987). Some of the cultures were incubated under near-ultraviolet (UV) light (12 h light, 12 h dark) on pine needle agar (PNA) (Smith et al., 1996, Su et al., 2012) to promote sporulation if necessary.
DNA extraction, PCR amplification and sequencing
Total genomic DNA was extracted from fresh mycelia harvested from cultures grown on OA for 7–14 d at room temperature using the Wizard® Genomic DNA purification kit (Promega, Madison, USA) following the manufacturers’ protocols. Four loci were amplified, including the internal transcribed spacer regions 1 and 2 and 5.8S nuclear ribosomal RNA gene (ITS), partial beta-tubulin gene (tub2), partial large subunit nrRNA gene (28S nrDNA; LSU) and partial DNA-directed RNA polymerase II second largest subunit (rpb2) gene using the primer pairs ITS5/ITS4 (White et al., 1990, De Hoog and Gerrits van den Ende, 1998), Btub2Fd/Btub4Rd (Woudenberg et al. 2009), LR0R/LR5 or LR0R/LR7 (Vilgalys and Hester, 1990, Rehner and Samuels, 1994), and RPB2-5F2/RPB2-7cR (Liu et al., 1999, Sung et al., 2007), respectively. The PCR amplifications, except for that of rpb2, were performed following the method of Chen et al. (2015). The amplification for rpb2 was performed according to an improved protocol with bovine serum albumin (BSA): a total volume of 12.5 μL containing 1.25 μL of EasyTaq Buffer (Bioline, Luckenwalde, Germany), 0.5 μL of dNTPs (40 μM), 0.5 μL of MgCl2 (2 mM), 0.5 μL of BSA (1 μg/μL), 0.25 μL of each primer (0.2 μM), 0.1 μL of Taq DNA polymerase (Bioline) and 1 μL of genomic DNA was used. The PCR conditions for rpb2 were: an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 45 s, annealing at 56 °C for 80 s and extension at 72 °C for 2 min, with a final extension step at 72 °C for 10 min. The PCR products for four loci were purified and sequenced in both directions as explained in Crous et al. (2013a). The consensus sequences were assembled from forward and reverse sequences using Seqman Pro v. 10.0.1 (DNASTAR, Madison, USA). Novel sequences generated in this study were deposited in GenBank (http://www.ncbi.nlm.nih.gov, Supplementary Table S1).
Morphology
Morphological studies of living cultures were conducted following the methods described by Chen et al., 2015, Chen et al., 2017. Isolates were grown on fresh oatmeal agar (OA), malt extract agar (MEA) and potato dextrose agar (PDA) (Crous et al. 2019b) and incubated at 25 °C, under near-ultraviolet (UV) light (12 h light/12 h dark) to induce sporulation. Colony diameter was measured and the NaOH spot test was performed by addition of a drop of 1N NaOH to determine the secretion of metabolite E on OA cultures after 7 d (Boerema et al. 2004). Colony characters (upper surface and reverse) and colours were rated following the colour charts of Rayner (1970) after 14 d of incubation. Micromorphological observation was carried out from cultures grown on OA after 7 d of incubation, and included the characteristics of conidiomata/ascomata, the presence of mycelium/setae outside of the conidiomata/ascomata, the number of ostioles, the thickness of the pycnidial/pseudothecial wall, the shape of cells of the pycnidial/ pseudothecial wall, and the length and width of conidiogenous cells/asci and conidia/ascospores. Slide preparations were mounted in distilled water to study the micromorphological structures of mature ascomata/conidiomata, ascospores/conidia and asci/conidiogenous cells (Aveskamp et al., 2010, Chen et al., 2015, Chen et al., 2017). At least 30 measurements were made for all morphologically informative features. Micromorphological observations were processed with a Nikon Eclipse 80i compound microscope with differential interference contrast (DIC) optics and a Nikon AZ100 dissecting microscope, both equipped with a Nikon DS-Ri2 high-definition colour digital camera. The structure of the mature pseudothecial/pycnidial wall was studied using microtome sections of 8–10 μm thickness, prepared with a Leica CM 1100 freezing cryostat microtome and mounted in lactic acid (Aveskamp et al., 2010, Chen et al., 2015). Descriptions of novelties and taxonomic recombinations were deposited in MycoBank, and specimens were deposited in the CBS fungarium.
Sequence alignment and molecular phylogenetic analysis
Among all the isolates in this study, partial genes of 431 isolates that were sequenced in previous studies and deposited in GenBank (Yarden et al., 2007, Aveskamp et al., 2009a, Aveskamp et al., 2010, De Gruyter et al., 2010, De Gruyter et al., 2013, Chen et al., 2015, Chen et al., 2017) were treated as reference sequences. Together with the sequences of related species described in recent studies (Thambugala et al., 2016, Wijayawardene et al., 2016) and outgroup species (Coniothyrium palmarum CBS 400.71, Neocucurbitaria aquatica CBS 297.74 and Pleiochaeta setosa CBS 496.63, CBS 118.25), all reference sequences were downloaded from GenBank and Q-Bank (Bonants et al. 2013), and listed in Table S1 and Table S2. Subsequent alignments for four individual loci (ITS, LSU, rpb2 and tub2) were generated with MAFFT v. 7 using the default settings on the web server of the European Bioinformatics Institute (EMBL-EBI) (http://www.ebi.ac.uk/Tools/msa/mafft/) (Katoh and Standley, 2013, Li et al., 2015), and were manually edited in MEGA v. 6.0 when necessary (Tamura et al. 2013). A multi-locus gene dataset was generated using SequenceMatrix v. 1.8 (Vaidya et al. 2011).
To investigate the phylogenetic relationships between different isolates and to establish the identity of the isolates at species level, both Bayesian inference (BI) and Maximum Likelihood (ML) methods were used for phylogenetic analyses of individual sequence alignments, followed by the concatenated alignments. The best substitution model of evolution for each of the four data partitions were estimated using jModeltest v. 2.1.4 (Darriba et al. 2012) according to the Akaike information criterion before the Bayesian analysis. Bayesian analyses were performed using MrBayes v. 3.2.6 (Ronquist et al. 2012) as described by Chen et al. (2015). Markov Chain Monte Carlo sampling (MCMC) analyses of four chains were started in parallel from a random tree topology. Four simultaneous Markov chains were run for 10 M generations with a sampling frequency set to the 1 000th generation (resulting in 10 000 total trees per parallel run) or until the run was stopped automatically when the average standard deviation of split frequencies fell below 0.01. The first 25 % of the trees were discarded as the burn-in phase of each analysis, and the remaining trees were used to calculate posterior probabilities (Chen et al. 2015). Maximum Likelihood analyses including 1 000 bootstrap replicates, which were conducted using RAxML v. 7.2.6 (Stamatakis & Alachiotis 2010). A general time reversible (GTR) model was applied with a gamma-distributed rate variation. The resulting trees were viewed using FigTree v. 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree). Alignments and the phylogenetic trees derived from this study were uploaded to TreeBASE (accession number: S25826; www.treebase.org). Three trees were generated: the phylogenetic tree with combined loci with a reduced set of isolates representing the different species (Fig. 1), the phylogenetic tree with combined loci with all isolates belonging to Didymellaceae included in this study (Supplementary Fig. S1), and a phylogenetic tree based only on the rpb2 locus with a reduced set of isolates representing the different species (Supplementary Fig. S2).
Phylogenetic tree inferred from a Maximum Likelihood analysis based on a concatenated alignment of LSU, ITS, rpb2 and tub2 sequences of 597 strains representing Didymellaceae and outgroup sequences. The RAxML bootstrap support values (MLBS) above 50 % and Bayesian posterior probabilities (BPP) above 0.80 are given at the nodes (BPP/MLBS). Some of the basal branches were shortened to facilitate layout (the fraction in round parentheses refers to the presented length compared to the actual length of the branch). The scale bar represents the expected number of changes per site. Genera are delimited in coloured boxes, with the genus name indicated to the right. Strains with special status are indicated with a superscript letter after the accession number (R: representative; T: ex-type). The new species are printed in red font and new combinations in green font. The tree is rooted to Coniothyrium palmarum culture CBS 400.71, Neocucurbitaria aquatica culture CBS 297.74 and Pleiochaeta setosa cultures CBS 496.63 and CBS 118.25.
Evaluation of loci for species resolution
Species names were applied to the trees based on the phylogenetic analyses performed in the present study but were also based on prior studies where, in some cases, more loci were employed for species recognition than the number of loci used here. Using loci to distinguish species was evaluated as described by Gomes et al. (2013) and Alvarez et al. (2016). RAxML trees with the GTR model were generated for each locus of the corresponding alignments. The trees were then manually evaluated as means to resolve species from one another. A species was counted only if it was distinct from its closest relatives and if the species clade contained all strains of that species included in the analysis.
Results
Phylogenetic analyses
Among the 1 124 isolates, 177 isolates were identified as not belonging to genera in Didymellaceae, but were distributed in 13 other families in Pleosporales (Table S2), which will be treated elsewhere. The phylogenetic tree based on the concatenated DNA sequence dataset (ITS, LSU, rpb2 and tub2) for all 947 Didymellaceae isolates examined in this study is showed in Fig. S1.
Among all the Didymellaceae isolates, 470 isolates with the complete dataset of genes together with 127 references isolates were selected to be included in the overview phylogenetic analyses (Fig. 1;Table S1 and the strain numbers showed in bold). The final concatenated DNA sequence dataset (ITS, LSU, rpb2 and tub2) used to infer delimitation at the family and genus levels comprised 593 isolates representing 301 ingroup Didymellaceae taxa, with Coniothyrium palmarum CBS 400.71, Neocucurbitaria aquatica CBS 297.74 and Pleiochaeta setosa CBS 496.63 and CBS 118.25 serving as outgroup. The concatenated aligned dataset consisted of 2 418 characters, including alignment gaps (gene boundaries ITS: 1–477, 477 bp; LSU: 483–1 444, 962 bp; rpb2: 1 450–2 048, 599 bp; tub2: 2 054–2 418, 365 bp). Among those, 1 581 characters are conserved sites and 811 are variable sites, including 95 characters that are parsimony-uninformative and 716 characters that are parsimony-informative. Based on the result of jModeltest, for BI, a TIM2ef+I+G model was selected for ITS, the GTR+I+G model for LSU and rpb2, and a TIM3+I+G model for tub2. The sequence dataset did not show conflict in the tree topologies for the 70 % reciprocal bootstrap trees, which allowed us to combine the four genes for the multi-locus analysis. The ML tree and the Bayesian consensus tree confirmed the congruent topologies. A total of 22 502 trees were generated after the BI analysis reached the stop value of 0.01, with the first 25 % trees discarded as the burn-in phase, and posterior probabilities (PP) were calculated from the remaining 16 877 trees. The topology of the BI tree was confirmed by ML analysis for the distinction of 36 well-supported monophyletic clades; therefore, only the ML tree based on the combined dataset was presented here with the bootstrap support values of the ML analysis (MLBS) and relevant Bayesian posterior probabilities (BPP) shown at the nodes (MLBS > 50 %, BPP > 0.80; Fig. 1).
A total of 593 ingroup isolates formed a fully-supported clade (BPP = 1; MLBS = 100 %) representing Didymellaceae, which included 36 monophyletic generic clades. Twenty-two genera were previously recognised, including Allophoma (Clade 2; BPP = 1; MLBS = 88 %), Ascochyta (Clade 28; BPP = 0.98; MLBS = 99 %), Boeremia (Clade 4; BPP = 1; MLBS = 100 %), Briansuttonomyces (Clade 27; BPP = 1; MLBS = 100 %), Calophoma (Clade 25; BPP = 0.97; MLBS = 68 %), Chaetasbolisia (Clade 5; BPP = 1; MLBS = 100 %), Didymella (Clade 15; BPP = 1; MLBS = 83 %), Epicoccum (Clade 12; BPP = 1; MLBS = 99 %), Heterophoma (Clade 3; BPP = 1; MLBS = 97 %), Leptosphaerulina (Clade 31; BPP = 1; MLBS = 100 %), Macroventuria (Clade 18; BPP = 1; MLBS = 100 %), Microsphaeropsis (Clade 22; BPP = 1; MLBS = 96 %), Neoascochyta (Clade 36; BPP = 1; MLBS = 99 %), Neodidymelliopsis (Clade 32; BPP = 1; MLBS = 99 %), Neomicrosphaeropsis (Clade 23; BPP = 1; MLBS = 94 %), Nothophoma (Clade 20; BPP = 0.94; MLBS = 92 %), Paraboeremia (Clade 16; BPP = 1; MLBS = 100 %), Phoma (Clade 30; BPP = 1; MLBS = 100 %), Phomatodes (Clade 29; BPP = 1; MLBS = 100 %), Pseudoascochyta (Clade 26; BPP = 1; MLBS = 100 %), Stagonosporopsis (Clade 1; BPP = 1; MLBS = 94 %), and Xenodidymella (Clade 33; BPP = 1; MLBS = 96 %). Several genera were recently added to this family, namely, Cumuliphoma (Clade 17; BPP = 1; MLBS = 100 %), Ectophoma (Clade 7; BPP = 1; MLBS = 100 %), Juxtiphoma (Clade 19; BPP = 1; MLBS = 100 %), Remotididymella (Clade 8; BPP = 1; MLBS = 100 %), Similiphoma (Clade 13; single strain lineage), Vacuiphoma (Clade 21; BPP = 0.95; MLBS = 100 %), and Vandijckomycella (Clade 35; BPP = 1; MLBS = 100 %). Seven new genera are recognised and described in the present study, namely Dimorphoma (Clade 34; BPP = 1; MLBS = 100 %), Ectodidymella (Clade 11; BPP = 1; MLBS = 100 %), Longididymella (Clade 10; BPP = 1; MLBS = 100 %), Macroascochyta (Clade 6; single strain lineage), Paramicrosphaeropsis (Clade 24; BPP = 1; MLBS = 100 %), Pseudopeyronellaea (Clade 9; BPP = 1; MLBS = 100 %) and Sclerotiophoma (Clade 14; BPP = 1; MLBS = 100 %), which were fully supported as independent clades.
Evaluation of loci for species resolution
Of the 947 Didymellaceae examined in this study, 194 isolates already had rpb2 sequences in GenBank and were downloaded as reference sequences, 715 isolates were successfully amplified and sequenced for rpb2, while the remaining 38 failed; the success rate for amplification of rpb2 is 94.95 %. Therefore, for the complete dataset in this study, 93.53 % of isolates have available rpb2 sequences. The RAxML overview rpb2 phylogenetic tree (Supplementary Fig. S2), including 573 selected representative ingroup isolates belonging to 286 taxa, had a topology highly similar to that of the four-gene tree (Fig. 1), and was able to discriminate 35 well-supported genera. Species of the genus Calophoma did not cluster in monophyletic lineages but formed two separate clades, although the discrimination for all species in this genus was successful. Among the 286 species, 279 species with rpb2 sequences could be distinguished in the rpb2 phylogeny, but failed for the following species: Boeremia heteromorpha, Boeremia populi, Didymella sinensis, Didymella aeria, Neomicrosphaeropsis elaeagni, Neomicrosphaeropsis alhagi-pseudalhagi and Neomicrosphaeropsis italica; while 15 other species represented by 24 isolates that were recognised in the four-gene tree lacked rpb2 sequence data. For the genus Boeremia, rpb2 did not satisfactorily resolve the species boundaries on its own; therefore, species recognition requires the use of additional loci, such as tub2 and ITS.
For the tub2 gene, a total of 366 isolates had tub2 sequences available in GenBank. For the rest of the isolates used, 565 isolates were successfully amplified and sequenced but failed for the remaining 16 isolates; the success rate was 97.25 %. Therefore, 96 % of isolates in the dataset have tub2 sequences. The phylogenetic analysis of tub2 comprised 582 selected isolates representing 291 taxa recognised in the four-gene tree. This locus could distinguish most genera except four genera: Calophoma separated into two clades, Epicoccum clustered in three different clades, Remotididymella clustered together with one part of Calophoma and Allophoma clustered in three lineages. Compared to the 301 species recognised in the four-gene tree, 15 isolates representing 10 species lacked tub2 sequence data. At species level, the single tub2 tree could recognise 285 species, but failed to distinguish six species, namely Stagonosporopsis papillata, St. bomiensis, Neodidymelliopsis longicolla, Neodidymelliopsis ranunculi, Ascochyta lentis and As. viciae-villosae.
The LSU phylogenetic tree was able to distinguish the Boeremia, Dimorphoma, Ectophoma, Juxtiphoma, Leptosphaerulina, Longididymella, Macroventuria, Neoascochyta, Neodidymelliopsis, Neomicrosphaeropsis, Paramicrosphaeropsis, and Pseudopeyronellaea clades and 32.48 % of the species.
The ITS phylogenetic tree failed to recognise 13 genera, namely, Allophoma, Ascochyta, Chaetasbolisia, Didymella, Ectophoma, Epicoccum, Heterophoma, Macroventuria, Microsphaeropsis, Nothophoma, Stagonosporopsis, Vacuiphoma, and Xenodidymella, while 61.92 % of the species could be recognised.
Taxonomy
The multi-locus sequence analysis of 597 strains delineated 36 clades, representing 301 taxa belonging to the Didymellaceae. After morphological examination, seven new genera and 40 new species were described. A further 21 new combinations, six epitypifications and six neotypifications were proposed, seven species were reduced to synonymy, and four Epicoccum species were resurrected. Three new species that proved to be sterile were described based on DNA sequence data, following the approach of Gomes et al. (2013) and Lombard et al. (2016). Taxa are arranged according to their position on the phylogenetic tree (Fig. 1).
Clade 1: Stagonosporopsis Died. emend. Aveskamp et al., Stud. Mycol. 65: 44. 2010.
Type species: Stagonosporopsis hortensis (Sacc. & Malbr.) Petr.
Stagonosporopsis ailanthicola Manawas. et al., Fungal Diversity 83: 82. 2017. Fig. 2.
Stagonosporopsis ailanthicola (CBS 140556). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H = 100 μm; I = 25 μm; J, N = 10 μm; K–M = 5 μm.
Description from culture CBS 140556: Conidiomata pycnidial, produced partly in the agar, solitary or aggregated, sometimes 2–4 confluent, scattered, mostly globose, subglobose to flask-shaped, pale brown to brown, thin-walled, glabrous, ostiolate, 520–830 × 420–550 μm. Ostioles 1–6, slightly papillate, increasing to eight when pycnidia confluent. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 3–5 layers, 15–40(–69) μm thick, outer two cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, subglobose, ampulliform or lageniform, 5–10 × 4–9 μm. Conidia oblong, with both ends rounded, smooth and thin-walled, aseptate, 4–7 × 2–2.5 μm, 2–3-guttulate. Conidial matrix whitish.
Culture characteristics: Colonies on OA, 65–70 mm diam after 7 d at 25 °C, margin regular, sparse aerial mycelium, grey olivaceous, pale luteous toward the periphery, abundant production of pycnidia; reverse concolourous. Colonies on MEA, 65–70 mm diam after 7 d, margin regular, aerial mycelium floccose, pale olivaceous grey; reverse concolourous. Colonies on PDA, 60–65 mm diam after 7 d, margin regular, covered by sparse felty aerial mycelium, concentric circles of different colours, centre hazel, buff, grey, olivaceous toward periphery; reverse olivaceous grey, salmon toward periphery. NaOH spot test: a pale greenish discolouration on OA.
Typus: Italy, Forlì Cesena (FC) Province, Via Pietro Nenni-Forlì, on dead branch of Ailanthus altissima (Simaroubaceae), 2 May 2015, E. Camporesi (holotype MFLU 16-2678, ex-type living culture MFLUCC 16-1439).
Materials examined: Australia, Tasmania, Antill Street, South Hobart, Hobart 7004, house dust, 31 Jan. 2009, B. Horton, culture CBS 140553. New Zealand, Wellington, Wyndham Rd., Silverstream, house dust, 3 May 2009, T. Atkinson, culture CBS 140556. USA, California, Koshland Hall, University of California Berkeley, Berkeley 94705, Alameda, house dust, 31 Mar. 2005, A. Amend, culture CBS 140554.
Notes: Four isolates from house dust in Australia, New Zealand and the USA were found to be genetically identical to St. ailanthicola, which was originally described from a dead branch of Ailanthus altissima in Italy (Tibpromma et al. 2017), suggesting St. ailanthicola to be highly cosmopolitan and plurivorous.
Stagonosporopsis cucumeris L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833477. Fig. 3.
Stagonosporopsis cucumeris (CBS 386.65). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pseudothecia forming on OA. H. Pseudothecium. I. Section through pseudothecium. J. Section of pseudothecial wall. K–L. Asci. M. Ascospores. N. Pycnidia forming on OA. O. Pycnidium. P–Q. Conidiogenous cells. R. Conidia. Scale bars: H = 100 μm; I = 20 μm; J–M = 10 μm; O = 50 μm; P–R = 5 μm.
Etymology: Name refers to the host genus from which it was isolated, Cucumis.
Description: Ascomata pseudothecial, superficial or semi-immersed, mostly solitary, scattered, globose to lageniform, dark brown, up to 350 μm diam, papillate, sometimes elongated with a short neck. Pseudothecial wall pseudoparenchymatous 24–41.5 μm thick, outer wall consisting of 2–4 layers of cells of textura angularis. Pseudoparaphyses not observed. Asci bitunicate, clavate to cylindrical, with stipe short or absent, 45–78 × 8.5–11.5 μm. Ascospores biseriate, sparsely uniseriately arranged, ellipsoidal, straight to slightly curved, 11.5–15 × 4.5–6.5 μm, hyaline, smooth, apex obtuse, base broadly obtuse to subobtuse, medianly 1-septate, upper cell often wider than lower cell, constricted at the septum. Conidiomata pycnidial, mostly solitary, sometimes confluent, scattered, globose and subglobose to flask-shaped, pale to dark brown, thin-walled, glabrous, semi-immersed, sometimes immersed or superficial on agar, ostiolate, 110–320 × 100–320 μm. Ostioles 1–3, slightly papillate, sometimes elongated as a short neck. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 3–5 layers, 11–29(–50.5) μm thick, outer 3 cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, globose to ampulliform, 5–8.5 × 4.5–6 μm. Conidia oblong or ovoid, smooth- and thin-walled, hyaline, aseptate, 4–7.5 × 3–4.5 μm, with 2–3 large guttules. Conidial matrix whitish.
Culture characteristics: Colonies on OA reaching 80–85 mm diam after 7 d 25 °C, margin regular, aerial mycelium flat, grey olivaceous, abundant production of buff pycnidia and ascomata; reverse concolourous. Colonies on MEA reaching 80–85 mm diam after 7 d, margin regular, aerial mycelium flat, pale olivaceous grey, olivaceous grey near centre; reverse concolourous. Colonies on PDA reaching 80–85 mm diam after 7 d, margin regular, covered by felty aerial mycelium, pale olivaceous grey; reverse olivaceous grey. NaOH spot test negative on OA.
Typus: The Netherlands, from greenhouse in Groningen, canker on Cucumis sativus (Cucurbitaceae), Dec. 1965, H.A. van Kesteren (holotype CBS H-23694, ex-type living culture CBS 386.65).
Notes: The isolates CBS 386.65 and CBS 214.65 were received as “Stagonosporopsis cucurbitacearum” (basionym: Sphaeria cucurbitacearum), and were originally isolated from Cucumis sativus, which represents a different host from that of St. cucurbitacearum (Cucurbitae and Momordicae; Fries 1823). Isolate CBS 214.65 was genetically identical to St. citrulli (holotype FLAS F-58996) that was morphologically similar with St. cucurbitacearum, based on ITS, LSU and tub2 sequences (Stewart et al. 2015). Therefore, CBS 214.65 was re-identified as St. citrulli. Besides, based on the multi-locus phylogenetic analyses, isolate CBS 386.65 formed a distinct lineage sister to St. citrulli and distant from the representative strain of St. cucurbitacearum (CBS 133.96). Morphologically, CBS 386.65 could be differentiated from St. cucurbitacearum by producing wider and aseptate conidia (aseptate, 4–7.5 × 3–4.5 μm vs. 0–1-septate, 4–8 × 2–3 μm) (Boerema et al. 2004). Although St. citrulli was originally introduced based on a phylogenetic analysis that showed it differs from the known Stagonosporopsis species, CBS 386.65 is distinct from St. citrulli (CBS 214.65) in 11 bp of rpb2 sequence and 6 bp of tub2 sequence (Stewart et al. 2015). We therefore introduced a new species St. cucumeris based on CBS 386.65.
Stagonosporopsis nemophilae (Neerg.) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833494.
Basionym: Phoma nemophilae Neerg., Bot. Tidsskr. 44(3): 361. 1938.
Description: De Gruyter et al. (2002).
Typus: Denmark, from seed of Nemophila insignis (Hydrophyllaceae), Oct. 1938, Rijksproefstation v. Zaadcontrole, culture CBS 249.38 (presumed ex-type, but sterile). The Netherlands, from seed of Nemophila insignis (Hydrophyllaceae), Nov. 1985, isolated by G.H. Boerema (neotype designated here CBS H-24316, MBT389690, ex-neotype living culture CBS 715.85 = PD 74/364).
Notes: Phoma nemophilae was described from seed of Nemophila insignis collected in Denmark, with cylindrical conidia measuring 4.5–9 × 1–2 μm (Neergaard 1938). However, the type specimen of Phoma nemophilae was not indicated in the original description and could not be traced in any fungarium after many efforts, and was therefore considered lost. In this study, CBS 249.38 was recognised as possible ex-type culture of Phoma nemophilae based on the information deposited in the CBS culture collection by the original collector and depositor of this species (Rijksproefstation v. Zaadcontrole). Unfortunately, this culture proved to be sterile. De Gruyter et al. (2002) described a representative culture of Phoma nemophilae (CBS 715.85), having morphological congruence with the original description of this species. In the present study, CBS 715.85 is genetically identical to the possible ex-type culture CBS 249.38, forming a well-supported distinct lineage in Stagonosporopsis (Fig. 1), and was therefore chosen as ex-neotype culture. Presently it is the only phoma-like species in Didymellaceae recorded on Nemophila insignis.
Stagonosporopsis sambucella L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833495.
Etymology: Name refers to the host genus Sambucus, from which this species was isolated.
Description: Culture sterile. Stagonosporopsis sambucella differs from its closest phylogenetic species St. astragali CBS 178.25 based on alignment of the concatenated four loci deposited in TreeBASE (S25826): ITS position 43(C), 50(A), 56(C), 293(C), 328(T), 347(C), 353(A), 423(A), deletion in position 444; LSU position 657(C), 879(C), 900(A); rpb2 positions: 1 469(T), 1 487(A), 1 514(T), 1 547(C), 1 559(C), 1 571(A), 1 610(T), 1 616(T), 1 634(T), 1 664(T), 1 700(G), 1 703(T), 1 712(C), 1 724(A), 1 745(A), 1 775(C), 1 829(T), 1 838(T), 1 841(G), 1 853(G), 1 878(T), 1 898(G), 1 913(C), 1 937(C), 1 940(G), 1 946(T), 1 949(T), 2 021(C), 2 033(T); tub2 positions: 2 062(C), 2 079(C), 2 124(C), 2 143(T), 2 167(T), 2 170(T), 2 209(T), 2 212(C), 2 269(T), 2 272(A), 2 275(T), 2 276(T), 2 277(T), 2 291(A), 2 400(C), 2 415(C).
Typus: Austria, Styria, Sambucus nigra (Caprifoliaceae), date unknown, A. Holler & D. Horvat (holotype CBS H-24317, ex-type living culture CBS 130003).
Notes: Isolate CBS 130003 formed a distinct lineage in the genus Stagonosporopsis (Fig. 1), but remained sterile in all culture media tested in this study. Considering that it is phylogenetically distinct, a new species, St. sambucella, was introduced here.
Clade 2: Allophoma Qian Chen & L. Cai, Stud. Mycol. 82: 162. 2015.
Type species: Allophoma tropica (R. Schneid. & Boerema) Qian Chen & L. Cai
Allophoma alba L.W. Hou, Pfenning, L. Cai & Crous, sp. nov. MycoBank MB833479. Fig. 4.
Allophoma alba (CBS 120422). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–N. Conidiogenous cells. O. Conidia. Scale bars: H = 100 μm; I = 50 μm; J = 10 μm; K–O = 5 μm.
Etymology: Latin, alba = white, referring to the whitish pycnidia.
Description: Conidiomata pycnidial, (semi-)immersed in the agar, mostly solitary, scattered towards periphery of colony, some arranged in concentric zones, (sub-)globose to ellipsoidal, whitish at onset, slightly brown around the ostioles with age, thin-walled, glabrous, ostiolate, (100–)205–635 × (100–)195–510 μm. Ostioles 1–3, non-papillate or slightly papillate. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 3–4 layers, 19–45 μm thick, without pigmented layers or the outer 1 cell layer slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, globose to ampulliform, 3.5–6.5(–8) × 4.5–9 μm. Conidia oblong, with both ends rounded, hyaline, smooth- and thin-walled, aseptate, 3–4.5 × 1.5–2.3 μm, with two large, polar guttules. Conidial matrix whitish.
Culture characteristics: Colonies on OA reaching 50–52 mm diam after 7 d at 25 °C, margin regular, aerial mycelium sparse, whitish to buff, with some olivaceous sterile sections, abundantly produced large pycnidia, conidial matrix visible; reverse concolourous. Colonies on MEA reaching 45–52 mm diam after 7 d, margin regular, aerial mycelium sparse, yellow to buff, pale olivaceous toward the periphery; reverse cinnamon, pale olivaceous near the margin. Colonies on PDA reaching 50–55 mm diam after 7 d, margin regular, covered by sparse floccose aerial mycelium, pale olivaceous, darker olivaceous near the centre; reverse concolourous. NaOH spot test negative on OA.
Typus: Brazil, Lavras, from the stem of Bambusa vulgaris (Poaceae), Oct. 2005, L.H. Pfenning (holotype CBS H-23661, ex-type living culture CBS 120422 = CML 660).
Notes: Isolate CBS 120422 was initially received as “Allophoma tropica”, but it formed an independent branch, being distant from the rest of the isolates including the ex-type strain of Al. tropica (CBS 436.75; Fig. 1). Morphologically, CBS 120422 produces larger conidiogenous cells [3.5–6.5(–8) × 4.5–9 μm vs. 2–6 × 3–6 μm] and shorter conidia than Al. tropica (3–4.5 × 1.5–2.3 μm vs. 3–5.9 × 1.2–2.1 μm). Furthermore, it produces white pycnidia, which is rare in this family. Thus Al. alba is introduced as a new species, based on isolate CBS 120422.
Allophoma anatii L.W. Hou & O. Yarden, sp. nov. MycoBank MB833478. Fig. 5.
Allophoma anatii (CBS 124673). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H = 100 μm; I = 20 μm; J, L, N = 10 μm; K, M = 5 μm
Etymology: The name was chosen to honour Prof. Anat Yarden for her contributions to integrate experiments using fungi as part of the high school curriculum in biology.
Description: Conidiomata pycnidial, produced on the agar surface, scattered, mostly solitary, globose, or subglobose to ellipsoidal, sometimes the confluent pycnidia merge with age, relatively large, pale brown, thin-walled, glabrous, (90–)130–400(–460) × (75–)120–370 μm. Ostioles 1–3, papillate or elongated with a short cylindrical neck. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 3–4 layers, 9.5–28.5 μm thick, outer two layers composed of pale brown cells. Conidiogenous cells phialidic, hyaline, smooth, ampulliform or lageniform, slightly papillate, 5–7 × 5.5–9 μm. Conidia oblong with both ends rounded or ovoid, smooth- and thin-walled, hyaline, aseptate, 3.5–5.5 × 2–3 μm, with 1–2 medium, polar guttules. Conidial matrix whitish.
Culture characteristics: Colonies on OA reaching 45–50 mm diam after 7 d 25 °C, margin regular, aerial mycelium sparse, pale cinnamon, abundant production of pycnidia, conidial matrix visible; reverse concolourous. Colonies on MEA reaching 45–55 mm diam after 7 d, margin regular, aerial mycelium sparse, vinaceous buff, with some radially furrowed zones near the centre; reverse brown, with white radial line near the centre, honey towards periphery. Colonies on PDA reaching 42–52 mm diam after 7 d, margin regular, aerial mycelium sparse, umber near the centre, buff toward periphery; reverse concolourous. NaOH spot test negative on OA.
Typus: Australia, Great Barrier Reef, from a brown band on Acropora formosa (Acroporidae; coral), Jul. 2006, O. Yarden (holotype CBS H-23688, ex-type living culture CBS 124673 = OY6506).
Notes: In the phylogenetic tree Al. anatii, which was isolated from Acropora formosa (Acroporidae; coral), formed an independent lineage basal to the genus Allophoma, and clearly separate from other species (Fig. 1). Morphologically, Al. anatii is characterised by larger pycnidia with thin and resilient pycnidial wall.
Allophoma anatii is phylogenetically distant from species in Didymellaceae that were recorded growing in marine habitats, such as Calophoma sandfjordenica, Didymella finnmarkica, Phoma antarctica, Phoma capitulum, Phoma hibernica, Phoma ostiolata, and Phoma multipora (World Register of Marine Species, WoRMS; Crous et al. 2019a). Most of these have been treated as synonyms of species in other families and were phylogenetically distant from Al. anatii. For example, Phoma ostiolata was treated as synonym of Westerdykella capitulum in Sporormiaceae, while Phoma multipora and Phoma capitulum were transferred to Coniothyriaceae and Sporormiaceae respectively (Pawar et al., 1967, De Gruyter et al., 2013). On the other hand, Allophoma anatii differed morphologically from Ca. sandfjordenica and Did. finnmarkica in its aseptate and smaller conidia [3.5–5.5 × 2–3 μm vs. (8–)10–14(–18) × (2–)3 μm in Ca. sandfjordenica and (6–)7–16(–18) × (2–)2.5(–4) μm in Did. finnmarkica, Crous et al. 2019a].
Allophoma oligotrophica Qian Chen et al., Stud. Mycol. 87: 119. 2017.
Typus: China, Guizhou, Shuanghe Cave National Geopark, from air, 8 May 2015, Z.F. Zhang (holotype HMAS 247035, ex-type living culture CGMCC 3.18114 = LC 6245).
Additional materials examined: China, Guizhou, Shuanghe Cave National Geopark, from air, 8 May 2015, Z.F. Zhang, culture CGMCC 3.18115 = LC 6246; ibid. culture CGMCC 3.18116 = LC 6247. The Netherlands, Wageningen, on Oncidium (Orchidaceae) in glasshouse, Jul. 1975, G.H. Boerema, specimen CBS H-16594, culture CBS 353.75. USA, South Carolina, Clemson, on Ipomoea batatas (Convolvulaceae), 7 Jun. 2005, A. Rossman, culture CBS 120112.
Notes: Allophoma oligotrophica was the first species isolated from air using carbon-free silica gel medium in a Chinese Cast cave (Jiang et al. 2017). In this study, two cultures isolated from different plants in the Netherlands and the USA grouped together with the ex-type strain of Al. oligotrophica (CGMCC 3.18114). This species was confirmed to be widely distributed and associated with diverse substrates. Two strains (CBS 353.75 and CBS 120112) clustered slightly apart from the other strains of Al. oligotrophica based on seven changes over the four genes; presently we refrain from introducing an additional species pending collection of more isolates.
Clade 3: Heterophoma Qian Chen & L. Cai, Stud. Mycol. 82: 165. 2015.
Type species: Heterophoma sylvatica (Sacc.) Qian Chen & L. Cai
Heterophoma nobilis (Kabát & Bubák) Qian Chen & L. Cai, Stud. Mycol. 82: 165. 2015. Fig. 6.
Heterophoma nobilis (CBS 507.91). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–L. Conidiogenous cells. M. Chlamydospores. N. Conidia. Scale bars: H–I = 100 μm; J = 10 μm; K–N = 5 μm.
Basionym: Ascochyta nobilis Kabát & Bubák, Oesterr. Bot. Z. 54: 3. 1904.
Synonym: Phoma dictamnicola Boerema et al., Persoonia 15: 90. 1992.
Description from ex-neotype (CBS 507.91): Conidiomata pycnidial, (semi-)immersed in the agar, under thick mycelial layers, mostly solitary, sometimes confluent, scattered, (sub-)globose to ellipsoidal, pale brown at onset, dark brown with age, thick-walled, glabrous, 170–415 × 170–350 μm. Ostioles inconspicuous. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 5–9 layers, 47–74 μm thick, the outer 5 cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, globose, lageniform to ampulliform, 6–8 × 5–7.5 μm. Conidia ellipsoidal to oblong with both ends rounded, hyaline, smooth- and thin-walled, aseptate, 3–4.5 × 1.5–2.5 μm, without guttules or with 2–5 minute guttules.
Culture characteristics: Colonies on OA reaching 65–70 mm diam after 7 d at 25 °C, margin regular, aerial mycelium woolly, pale grey; reverse pale olivaceous to olivaceous. Colonies on MEA reaching 65–70 mm diam after 7 d, margin regular, aerial mycelium woolly, pale grey; reverse orange to dark brown. Colonies on PDA reaching 65–70 mm diam after 7 d, margin regular, covered by floccose aerial mycelium, pale grey; reverse buff to dark brown. NaOH spot test negative on OA.
Typus: The Netherlands, Gelderland Province, Arnhem, from a stem of Dictamnus albus (Rutaceae), Sep. 1991, J. de Gruyter (neotype designated here MBT389692, CBS H-24313, ex-neotype living culture CBS 507.91 = PD 74/148).
Notes: Heterophoma nobilis was originally described from dying leaves of Dictamnus fraxinella in Anlagern Turnau, Czechia (conidia aseptate, later becoming medially 1-septate). However, Bubák & Kabät (1904) did not designate a holotype specimen. Later a representative culture of H. nobilis (CBS 507.91) was described by Boerema et al. (2004), which was isolated from same host in Europe. The morphology of CBS 507.91 agreed with the original description of H. nobilis (De Gruyter & Noordeloos 1992). Since no holotype specimen could be located in this study, we designated CBS 507.91 as ex-neotype culture.
Heterophoma verbasci-densiflori L.W. Hou, L. Cai & Crous, nom. nov. MB834017. Fig. 7.
Heterophoma verbasci-densiflori (CBS 127.93). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H–I. Pycnidia. J. Section of pycnidial wall. K–N. Conidiogenous cells. O–P. Conidia. Scale bars: H, I = 100 μm; J, O, P = 10 μm; K–N = 5 μm.
Basionym: Phyllosticta verbascicola Ellis & Kellerm., Bull. Torrey Bot. Club 11: 115. 1884.
Synonyms: Phoma poolensis var. verbascicola (Ellis & Kellerm.) Aa & Boerema, Persoonia 15: 385. 1993.
Phoma novae-verbascicola Aveskamp et al., Stud. Mycol. 65: 41. 2010.
Heterophoma novae-verbascicola (Aveskamp et al.) Qian Chen et al., Stud. Mycol. 82: 165. 2015. Nom. illegit., Art. 52.1.
Description from ex-epitype (CBS 127.93): Conidiomata pycnidial, semi-immersed in the agar, mostly solitary, sometimes confluent, scattered, (sub-)globose, whitish to buff at beginning, irregular-shaped and pale brownish with age, ostiolate, 160–390 × 155–345 μm. Ostioles 1–6, up to 12 with age, papillate or elongated into a short neck. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 3–5 layers, 10–17 μm thick, the outer two cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, subglobose, lageniform to ampulliform, or doliiform, 5–7.5(–10.5) × 3.5–8 μm. Conidia two types, both originating from the same pycnidium. Conidia of type 1: oblong, smooth- and thin-walled, hyaline, aseptate, 3.5–5.5 × 2–3 μm, two polar guttulate, medium. Conidia of type 2: globose to sub-globose, smooth- and thin-walled, aseptate, 3–4 × 2.5–3.5 μm, guttulate. Chlamydospores not observed.
Culture characteristics: Colonies on OA reaching 60–65 mm diam after 7 d at 25 °C, margin regular, aerial mycelium sparsely, buff to pale brown; reverse rosy buff to pale brown. Colonies on MEA reaching 60–65 mm diam after 7 d, margin regular, aerial mycelium woolly, whitish to buff; reverse orange to pale brown. Colonies on PDA reaching 65–70 mm diam after 7 d, margin regular, covered by floccose aerial mycelium, whitish to buff; reverse buff to pale brown. NaOH spot test negative on OA.
Typus: The Netherlands, Utrecht Province, Zeist, Abburg nursery, from Verbascum sp. (Scrophulariaceae) (holotype L 9893.00.134); North Holland Province, Haarlem, from dead stem of Verbascum densiflorum, Jan. 1993, J. de Gruyter (epitype designated here MBT389693, CBS H-24314, ex-epitype living culture CBS 127.93 = PD 92/347).
Additional material examined: The Netherlands, Baarn, from dead stem of Verbascum sp., 29 Jun. 1981, H.A. van der Aa, culture CBS 449.81.
Notes: Isolates CBS 127.93 and CBS 114.93 were originally received as “Phoma poolensis var. verbascicola” (De Gruyter et al. 1993). Later, Boerema et al. (2004) described CBS 127.93 as a representative culture of Phoma poolensis var. verbascicola, as it agreed well with the original description with regards to morphology, host and location. Subsequently, a new name, Phoma novae-verbascicola, was proposed for this species as it is distinguishable from Phoma poolensis and could not be elevated to species level as Phoma verbascicola has already occupied. When Chen et al. (2015) allocated this name to Heterophoma, they should have given preference to the older epithet of Phyllosticta verbascicola, thus rendering their new combination illegitimate. The name is herewith validated with the introduction of a new name, H. verbasci-densiflori, as H. verbascicola is already occupied. In the present study, we followed Boerema’s suggestion and designated CBS 127.93 as ex-epitype of this species.
Phylogenetically, isolate CBS 114.93 formed an intermediate clade between H. verbasci-densiflori and H. verbascicola (described from leaves of Verbascum thapsus in China) and appeared to represent an undescribed species (Fig. 1).
Clade 4: Boeremia Aveskamp et al., Stud. Mycol. 65: 36. 2010.
Type species: Boeremia exigua (Desm.) Aveskamp et al.
Boeremia foveata (Foister) Aveskamp et al., Stud. Mycol. 65: 40. 2010.
Basionym: Phoma foveata Foister, Trans. & Proc. Bot. Soc. Edinburgh 33: 66. 1940.
Typus: UK, from a tuber canker on Solanum tuberosum (Solanaceae), unknown date, C.E. Foister, ex-isotype culture CBS 200.37.
Additional material examined:Bulgaria, from a tuber of Solanum tuberosum (Solanaceae), Jan. 2001, H. de Gruyter, culture CBS 109176 = CECT 2828 = PD 94/1394.
Note: In this paper, the ex-isotype strain of Boeremia foveata (CBS 200.37) was examined, LSU and rpb2 sequences were added, and a more complete tub2 sequence was provided.
Clade 5: Chaetasbolisia Speg., Physis Rev. Soc. Arg. Cienc. Nat. 4: 293. 1918.
Conidiomata pycnidial, solitary, globose to subglobose, superficial, black, ostiolate. Pycnidial wall textura angularis, with long branched septate fuliginous hyphal outgrowths or glabrous. Conidiogenous cells phialidic, hyaline, smooth, globose, ampulliform to lageniform. Conidia globose, broad ovoid, cylindrical or oblong, hyaline, smooth- and thin-walled, aseptate, eguttulate or guttulate. (Griffon and Maublanc, 1909, Spegazzini, 1918).
Type species: Chaetasbolisia erysiphoides (Griffon & Maubl.) Griffon & Maubl.
Notes: Chaetasbolisia was introduced by Spegazzini in 1918 to accommodate Chaetophoma erysiphoides with a brief generic description (Spegazzini 1918). Subsequently, additional species were introduced in this genus, but most lacking molecular data. In the present study, Chaetasbolisia is represented by two species and forms a well-supported separate clade from other genera of Didymellaceae. The generic circumscription of Chaetasbolisia is emended to incorporate the morphological features of these newly added species, such as glabrous pycnidial conidiomata and cylindrical or oblong shaped conidia. However, the taxonomy of the genus Chaetasbolisia remains unresolved.
Chaetasbolisia argentina L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833551. Fig. 8.
Chaetasbolisia argentina (CBS 148.94). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H–I = 50 μm; J, N = 10 μm; K–M = 5 μm.
Etymology: Epithet derived from the country of origin, Argentina.
Description: Conidiomata pycnidial, (semi-)immersed, solitary, sometimes 2–4 confluent, scattered or aggregated, mostly (sub-)globose or flask-shaped, pale brown, darker brown around the ostioles, thin-walled, glabrous, ostiolate, 110–460 × 110–310 μm. Ostioles mostly 1–2, increasing to 8 when confluent, slightly papillate. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 2–4 layers, 10–28.5 μm thick, outer 2 cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, globose, lageniform, ampulliform or doliiform, 5–8.5 × 4.5–6.5 μm. Conidia cylindrical or oblong, hyaline, smooth- and thin-walled, aseptate, 4–7.5 × 2–2.5 μm, with two small, polar guttules. Conidial matrix whitish.
Culture characteristics: Colonies on OA reaching 80–85 mm diam after 7 d at 25 °C, margin regular, aerial mycelium flat, grey olivaceous, pale brown near the centre, abundant production of pycnidia; reverse olivaceous grey, orange near the centre. Colonies on MEA reaching 80–85 mm diam after 7 d, margin regular, aerial mycelium felty, pale olivaceous grey to olivaceous grey; reverse olivaceous, orange near the margin. Colonies on PDA reaching 80–85 mm diam after 7 d, margin regular, covered by felty aerial mycelium, pale olivaceous grey, olivaceous black towards the periphery; reverse olivaceous grey. NaOH spot test negative on OA.
Typus: Argentina, from soil, Apr. 1994, A. Arambarri (holotype CBS H-24310, ex-type living culture CBS 148.94).
Notes: Chaetophoma erysiphoides was originally described from Quercus ilicis in Cadillac, Gironde, France (Griffon & Maublanc 1909). Later a new genus, Chaetasbolisia, was proposed to accommodate this species (Spegazzini 1918). In the present study, isolate CBS 148.94 which was initially identified as Chaeta. erysiphoides from soil in Argentina was examined. It differed from the original description of Chaeta. erysiphoides by its larger guttulate conidia (4–7.5 × 2–2.5 μm, 2–3 guttules vs. 3–5.5 × 2.5–4 μm, eguttulate), and was therefore described as a new species.
Chaetasbolisia eupatorii (Died.) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833496. Fig. 9.
Chaetasbolisia eupatorii (CBS 123.93). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H = 100 μm; I = 50 μm; J = 10 μm; K–N = 5 μm
Basionym: Phoma eupatorii Died., Ann. Mycol. 10: 447. 1912.
Description from ex-epitype (CBS 123.93): Conidiomata pycnidial, superficial on the agar or (semi-)immersed in the agar, solitary, sometimes confluent, scattered or aggregated, mostly (sub-)globose to flask-shaped, whitish to buff at beginning, brownish with age, with white short hyphal outgrowths, ostiolate, 250–400 × 250–350 μm. Ostioles 1–6(–10), non-papillate or slightly papillate. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 4–5 layers, 16–42.5(–52) μm thick, without pigmented layers or outer 1–3 cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, subglobose, ampulliform, lageniform to doliiform, with periclinal thickening, 4.5–10.5 × 4–9.5 μm. Conidia cylindrical, oblong or ovoid, hyaline, smooth- and thin-walled, aseptate, 4–6 × 2–2.5 μm, with two medium, polar guttules. Conidial matrix buff.
Culture characteristics: Colonies on OA reaching 55–60 mm diam after 7 d at 25 °C, margin irregular, aerial mycelium flat, abundant production of pycnidia, scattered near the centre, in the beginning whitish, but turning saffron because of conidial mass exuding from pycnidia, umber towards the periphery; reverse concolourous. Colonies on MEA reaching 60–65 mm diam after 7 d, margin irregular, covered by floccose aerial mycelium, buff, whitish towards the periphery, dark brown conidiomata formed in centre; reverse concentric circles of different colours, centre buff, olivaceous black, yellow towards periphery, a dark brown circle formed in the middle. Colonies on PDA reaching 50–55 mm diam after 7 d, margin regular, aerial mycelium floccose, brown, whitish near the centre and buff towards periphery; reverse dark brown, whitish towards periphery. NaOH spot test negative on OA.
Typus: Germany, Sperenberg bei Zossen, from stems of Eupatorium cannabinum (Asteraceae), 16 May 1912, H. Sydow (lectotype designated here MBT389670, F56137). The Netherlands, Gelderland Province, Arnhem, Openlucht museum, from dead stem of Eupatorium cannabinum (Asteraceae), Jan. 1993, J. de Gruyter (epitype designated here, CBS H-23676, MBT389671, ex-epitype living culture CBS 123.93 = PD 77/1148).
Notes: This species was initially described from dead stems of Eupatorium cannabinum in Germany, with conidia being oblong-elliptical, usually with two small oil droplets, 4–5.5 × 1.5–2 μm (Sydow & Sydow 1912). However, no holotype specimen was cited in the original description. In the present study, we located two type specimens in the Swedish Museum of Natural History (S), and both properly matched the information in the protologue, including one (F49345) from Sydow’s own herbarium. Since the holotype specimen was not cited in the original paper, we designated F49345 as lectotype. Furthermore, in this study, the isolate (CBS 123.93) from Eu. cannabinum in the Netherlands agreed well in morphology with the protologue, as conidia were aseptate, measuring 4–6 × 2–2.5 μm, with two small-sized polar guttules. Thus CBS 123.93 was designated as ex-epitype culture and a new combination Chaetasbolisia eupatorii was therefore introduced. Chaetasbolisia eupatorii differed from other species in this family by producing whitish pycnidia covered in whitish mycelium.
Clade 6: Macroascochyta L.W. Hou, L. Cai & Crous, gen. nov. MycoBank MB833497.
Etymology: Morphologically resembling the genus Ascochyta, but distinct in having larger conidia.
Conidiomata pycnidial, solitary or confluent, globose to subglobose, semi-immersed or immersed in the agar, ostiolate. Pycnidial wall pseudoparenchymatous, multi-layered, outer layers pigmented. Conidiogenous cells phialidic, hyaline, smooth, globose, ampulliform to lageniform. Conidia cylindrical, bacilliform to fusiform, sometimes curved, hyaline, smooth- and thin-walled, aseptate, guttulate. Chlamydospores hyaline or pale brown, intercalary, unicellular, globose to subglobose, solitary, eguttulate. Sexual morph unknown.
Notes: A new asexual species clustered in Didymellaceae as a distinct monophyletic clade (Fig. 1) in both ML and Bayesian analyses. This species was collected from Tradescantia sp. in post entry quarantine in New Zealand. Since fungi collected herein clearly form an independent lineage and are phylogenetically segregated from other genera, we introduce Macroascochyta as a new genus to accommodate this species.
Type species: Macroascochyta grandis L.W. Hou, L. Cai & Crous
Macroascochyta grandis L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833498. Fig. 10.
Macroascochyta grandis (CBS 100409). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N–O. Chlamydospores. P. Conidia. Scale bars: H–I = 50 μm; J–P = 10 μm.
Etymology: Name reflects the large (= grandis in Latin) conidia produced by this species.
Description: Conidiomata pycnidial, (semi-)immersed in the agar, solitary, scattered or aggregated, mostly (sub-)globose to flask-shaped, pale to dark brown, with hyphal outgrowths, ostiolate, 175–400 × 160–375 μm. Ostioles single, central, slightly papillate. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 4–6 layers, 18.5–35.5 μm thick, without pigmented layers or outer 1–4 cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, (sub-)globose, ampulliform to lageniform, with minute periclinal thickening, 7–13.5 × 6.5–12.5 μm. Conidia cylindrical, bacilliform to fusiform, hyaline, smooth- and thin-walled, aseptate, 12.5–21 × 4.5–7 μm, with 6–10 small guttules each end. Chlamydospores hyaline or pale brown, intercalary, unicellular, (sub-)globose, solitary, eguttulate, thin-walled. Conidial matrix orange.
Culture characteristics: Colonies on OA reaching 70–75 mm diam after 7 d at 25 °C, margin irregular, aerial mycelium flat, buff to vinaceous buff, abundant production of pycnidia, orange conidial mass exuding from pycnidia; reverse pale cinnamon. Colonies on MEA reaching 75–80 mm diam after 7 d, margin irregular, covered by woolly aerial mycelium, whitish to pale grey; reverse orange to leaden black, yellow towards periphery. Colonies on PDA reaching 75–80 mm diam after 7 d, margin regular, aerial mycelium woolly, whitish to buff; reverse buff to pale brown. NaOH spot test negative on OA.
Typus: New Zealand, South Auckland, Alfriston, from Tradescantia sp. (Commelinaceae; in post entry quarantine), date unknown, coll. K. Ramsay, isol. C.F. Hill (holotype CBS H-23675, ex-type living culture CBS 100409).
Notes: Phoma commelinicola (basionym: Phyllosticta commelinicola) was originally reported from Commelina nudiflora in Puerto Rico, with conidia measuring 9.6–14.4 × 4.8–7.2 μm (Young 1915). The isolate CBS 100409 was collected from Tradescantia sp., the same host family as Phoma commelinicola (Commelinaceae), and therefore it was initially identified as “Phoma commelinicola” (De Gruyter 2002). However, its conidia [(10.5–)13–17(–21) × (4–)5–6.5 μm] were found to differ from the original description of Phoma commelinicola. Phylogenetically, CBS 100409 formed a distinct lineage, separated from all genera previously described in Didymellaceae. Thus, a new genus and species, Macroascochyta grandis, was introduced to accommodate this isolate.
Clade 7: Ectophoma Valenz.-Lopez et al., Stud. Mycol. 90: 34. 2017 (2018).
Type species: Ectophoma multirostrata (P.N. Mathur et al.) Valenz.-Lopez et al.
Ectophoma insulana (Mont.) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833480. Fig. 11
Ectophoma insulana (CBS 252.92). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–N. Conidiogenous cells. O. Conidia. Scale bars: H–I = 50 μm; J, O = 10 μm; K–N = 5 μm.
Basionym: Phyllosticta insulana Mont., Ann. Sci. Nat., Bot. IV 5: 343. 1856.
Synonym: Phoma insulana (Mont.) Boerema & Malathr., Versl. Meded. Plziektenk. Dienst Wageningen (Jaarb. 1981) 158: 28. 1982.
Description from ex-neotype (CBS 252.92): Conidiomata pycnidial, (semi-)immersed in the agar, solitary, or 2–5 confluent, scattered or aggregated, (sub-)globose, at the beginning buff, thin-walled, becoming dark brown with age, glabrous, ostiolate, 100–300(–510) × 100–270(–370) μm. Ostioles 1–4, increasing to 2–6(–12) when confluent, non-papillate or distinctly papillate. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 4–7 layers, 13–50 μm thick, outer 2–3 cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, ampulliform, lageniform or doliiform, 5–8 × 4–6.5 μm. Conidia ellipsoidal or oblong with both ends rounded, hyaline, smooth- and thin-walled, aseptate, 4–6.5 × 2–3 μm, with two large, polar guttules or several small scattered guttules. Conidial matrix whitish to buff.
Culture characteristics: Colonies on OA reaching 70–75 mm diam after 7 d at 25 °C, margin regular, aerial mycelium flat, olivaceous black; reverse concolourous. Colonies on MEA reaching 65–70 mm diam after 7 d, margin regular, covered by floccose aerial mycelium, with some radially furrowed zones, concentric circles of different colours, centre olivaceous, continued by a black circle formed by abundant pycnidia, buff towards periphery; reverse vinaceous buff to hazel. Colonies on PDA reaching 75–80 mm diam after 7 d, margin regular, covered by sparsely felty aerial mycelium, vinaceous buff; reverse olivaceous greenish. NaOH spot test negative on OA.
Typus: Greece, from fruit of Olea europaea (Oleaceae), deposited in May 1992, J. de Gruyter (neotype designated here CBS H-23679, MBT389673, ex-neotype living culture CBS 252.92 = PD 80/1144).
Additional material examined: South Africa, Western Cape Province, Kuilsrivier, Tarentaal Street, from house dust, 24 Jul. 2009, K. Jacobs, culture CBS 140548.
Notes: The holotype of Phoma insulana (basionym: Phyllosticta insulana), which was originally described from leaves of Olea europaea in Montaud-lès-Miramas, France, could not be located in PC (Muséum d'histoire naturelle; Montagne 1856), and is considered lost. In this study, isolate CBS 252.92 was collected from the same host in the Netherlands, and was described as a representative culture of Phoma insulana (De Gruyter et al. 1993). Here we designated CBS 252.92 as ex-neotype strain, as its morphological characters (conidial size) agreed well with the original description of Phyllosticta insulana (4–6.5 × 2–3 μm vs. 6 μm in length, Montagne 1856). Based on our phylogenetic result, culture CBS 252.92 clustered within the Ectophoma clade, forming a distinct lineage. Hence, we introduced a new combination for this taxon, Ectophoma insulana.
Ectophoma multirostrata (P.N. Mathur et al.) Valenz.-Lopez et al., Stud. Mycol. 90: 34. 2017 (2018).
Basionym: Sphaeronaema multirostratum P.N. Mathur et al., Sydowia 13: 146. 1959. (as “Sphaeronema”)
Synonyms: Phoma multirostrata (P.N. Mathur et al.) Dorenb. & Boerema, Mycopathol. Mycol. Appl. 50: 256. 1973, emend. Aveskamp et al. Mycologia 101: 375. 2009.
Phoma liliana S. Chandra & Tandon, Curr. Sci. 34: 566. 1965.
Phoma ehretiae S. Chandra & Tandon, Mycopathol. Mycol. Appl. 29 (3–4): 276. 1966.
Phoma terrestris Saksena et al., Mycopathol. Mycol. Appl. 29: 86. 1966 (as “P. terrestre”); non P. terrestris Hansen.
Description: Boerema et al. (2004).
Typus: India, Maharashtra, Poona, Talegaon, from poultry farm soil, Mar. 1959, M.J. Thirumalachar (isotype CBS H-7616, ex-isotype living culture CBS 274.60 = IMI 081598 = FMR 15335).
Additional materials examined : India, Maharashtra, Poona, Talegaon, from soil, Mar. 1959, M.J. Thirumalachar, culture CBS 368.65 = PD 92/1757 = FMR 15336; host unknown, M.J. Thirumalachar, culture CBS 383.65 (ex-isotype living culture of “Labyrinthophoma pimprina P.N. Mathur & Thirumalachar, ined.”); University of Allahabad, Botanical Garden, from leaves of Lilium sp. (Liliaceae), Nov. 1963, S. Chandra & R.N. Tandon, culture CBS 380.67 = IMI 105607 (ex-isotype living culture of Phoma liliana); host unknown, Jun. 1965, M.J. Thirumalachar, culture CBS 340.65 (ex-isotype living culture of “Sphaeronaema roseum P.N. Mathur & Thirumalachar, ined.”). The Netherlands, Hoorn, greenhouse, from the stem of Cucumis sativus (Cucurbitaceae), Aug. 1967, G.H. Boerema, culture CBS 110.79 = PD 65/8875 = FMR 15342.
Notes: CBS 380.67 and CBS 340.65 were received as ex-isotype strains of “Phoma liliana” and “Sphaeronaema roseum” respectively. Phoma liliana was originally described from leaves of Lilium sp. in India (Chandra & Tandon 1965). A subsequent study treated both species as synonyms of Phoma multirostrata (currently Ectophoma multirostrata) based on morphological characters (Dorenbosch & Boerema 1973). On the other hand, isolate CBS 383.65 was deposited in the CBS collection in 1965 as ex-isotype of “Labyrinthophoma pimprina”. However, together with Sp. roseum, these two names were never published in a resource indexed in Index Fungorum or MycoBank. In the present study, the ex-isotype strains of Phoma liliana, Sp. roseum and La. pimprina were revealed to be genetically identical to Ec. multirostrata in all sequenced loci, which verified the treatment of Dorenbosch & Boerema (1973).
Clade 8: Remotididymella Valenz.-Lopez et al., Stud. Mycol. 90: 35. 2017 (2018).
Conidiomata pycnidial, brown to dark brown, mostly confluent; pycnidial wall of textura angularis, mostly glabrous, globose or irregularly-shaped, with a single ostiole. Conidiogenous cells phialidic, hyaline, smooth-walled, globose or ampulliform. Conidia aseptate, hyaline, smooth- and thin-walled, allantoid or cylindrical, guttulate. (Valenzuela-Lopez et al. 2018). Chlamydospores intercalary or terminal, solitary, subhyaline to dark brown, variable, irregular, verruculose or incidentally tuberculate, 1–2 cells, smooth. Ascomata immersed or superficial, globose, conical globose to lenticular, scattered or clustered, papillate or apapillate, ostiolate. Peridium composed of several layers of brown to hyaline cells of textura angularis, fusing at the outside with the host tissue. Hamathecium dense, filamentous, septate, branching and hyaline, cellular pseudoparaphyses. Asci 8-spored, bitunicate, fissitunicate, clavate to cylindrical, short-pedicellate. Ascospores overlapping 2–3-seriate, hyaline, fusiform, 1–3-septate, constricted at middle septum (Jayasiri et al. 2019).
Type species: Remotididymella destructiva (Plowr.) Valenz.-Lopez et al.
Notes: Remotididymella was typified by R. destructiva (basionym Phoma destructiva) that is notorious by infecting tomato crops and causing light brown necroses (Boerema et al., 2004, Valenzuela-Lopez et al., 2018). Later, several new species were introduced in this genus, including R. bauhiniae that is known from its sexual morph. Remotididymella bauhiniae is the first record of a sexual morph for this genus, and therefore the generic description of Remotididymella is emended with the addition of sexual morph characters.
Remotididymella brunnea L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833555. Fig. 12.
Remotididymella brunnea. (CBS 993.95). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–N. Conidiogenous cells. O. Conidia. Scale bars: H, I = 20 μm; J = 10 μm; K–O = 5 μm.
Etymology: Name reflects the brown (= brunus in Latin) pycnidia produced by this species.
Description: Conidiomata pycnidial, immersed or semi-immersed, solitary, sometimes confluent, scatter or aggregated, (sub-)globose, dark brown at beginning, thin-walled, with mycelial outgrowths, ostiolate, become pale brown with age, 90–240 × 100–190 μm. Ostiole 0–1, slightly papillate or non-papillate. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 2–3 layers, 6.5–26 μm thick, outer 1–2 layers pigmented. Conidiogenous cells phialidic, hyaline, smooth, globose, ampulliform or lageniform, 5.5–8.5 × 4.5–7.5 μm. Conidia cylindrical to oblong with both ends rounded, hyaline, smooth- and thin-walled, aseptate, sometimes constricted at the middle part, 4.5–6.5 × 2–3 μm, with 2–3 large guttules. Conidial matrix white.
Culture characteristics: Colonies on OA reaching 65 mm diam after 7 d at 25 °C, margin regular, the section produced pycnidia without aerial mycelium, olivaceous, and the sterile sector covered by flat and grey to darker grey mycelium; reverse concolourous. Colonies on MEA reaching 50–55 mm diam after 7 d, margin regular, aerial mycelium floccose, buff; reverse dark brown with orange edge. Colonies on PDA reaching 65–70 mm diam after 7 d, margin regular, densely covered by felty buff aerial mycelium near the center, flat and pale olivaceous grey towards the periphery; reverse pale greenish grey. NaOH spot test negative on OA.
Typus: Papua New Guinea, Madang Provence, Brahman, from soil in tropical forest, Nov. 1995, A. Aptroot (holotype CBS H-23685, ex-type living culture CBS 993.95).
Notes: Phylogenetically, Remotididymella brunnea forms a sister clade to R. bauhiniae with moderate support. Remotididymella brunnea differs in 9 bp in ITS, 33 bp in rpb2 from R. bauhiniae (tub2 is missing in R. bauhiniae). However, the morphological comparison is difficult as R. bauhiniae was only described based on a sexual morph with production of chlamydospores (Jayasiri et al. 2019), while we only observed the asexual morph of R. brunnea in culture, lacking chlamydospores. Besides, morphological comparisons revealed that R. brunnea was similar to R. humicola, another novel species collected from soil and described in the present study, in the shape of its conidia and conidiogenous cells, but was clearly distinguished from the latter by producing shorter conidia [4.5–6.5 × 2–3 μm vs. 6–8.5(–11.5) × 2–3 μm].
Remotididymella capsici (Bond.-Mont.) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB834117.
Basionym: Ascochyta capsici Bond.-Mont., Monitor Phytopath. Sect. Chief Bot. Gard. R.S.F.S.R. 12: 72. 1923.
Description: Culture sterile.
Culture characteristics: Colonies on OA reaching 70–75 mm diam after 7 d at 25 °C, margin regular, pale brown without aerial mycelium near the centre, buff with flat aerial mycelium toward periphery; reverse buff to honey. Colonies on MEA reaching 65–70 mm diam after 7 d, margin regular, aerial mycelium floccose, whitish with some black zones; reverse pale brown near the centre and brown toward periphery. Colonies on PDA reaching 80–85 mm diam after 7 d, margin regular, covered by felty aerial mycelium, whitish, with olivaceous grey edge; reverse iron-black with buff edge. NaOH spot test a pale olivaceous discolouration on OA.
Material examined: The Republic of Fiji, from leaf spot on Capsicum annuum (Solanaceae), 9 Jul. 1977, isol. J.D. Baird, dep. G.F. Laundon, specimen CBS H-24340, culture CBS 679.77 = LEV 11926b.
Notes: In this study, isolate CBS 679.77 was originally received as Ascochyta capsici, isolated from the same host as As. capsici (from the living leaves of Capsicum annuum in Russia) in Fiji. Phylogenetically, CBS 679.77 clustered in Remotididymella, forming an independent lineage which was distinct from other species. Although we introduce a new combination for this taxon in Remotididymella, future collections from Capsicum annuum in Russia may well show CBS 679.77 to represent a new species.
Remotididymella humicola L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833499. Fig. 13.
Remotididymella humicola (CBS 120117). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H = 50 μm; I = 20 μm; J–N = 10 μm.
Etymology: Name derived from the substrate this species was collected from, soil.
Description: Conidiomata pycnidial, (semi-)immersed, solitary, scattered on the agar, (sub-)globose, pale brown, thin-walled, glabrous, ostiolate, 110–410 × 110–400 μm. Ostioles 1–5, papillate, sometimes elongated with a long neck, up to 100 μm. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 2–4 layers, 6–21(–30) μm thick, outer cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, globose to ampulliform, 5.5–9.5 × 5–8 μm. Conidia cylindrical or oblong, with both ends rounded, hyaline, smooth- and thin-walled, aseptate, sometimes constricted in the middle, 6–8.5(–11.5) × 2–3 μm, with 2–4 large, polar guttules. Conidial matrix whitish to buff.
Culture characteristics: Colonies on OA reaching 70 mm diam after 7 d at 25 °C, margin regular, without aerial mycelium, buff, abundant production of pycnidia; reverse concolourous. Colonies on MEA reaching 70–75 mm diam after 7 d, margin regular, without aerial mycelium, pinkish with some black zones, brown towards periphery, becoming flesh with age because of the conidial matrix exuding from pycnidia; reverse brownish. Colonies on PDA reaching 70–75 mm diam after 7 d, margin regular, covered by sparsely felty aerial mycelium, greyish brown with olivaceous margin; reverse concolourous. NaOH spot test negative on OA.
Typus: Puerto Rico, from soil in onion field, 1 Jul. 2004, A.R. Rossman (holotype CBS H-23687, ex-type living culture CBS 120117).
Notes: The genus Remotididymella was originally introduced to accommodate R. anthropophila and R. destructiva (Valenzuela-Lopez et al. 2018). Remotididymella humicola was represented by an isolate from soil collected in Puerto Rico, which formed a distinct lineage clearly different from other species in Remotididymella. Morphologically, R. humicola was distinguished from its closest neighbour R. anthropophila by having larger conidia [6–8.5(–11.5) × 2–3 μm vs. 5.5–7.5 × 1.5–2.5 μm] and conidiogenous cells (5.5–9.5 × 5–8 μm vs. 5–6 μm; Valenzuela-Lopez et al. 2018). Moreover, R. humicola and R. anthropophila also differed in the colour of their pycnidia and number of ostioles: R. anthropophila produced apricot to pale brown pycnidia with single ostioles, while pycnidia of R. humicola were brown with 1–6 ostioles.
Clade 9: Pseudopeyronellaea L.W. Hou, L. Cai & Crous, gen. nov. MycoBank MB833500.
Etymology: Name refers to the morphological similarity with the genus Peyronellaea.
Ascomata pseudothecial, globose, pyriform, superficial, with central papillate ostiole, lacking setae; wall of multi-layered. Pseudoparaphyses absent. Asci bitunicate, narrowly ellipsoid to subcylindrical, 8-spored. Ascospores bi- to triseriate, hyaline, ovate to fusoid, prominently guttulate with mucoid sheath, widest just above septum, ends subobtusely rounded. Asexual morph unknown.
Notes: Species of Peyronellaea are characterised by producing pycnidial conidiomata with phialidic conidiogenous cells as well as dictyochlamydospores, having both transverse and longitudinal septa (Aveskamp et al. 2009a). Later, Peyronellaea was synonymised with Didymella (Chen et al. 2015) after phylogenetic analysis. In the present study, Peyronellaea eucalypti that was collected from leaves of Eucalyptus pellita (Myrtaceae), with only a sexual morph observed in culture, is phylogenetically distant from the Didymella clade and other known genera of Didymellaceae. Therefore, a new genus Pseudopeyronellaea is introduced to accommodate Peyronellaea eucalypti. Morphologically, Pseudopeyronellaea differs from Didymella by producing bi- to triseriate, ovate to fusoid and prominently guttulate ascospores with mucoid sheaths, while ascospores of Didymella species are biseriate, ellipsoidal to cymbiform (Chen et al. 2015).
Type species: Pseudopeyronellaea eucalypti (Crous & M.J. Wingf.) L.W. Hou et al.
Pseudopeyronellaea eucalypti (Crous & M.J. Wingf.) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833501.
Basionym: Peyronellaea eucalypti Crous & M.J. Wingf., Persoonia 38: 277. 2017.
Description: Crous et al. (2017a).
Typus: Malaysia, Sabah, from leaves of Eucalyptus pellita (Myrtaceae), May 2015, M.J. Wingfield (holotype CBS H-23085, ex-type living culture CBS 142522 = CPC 27678); ibid., culture CPC 27682.
Notes: The holotype of Peyronellaea eucalypti was described from leaves of Eucalyptus pellita collected in Sabah, Malaysia (Crous et al. 2017a). Based on the phylogenetic analysis of the ITS gene, two isolates of Pe. eucalypti (CBS 142522 and CPC 27682) were phylogenetically related to Didymella glomerata (Crous et al. 2017a). However, in the multigene analysis these isolates formed an independent lineage distant from Peyronellaea (currently: Didymella). Consequently, Pseudopeyronellaea was introduced as a new genus to accommodate this taxon.
Clade 10: Longididymella L.W. Hou, L. Cai & Crous, gen. nov. MycoBank MB833502.
Etymology: Name reflects the longer (= Longi, in Latin) conidia characteristic of these fungi.
Ascomata pseudothecial, globose, subglobose to pyriform, superficial, solitary or clustered, ostiolate, with prominent elongated neck, lacking setae; wall black, textura globulosa, multi-layered. Pseudoparaphyses absent. Asci bitunicate, cylindrical with club-shaped base, 8-spored. Ascospores hyaline, septate, ovate to obpyriform, smooth. Ascospore mass white. Conidiomata pycnidial, superficial or semi-immersed, also immersed in the agar or on aerial mycelium, (sub-)globose, flask-shaped or irregular, glabrous, scattered, solitary or confluent, ostiolate. Ostioles on an elongated neck. Pycnidial wall pseudoparenchymatous, 2–5 layers, outer layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, variable in shape and size, globose, flask-shaped, ampulliform or doliiform, with minute periclinal thickening. Conidia ellipsoidal, elongated, allantoid, smooth- and thin-walled, 0–1-septate, mostly guttulate, incidentally smaller, aseptate conidia present.
Notes: Longididymella was proposed to accommodate two species collected from leaves of Clematis spp. Both species were originally received as Phoma clematidina. However, their sexual morphs were observed to be similar to Didymella (Woudenberg et al. 2009). By means of DNA sequence comparisons, they were described as members of Didymella (Woudenberg et al. 2009). In the present study, Didymella clematidis and Did. vitalbina formed a fully supported clade that is distant from Didymella and distinct from other known genera in family Didymellaceae. Considering that both species produced relatively long conidia, a new genus Longididymella is introduced.
Type species: Longididymella vitalbae (Crous & A.R. Wood) L.W. Hou et al.
Longididymella clematidis (Woudenb. et al.) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833503.
Basionym: Didymella clematidis Woudenb. et al., Persoonia 22: 60. 2009.
Description: Woudenberg et al. (2009).
Typus: USA, Washington State, Toppenish, on leaves of Clematis ligusticifolia (Ranunculaceae), 7 Sep. 1991, A.G. Spiers (holotype PDD69379, ex-type living culture ICMP 13664 = AGS 30/32 = CBS 123705 = PD 97/13460-1).
Notes: This species was originally described by Woudenberg et al. (2009) as Didymella clematidis, infecting leaves of Clematis ligusticifolia in Toppenish, Washington State, USA. However, in our phylogenetic analysis of more loci, the ex-type strain of Did. clematidis (CBS 123705) clustered within the new genus Longididymella introduced in the present study. Both species of Longididymella could produce sexual and asexual morphs in vitro.
Longididymella vitalbae (Briard & Har.) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833505.
Basionym: Ascochyta vitalbae Briard & Har., Rev. Mycol. (Toulouse) 13: 17. 1891.
Synonyms: Diplodina vitalbae (Briard & Har.) Allesch., Rabenh. Krypt.-Fl., ed. 2. Pilze 6 (Lief. 69): 683. 1900 (vol. dated1901).
Diplodina clematidina Fautrey & Roum., Rev. Mycol. (Toulouse) 14: 105. 1892.
Didymella vitalbina Petr., Ann. Mycol. 38: 348. 1940.
Description: Woudenberg et al. (2009).
Typus: Switzerland, Gampel-Steg, on leaves of C. vitalba, 10 Oct. 1991, A.G. Spiers, epitype of Didymella vitalbina PDD69378, ex-epitype living culture ICMP 13663 = AGS 9 = PD 97/13460-2 = CBS 123707.
Additional materials examined: France, Var, Jouques, on leaves of Clematis vitalba (Ranunculaceae), Dec. 1964, E. Müller, specimen CBS H-11972, culture ETH 2672 = CBS 454.64. Germany, Alf a. d. Mosel, on leaves of C. vitalba, 3 Oct. 1987, H.A. van der Aa, specimen CBS H-16192, culture CBS 911.87. The Netherlands, Renkum, Jufferswaard, on leaves of C. vitalba, 9 Apr. 2008, J. de Gruyter, culture CBS 123706.
Notes: The holotype of Didymella vitalbina (basionym: Ascochyta vitalbae) was originally isolated from Clematidis vitalbae in Gaisberg, Austria (Petrak 1940). Woudenberg et al. (2009) designated CBS 123707 as ex-epitype for this species based on its similar morphological characters. In this study, Did. vitalbina was represented by five strains including the ex-epitype (CBS 123707), which together with the ex-type strain of Did. clematidis (CBS 123705), formed a fully supported independent lineage that was distinct from its closest genus Ectophoma. Therefore, Longididymella was proposed as a new genus to accommodate these two species.
Clade 11: Ectodidymella L.W. Hou, L. Cai & Crous, gen. nov. MycoBank MB833506.
Etymology: From the Greek εκτοs, outside, because it is phylogenetically distant from Didymella.
Conidiomata pycnidial, scattered, globose, depressed globose, black, large, usually with a conspicuous neck. Pycnidial wall thick, pseudoparenchymatous, outer layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, ampulliform to doliiform, with minute periclinal thickening. Conidia oblong to subcylindrical, eguttulate or with a small guttule at each end. Ascomata pseudothecial, only recorded in one species in vivo, erumpent, globose to irregular-shaped, solitary or confluent, scattered or aggregated, ostiolate, with a small papilla-shaped ostioles, lacking setae. Pseudothecial wall rough, multi-layered, composed of pseudoparenchymatous cells, outer layers pigmented, black-brown. Asci narrow, cylindrical to clavate, 4–8-spored, coarse and thick-walled. Pseudoparaphyses scanty and atypical, coarse, branched. Ascospores hyaline, biseriate, elongated clavate to fusiform, uniseptate, slightly or barely constricted at the septum, upper cell slightly wider than the lower cell (Petrak 1928).
Notes: Ectodidymella is typified by Phoma nigrificans. The sexual morph of Phoma nigrificans (Didymella macropodii) is morphologically similar with species of Didymella. However, phylogenetically it forms a distinct clade that is distant from Didymella and separated from all genera previously described in Didymellaceae. Morphologically, Ectodidymella differs from Didymella by occasionally producing four ascospores in a single ascus, which is rare in Didymellaceae.
Type species: Ectodidymella nigrificans (P. Karst.) L.W. Hou et al.
Ectodidymella nigrificans (P. Karst.) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833507. Fig. 14.
Ectodidymella nigrificans (CBS 100190). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–N. Conidiogenous cells. O. Conidia. Scale bars: H–I = 50 μm; J, O = 10 μm; K–N = 5 μm.
Basionym: Sphaeronaema nigrificans P. Karst., Meddeland. Soc. Fauna Fl. Fenn. 16: 17. 1888. (as “Sphaeronema”).
Synonyms: Phoma nigrificans (P. Karst.) Boerema et al., J. Phytopathol. 115(3): 270. 1986.
Didymella macropodii Petr., Hedwigia 68: 219. 1928.
Plenodomus macropodii Petr., Hedwigia 68: 237. 1928.
Description: Boerema & de Gruyter (1998) and Petrak 1928.
Materials examined: Germany, from Brassica napus (Brassicaceae), 1982, G.H. Boerema, culture CBS 100190 = PD 82/736. The Netherlands, from an unidentified crucifer (Cruciferae sp., Brassicaceae), 1984, G.H. Boerema, culture PD 84/512.
Notes: Sphaeronaema nigrificans was described from Armoraciae rusticanae in Finland, with conidia measuring 6–9 × 2–3 μm (Karsten 1888). This species was later recombined into Phoma as Phoma nigrificans, while its connection with Didymella macropodii was established by Boerema et al. (1986). Later Boerema et al. (2004) described CBS 100190 and CBS 100191 as representative cultures of Phoma nigrificans based on morphology. However, CBS 100191 clustered with Phomatodes nebulosa (Chen et al. 2015), distinct from CBS 100190, which was also examined in the present study (Fig. 1). Isolate CBS 100190, together with PD 84/512 formed an independent clade which was distant from other genera in Didymellaceae. Morphologically CBS 100190 agreed well with the original description of Sp. nigrificans by producing comparably sized conidia (6–9 × 2–3 μm vs. 4.5–8 × 2–3.5 μm). Hence, a new genus Ectodidymella was introduced to accommodate this species.
Clade 12: Epicoccum Link, Mag. Neuesten Entdeck. Gesammten Naturk. Ges. Naturf. Freunde Berlin 7: 32. 1815, emend. Q. Chen & L. Cai, Stud. Mycol. 82: 171. 2015.
Type species: Epicoccum nigrum Link
Epicoccum brahmansense L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833508. Fig. 15.
Epicoccum brahmansense (CBS 990.95). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–L. Conidiogenous cells. M. Conidia. Scale bars: H = 50 μm; I = 20 μm; J = 10 μm; K–M = 5 μm.
Etymology: Epithet derived from the location of origin, Brahman, Madang Province in Papua New Guinea.
Description: Conidiomata pycnidial, (semi-)immersed in agar, solitary or confluent, scattered, mostly (sub-)globose to flask-shaped, pale brown, thin-walled, with hyphal outgrowths, ostiolate, 150–350 × 135–265 μm. Ostioles 1–4(–8), non-papillate or slightly papillate and forming a collarette. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 3–6 layers, (8.5–)12.5–29 μm thick, outer 1–3 cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, globose to ampulliform, 5–9 × 3.5–8 μm. Conidia ovoid to ellipsoidal, hyaline, smooth- and thin-walled, aseptate, 3.5–6 × 2.5–3.5 μm, with two medium, polar guttules. Conidial matrix whitish.
Culture characteristics: Colonies on OA reaching 50–55 mm diam after 7 d at 25 °C, margin regular, aerial mycelium felty, whitish to pink; reverse olivaceous to dark brick. Colonies on MEA reaching 50–55 mm diam after 7 d, margin regular, aerial mycelium abundant, woolly, buff, mycelium flat and dark brown near the centre; reverse brown with orange edge. Colonies on PDA reaching 40–45 mm diam after 7 d, margin regular, covered by felty aerial mycelium, floccose near the centre, buff to pink, smoke grey toward periphery; reverse olivaceous with brownish centre, abundant dark brown secondary metabolite visible. NaOH spot test: olivaceous with red margin on OA.
Typus: Papua New Guinea, Madang Province, Brahman, from soil in tropical forest, Nov. 1995, A. Aptroot (holotype CBS H-23667, ex-type living culture TA10NZ-306 = CBS 990.95).
Additional material examined: Papua New Guinea, Madang Province, Brahman, from soil in tropical forest, Nov. 1995, A. Aptroot, culture CBS 985.95.
Notes: In the phylogenetic tree, Epicoccum brahmansense was represented by two isolates (CBS 990.95 and CBS 985.95; from soil in Papua New Guinea) which clustered in a distinct clade clearly distinguished from other species in this genus. Morphologically, Ep. brahmansense could be differentiated from its closest neighbours Ep. draconis and Ep. multiceps in its conidial size and the numbers of ostioles: Ep. brahmansense differed from Ep. draconis in its larger pycnidia (150–350 × 135–265 μm vs. 90–220 μm) and smaller conidia (3.5–6 × 2.5–3.5 μm vs. 4–8.5 × 2–4 μm). Although Ep. brahmansense is similar to Ep. multiceps in conidial size, the latter usually produces larger pycnidia with more ostioles [150–350 × 135–265 μm, 1–4(–8) ostioles vs. 135–540 × 128–465 μm, 3–13 ostioles].
Epicoccum dickmanii L.W. Hou & O. Yarden, sp. nov. MycoBank MB834682. Fig. 16.
Epicoccum dickmanii (CBS 124671). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H–I. Pycnidia. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H–I = 100 μm; J–N = 10 μm.
Etymology: Name in memory of Prof. Martin B. Dickman, a leader in the study of fungal biology and fungal-host interactions.
Description: Conidiomata pycnidial, immersed in agar, scattered, mostly solitary, (sub-)globose, or ellipsoidal, rosy buff to buff, thin-walled, with hyphal outgrowths, 100–310 × 95–260 μm. Ostioles inconspicuous. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 3–4 layers, 8–10 μm thick, without pigmented layers. Conidiogenous cells phialidic, hyaline, smooth, ampulliform or lageniform, 7.5–12 × 7.5–10 μm. Conidia variable in shape and size, oblong, ovoid or broad ellipsoidal, smooth- and thin-walled, aseptate, 4.5–10.5 × 3–4.5 μm, with 2–3 minute guttules. Conidial matrix rosy buff.
Culture characteristics: Colonies on OA reaching 45–50 mm diam after 7 d 25 °C, margin regular, aerial mycelium floccose, whitish, aerial mycelium flat and buff towards periphery; reverse buff to pale olivaceous. Colonies on MEA reaching 45–55 mm diam after 7 d, margin regular, aerial mycelium woolly, rosy buff; reverse orange, buff towards periphery. Colonies on PDA reaching 50–55 mm diam after 7 d, margin regular, aerial mycelium floccose, buff near the centre, aerial mycelium sparse, pale brown to whitish toward periphery, abundant production of pycnidia; reverse buff, pale brown to brown. NaOH spot test negative on OA.
Typus: Australia, Great Barrier Reef, from a brown band on Acropora formosa (Acroporidae; coral), Jul. 2006, O. Yarden (holotype CBS H-24342, ex-type living culture CBS 124671 = OY8406).
Additional material examined: Australia, Great Barrier Reef, from a brown band on Acropora formosa, Jul. 2006, O. Yarden, culture CBS 124672.
Notes: Two strains of Epicoccum dickmanii that were isolated from Acropora formosa formed a distinct branch on the multi-locus tree (Fig. 1). Epicoccum dickmanii is phylogenetically close to Ep. duchesneae, a species reported from leaves of Duchesnea indica in China. However, it differs from the latter in producing pycnidia without conspicuous ostioles while Ep. duchesneae produces pycnidia with single, sometimes elongated ostioles. Besides, the conidia and conidiogenous cells of Epicoccum dickmanii (conidiogenous cells 7.5–12 × 7.5–10 μm, and conidia 4.5–10.5 × 3–4.5 μm) are larger than those of Ep. duchesneae (conidiogenous cells 4.5–9.5 × 3.5–7 μm, and conidia 2.5–3.5 × 1.5–2 μm).
Epicoccum longiostiolatum L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833510. Fig. 17.
Epicoccum longiostiolatum (CBS 886.95). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H–I = 50 μm; J = 10 μm; K–N = 5 μm.
Etymology: Name refers to the long ostiolate of conidiomata.
Description: Conidiomata pycnidial, produced superficially on agar, as well as on aerial mycelium, solitary, sometimes 2–3 confluent, scattered or aggregated, mostly (sub-)globose or lageniform, with some mycelium outgrowths, pale brown, thin-walled, ostiolate, 165–435 × 140–325 μm. Ostioles 1–2, up to four when confluent, papillate, sometimes elongated to a short neck. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 4–5 layers, 10–34 μm thick, outer three cell layers pigmented. Conidiogenous cells phialidic, hyaline, smooth, ampulliform to lageniform, or irregular-shaped, 6.5–11.5 × 4–9 μm. Conidia variable in shape and size, oblong, ovoid or ellipsoidal, smooth- and thin-walled, aseptate, 5–9(–12) × 2–4 μm, with two small, polar guttules; globose conidia smooth- and thin-walled, aseptate, 3.5–4 × 3–3.5 μm. Conidial matrix whitish.
Culture characteristics: Colonies on OA reaching 70–75 mm diam after 7 d at 25 °C, margin regular, covered by woolly aerial mycelium, buff with pale olivaceous margin; reverse buff with pale olivaceous zones. Colonies on MEA reaching 60–65 mm diam after 7 d, margin irregular, covered by woolly aerial mycelium, with feathered edge, slightly pinkish and buff near the centre, with whitish edge; reverse orange. Colonies on PDA reaching 40–45 mm diam after 7 d, margin irregular, densely covered by felty aerial mycelium, whitish with olivaceous buff zones; reverse gradually changed from brown to yellow, buff towards periphery. NaOH test on OA changes from buff to herbage green with red margin.
Typus: Papua New Guinea, Northern Province, Myola, from Stellaria sp. (Caryophyllaceae), Oct. 1995, A. Aptroot (holotype CBS H-23666, ex-type living culture CBS 886.95).
Additional material examined: Papua New Guinea, Northern Province, Myola, Owen Stanley Range, near guesthouse, alt. 2 100 m, from Cyperus sp. (Cyperaceae), 19 Oct. 1995, A. Aptroot, specimen CBS H-6369, culture CBS 902.96.
Notes: The isolates CBS 886.95 and CBS 902.96 were originally identified as Phoma sorghina, but formed an obviously distinct clade from other strains of this species (Aveskamp et al. 2009a). Phoma sorghina was subsequently transferred to the genus Epicoccum after phylogenetic analyses, excluding the isolates CBS 886.95 and CBS 905.96 (Aveskamp et al. 2010). In our phylogenetic study, these two isolates formed a well-supported clade distant from Phoma sorghina (currently Ep. sorghinum). Thus Ep. longiostiolatum was introduced to accommodate these isolates. Morphologically, Ep. longiostiolatum differs from its most closely related species, Ep. polychromum, by producing larger conidia [5–9(–12) × 2–4 μm vs. 4.5–8 × 2–4 μm] and having larger conidiogenous cells (6.5–11.5 × 4–9 μm vs. 4.5–8.5 × 3.5–7 μm).
Epicoccum multiceps L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833557. Fig. 18.
Epicoccum multiceps (CBS 119734). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H–I. Pycnidia. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H = 100 μm; I = 50 μm; J = 10 μm; K–N = 5 μm.
Etymology: Name derived from the characteristic that many ostioles are produced on the pycnidia by this species.
Description: Conidiomata pycnidial, produced partly or fully immersed in the agar, mostly solitary, sometimes two or more aggregated, variable in shape and size, (sub-)globose, flask-shaped, irregular-shaped with age, pale brown, dark brown around the ostioles, thin-walled, glabrous, ostiolate, 135–540 × 128–465 μm. Ostioles 3–11(–13), slightly papillate or non-papillate. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 2–4 layers, 11–25 μm thick, outer two cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, globose, ampulliform to lageniform, 3.5–7.5 × 3.5–6 μm. Conidia ellipsoidal, oblong or ovoid, smooth- and thin-walled, aseptate, 3.5–5.5 × 2–3 μm, with 2–3 medium, polar guttules. Conidial matrix whitish.
Culture characteristics: Colonies on OA reaching 60–65 mm diam after 7 d at 25 °C, margin regular, aerial mycelium flat, concentric circles of different colour, centre buff and olivaceous, pale olivaceous grey, dark brick, buff towards periphery; reverse brown to darker brown. Colonies on MEA reaching 60–65 mm diam after 7 d, margin regular, aerial mycelium felty, pale olivaceous grey, whitish near the centre and buff towards periphery; reverse concentric circles of different colour, centre black, dark brown, orange to yellow. Colonies on PDA reaching 60–65 mm diam after 7 d, margin regular, covered by sparse felty aerial mycelium, buff to pale honey, flat and pale brick coloured towards periphery; reverse olivaceous black, pale brick towards periphery. NaOH spot test negative on OA.
Typus: Brazil, São Paulo, Araraquara, from leaf of Cupania vernalis (Sapindaceae), Jun. 2003, L.H. Pfenning (holotype CBS H-23678, ex-type living culture CBS 119734 = CML 289).
Notes: Phylogenetically, Epicoccum multiceps clustered with the representative strain of Ep. draconis (CBS 186.83; Fig. 1), but could be differentiated from the latter in producing larger pycnidia (135–540 × 128–465 μm vs. 90–220 μm diam) with more ostioles [3–11(–13) vs. 1–2] and smaller conidia (3.5–5.5 × 2–3 μm vs. 4–8.5 × 2–4 μm). In addition, the test of metabolite E production was negative for Ep. multiceps while a positive reaction as a greenish, then red (E+ reaction) discolouration was observed in Ep. draconis (De Gruyter et al. 1998). This is the first and only report of a Didymellaceae species infecting a member of Cupania (Sapindaceae).
Epicoccum mezzettii Goid., Boll. Staz. Patol. Veg. Roma, N.S. 17: 452. 1937.
Description: Mycelium hyaline at beginning, 1.8 μm in diam, later become brownish, regular thickened at septa, branched, 2.5–5.2 in diam. Colony dull yellow to brown. Sporodochia black, stromata formed from the irregularly rounded rectangular cells. Conidiophores short, unicellular, bearing conidia at the apex. Conidia spherical, obscure, septate, thickened with age, red-brown, with or without few but large incrustations, conidia size constant, 9–11 μm (Goidànich 1937).
Typus: Italy, from Populus sp. (Salicaceae) wood pulp in paper factory, Apr. 1938, isol. G. Goidánich, culture CBS 173.38 (ex-type living culture of Epicoccum mezzettii).
Additional materials examined: Germany, former West-Germany, Solanum tuberosum (Solanaceae), Dec. 1922, H.W. Wollenweber, culture CBS 120.22. India, Madhya Pradesh, Balaghat, from leaf of Artocarpus heterophylla (Moraceae), Jul. 1972, S.M. Singh, culture CBS 873.72 = IFO 32913. Unknown, from leaf of Malus sylvestris (Rosaceae), Nov. 1959, D. Mulder, culture CBS 238.59 = IMI 079498; from Triticum aestivum, unknown date and collector, culture CBS 169.33.
Notes: Epicoccum mezzettii was originally isolated from wood pulp for paper making in Italy (Goidànich 1937). However, together with several other Epicoccum species, it was incorrectly treated as synonym of Ep. nigrum by Schol-Schwarz (1959). In this study, CBS 173.38 (ex-type living culture of Ep. mezzettii), together with four other plant-associated isolates, formed a distinct clade that was phylogenetically distant from the ex-type strain of Ep. nigrum (CBS 173.73). Morphologically, Ep. mezzettii is characterised by producing consistently smaller conidia compared with Ep. nigrum (9–11 μm vs. 13–31 μm) and less verrucose on conidia (Goidànich, 1937, Punithalingam et al., 1972). Besides, conidiophores of Ep. mezzettii are unicellular, while that of Ep. nigrum are usually 2-septate. Therefore, Ep. mezzettii was resurrected as a separate species.
Epicoccum oryzae S. Ito & Iwadare, Rep. Hokkaido Prefect. Agric. Exp. Sta. 31: 1. 1934.
Description: Culture sterile.
Culture characteristics: Colonies on OA reaching 65–70 mm diam after 7 d 25 °C, margin regular, aerial mycelium felty, rosy buff, greyish near the centre; reverse brick to dark brown. Colonies on MEA 60–65 mm diam after 7 d, margin regular, densely covered by felty aerial mycelium, rosy buff, with some brick or pinkish zones; reverse brick. Colonies on PDA, 65–70 mm diam after 7 d, margin regular, covered by felty aerial mycelium, rosy buff to brick with pale olivaceous margin; reverse brick near the centre, coral towards periphery. NaOH spot test negative on OA.
Typus: Japan, from Oryza sativa (Poaceae), Jan. 1934, deposited by S. Ito (neotype designated here CBS H-24312, MBT390301, ex-neotype living cultures CBS 173.34 = ATCC 24428 = IMI 164070); ibid., culture CBS 174.34.
Notes: Epicoccum oryzae was initially described from harvested rice grains causing “red blotch” disease in Hokkaido, Japan (Ito & Iwadare 1934), which is characterised by “olivaceous hyphae, globose or subglobose sporodochia, and globose, subglobose, or pyriform, granular, verrucose, olivaceous conidia, 9.9–23.1 × 6.6–16.5 μm, consisting of one to five cells”. However, Ep. oryzae was synonymised as Ep. nigrum (Schol-Schwarz 1959). The isolates CBS 173.34 and CBS 174.34 were recorded as Ep. oryzae, which were deposited in CBS collection by the original author of the species (S. Ito). Both isolates agreed well with the original description of Ep. oryzae with regard to the date, host and location. Archived correspondence revealed that they could be ex-isotype strains of Ep. oryzae. Since no holotype specimen is known, we proposed CBS 173.34 as ex-neotype of Ep. oryzae. Unfortunately, both isolates proved to be sterile, but still produced the red pigment on OA plates as reported in the original description. Phylogenetically, Epicoccum oryzae clustered in a sister clade to Ep. mackenziei, distant from the ex-type strain of Ep. nigrum (CBS 173.73). Therefore, Ep. oryzae was resurrected as a separate species.
Epicoccum polychromum L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833509. Fig. 19.
Epicoccum polychromum (CBS 141502). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–L. Conidiogenous cells. M. Chlamydospores. N. Conidia. Scale bars: H–I = 50 μm; J, M = 10 μm; K–L, N = 5 μm.
Etymology: Epithet derived from the colony colour.
Description: Conidiomata pycnidial, produced on the agar surface, semi-immersed as well as produced on aerial mycelium, solitary, scattered or aggregated, (sub-)globose or flask-shaped, irregular-shaped with age, brown to dark brown, thin-walled, with hyphal outgrowths, ostiolate, 125–695 × 90–535 μm. Ostioles 1–4(–8), slightly papillate. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 2–3 layers, 10–41 μm thick, outer 1–2 cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, globose to ampulliform, 4.5–8.5 × 3.5–7 μm. Conidia cylindrical or oblong with both ends obtusely rounded, smooth and thin-walled, aseptate, 4.5–8 × 2–4 μm, with 0–2 min guttules. Chlamydospores aseptate, brown, in chains or singly, (sub-)globose, 5.5–10 × 4.5–8.5 μm. Conidial matrix buff.
Culture characteristics: Colonies on OA reaching 50–55 mm diam after 7 d at 25 °C, margin regular, densely covered by felty aerial mycelium, concentric circles of different colour, centre buff, flesh, whitish, pale olivaceous grey towards periphery; reverse olivaceous grey with pinkish circles. Colonies on MEA reaching 45–50 mm diam after 7 d, margin regular, densely covered by felty aerial mycelium, rosy buff to whitish, pale grey near the centre, some radially furrowed zones near the centre; reverse dark brown, brick or orange towards the periphery, with some radial lines near the centre. Colonies on PDA reaching 50–55 mm diam after 7 d, margin regular, covered by felty aerial mycelium, buff, with flat mycelium towards the periphery and in blood colour; reverse orange, dark brown near the centre. NaOH spot test results in a dark greenish discolouration on OA.
Typus: France, La Réunion island, from leaves of Paspalum dilinateum (Poaceae), 7 Mar. 2015, P.W. Crous (holotype CBS H-23668, ex-type living culture CPC 26197 = CBS 141502).
Notes: Isolate CBS 141502 was isolated from leaves of Paspalum dilinateum in La Réunion island, France, and identified as Epicoccum sorghinum (Hernández-Restrepo et al. 2016) based on its ITS sequence data. Currently, five species are known to occur on Paspalum spp., namely, Ascochyta paspali, Ep. andropogonis, Ep. nigrum, Ep. sorghinum and Phoma paspali. However, CBS 141502 formed a phylogenetic lineage distinct from the ex-type strain of Ep. nigrum (CBS 173.73) and the representative strain of Ep. sorghinum (CBS 179.80). Although molecular data of As. paspali, Ep. andropogonis and Phoma paspali were unavailable, CBS 141502 could be differentiated from Ep. andropogonis by only producing aseptate conidia, while Ep. andropogonis only produced sporodochia and multi-septate conidia; from Phoma paspali in its larger pycnidia with more ostioles [125–695 × 90–535 μm, 1–4(–8) ostioles vs. 120–250 μm, single ostiole] and smaller conidia [4.5–8 × 2–4 μm vs. 6.5–9(–11) × 3–4 μm]; from As. paspali in its larger pycnidia (125–695 × 90–535 μm vs. 100–200 μm) with aseptate, smaller and hyaline conidia (4.5–8 × 2–4 μm), while As. paspali produced conidia that were 0–3-septate, pale brown, measuring 13–33 × 4–7.5 μm.
Epicoccum purpurascens Ehrenb. ex Schltdl., Fl. Berol. (Berlin) 2: 136. 1824. Fig. 20.
Epicoccum purpurascens (CBS 128906). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G–H. Sporodochia on OA. I–J. Sporodochium. K. Conidiogenous cells. L. Conidia. Scale bars: J = 50 μm; K = 20 μm; L = 10 μm.
Description from reference culture (CBS 128906): Conidiomata sporodochial, abundant, scattered or aggregated, superficial on the agar, pale brown initially, turning black with age. Vegetative hyphae septate, frequently branched. Conidiophores macronematous or semi-macronematous, unbranched, sometimes elongated and covered in mycelial hairs, yellow to pale brown. Conidia solitary, acrogenous, globose, aseptate and smooth when young, later becoming multicellular-phragmosporous, verrucose, subglobose to pyriform, brown, 9.5–24 μm diam.
Culture characteristics: Colonies on OA, 55–60 mm diam after 7 d 25 °C, margin regular, without aerial mycelium, olivaceous to brown, conidiophores and conidia abundant; reverse vinaceous buff, sienna near the centre. Colonies on MEA, 45–50 mm diam after 7 d, margin irregular, aerial mycelium flat, conidiophores and conidia abundant, brick with red edge, reverse fuscous black with yellow edge. Colonies on PDA, 65–75 mm diam after 7 d, margin regular, aerial mycelium flat, with feathery margin, dark brown, with buff to cinnamon elongated conidiophores on the surface; reverse olivaceous to olivaceous black. NaOH spot test: a slight olivaceous discolouration on OA.
Materials examined:China, from Poa annua (Poaceae), unknown date and collector, culture LC 8159. Denmark, from human toenail, date unknown, D.M. Saunte, culture CBS 124435. Unknown, unknown host, 1932, G.D. Ruehle, culture CBS 166.32. USA, Dubois, Wyoming, soil, 1997, M. Christensen, culture CBS 128906.
Notes: Epicoccum purpurascens was originally introduced by Von Schlechtendal (1824). According to Saccardo’s records, this species has a diverse host range and is mainly recorded in Europe. However, this species, together with several other species in Epicoccum, were formerly regarded as synonyms of Ep. nigrum (Schol-Schwarz 1959). Isolate CBS 128906 was originally received as Ep. purpurascens, with which it agreed well morphologically, and therefore we regarded CBS 128906 to represent Ep. purpurascens. In the present study, four isolates of Ep. purpurascens formed a well-supported clade, which was clearly distant from the ex-epitype strain of Ep. nigrum (CBS 173.73).
Epicoccum thailandicum Goonas. et al., Mycosphere 8: 713. 2017.
Synonym: Epicoccum pneumoniae Valenz.-Lopez et al. Stud. Mycol. 90: 32. 2017 (2018).
Description: Thambugala et al. (2017).
Typus: Thailand, Chiang Mai Province, Mae Taeng, Mushroom Research Centre, on stems of grass litter (Poaceae), 24 Mar. 2016, I.D. Goonasekara (holotype MFLU 16-2855, ex-type living culture MFLUCC 16-0892 = KUMCC 17-0026).
Material examined: USA, from human sputum sample, 2008, D.A. Sutton (holotype of Ep. pneumoniae FMR H-13747, ex-type living culture UTHSC DI16-257).
Notes: Epicoccum thailandicum (holotype: MFLU 16-2855) was introduced by Thambugala et al. based on phylogenetic analyses of ITS and LSU and was reported on the stems of grass litter (Poaceae) in Thailand (Thambugala et al. 2017; published 30 Apr. 2017), while Ep. pneumoniae (UTHSC DI16-257) was isolated from a human sputum sample in the USA (Valenzuela-Lopez et al. 2018; published 21 Nov. 2017). However, the ITS and LSU sequence data of Ep. pneumoniae were identical to those of Ep. thailandicum that was earlier published. Unfortunately, Epicoccum pneumoniae was sterile. Further loci should be sequenced of Ep. thailandicum to confirm the synonymy.
Epicoccum tobaicum (Szilv.) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB834115.
Basionym: Toruloidea tobaica Szilv., Arch. Hydrobiol. Suppl. 14: 519. 1936.
Synonym: Epicoccum layuense Qian Chen et al., Stud. Mycol. 87: 145. 2017.
Description: Von Szilvinyi (1936).
Typus: Indonesia, Sumatra, Toba Heath, heath soil, Jun. 1936, A. von Szilvinyi, specimen CBS H-24336, ex-type living culture CBS 384.36 = MUCL 9832.
Additional materials examined: China, Tibet, Lulang, on leaves of Perilla sp. (Lamiaceae), 15 Jun. 2015, Q. Chen (holotype of Epicoccum layuense HMAS 247165, ex-type living culture CGMCC 3.18362 = LC 8155); ibid., culture LC 8156. France, Loire, host unknown, 1955, J. Nicot, culture CBS 235.59. USA, Ohio, Cincinnati, air, date unknown, W.B. Cooke, culture CBS 232.59; ibid., culture CBS 233.59 = IMI 079496.
Notes: In this study the ex-type isolate of Toruloidea tobaica (CBS 384.36) was examined. This species was originally described from heath soil in Sumatra (Von Szilvinyi 1936), but later synonymised with Epicoccum nigrum. In our phylogenetic study, the ex-type strain of T. tobaica formed a distinct clade in the genus Epicoccum, distant from Ep. nigrum (CBS 173.73). The ex-type strain of the more recently described Ep. layuense (CGMCC 3.18362; Chen et al. 2017) was found to be genetically identical to that of Ep. tobaicum (CBS 384.36) and was therefore reduced to synonymy.
Epicoccum variabile L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833511. Fig. 21.
Epicoccum variabile (CBS 119733). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–N. Conidiogenous cells. O. Conidia. P. Globose conidia. Scale bars: H–I = 50 μm; J = 10 μm; K–P = 5 μm.
Etymology: From Latin variabilis, due to the variable shape of the conidia.
Description: Conidiomata pycnidial, produced superficially on agar and covered by sparse aerial mycelium, scattered or aggregated, solitary or confluent, brown, thin-walled, mostly (sub-)globose, glabrous or with some hyphal outgrowths, 110–450 × 110–350 μm. Ostioles inconspicuous. Pycnidial wall pseudoparenchymatous, thin, composed of oblong to isodiametric cells, 9–21.5 μm thick, 2–3 layers, outer cell layer pigmented. Conidiogenous cells phialidic, hyaline, smooth, globose, ampulliform or doliiform, 5–8 × 4–6.5 μm. Conidia variable in shape and size, ovoid or oblong, hyaline, smooth- and thin-walled, aseptate, with 2 minute, polar guttules, 4.5–6 × 2.5–3.5 μm; globose conidia present, smooth- and thin-walled, aseptate, 3–4 × 2.5–3.5 μm, with one medium-sized guttule. Conidial matrix pinkish.
Culture characteristics: Colonies on OA reaching 75–80 mm diam after 7 d at 25 °C, margin regular, densely covered by felty aerial mycelium, pale smoke grey, buff towards periphery; reverse concolourous. Colonies on MEA reaching 75–80 mm diam after 7 d, margin regular, densely covered by floccose aerial mycelium, whitish to buff, pink conidial matrix visible; reverse yellow, pale brown near the centre. Colonies on PDA reaching 70–75 mm diam after 7 d, margin regular, covered by flat aerial mycelium, floccose near the centre, grey to buff near the centre, vinaceous buff towards periphery; reverse dark brown, vinaceous buff towards periphery. NaOH spot test negative on OA.
Typus: Brazil, Minas Gerais, Capelinha, from leaf of Coffea arabica (Rubiaceae), Sep. 1999, L.H. Pfenning (holotype CBS H-23677, ex-type living culture CBS 119733 = CML 190).
Notes: Phylogenetically, Epicoccum variabile grouped in a distinct clade closely related to Ep. camelliae and Ep. viticis. Morphologically, Epicoccum variabile could be clearly differentiated from Ep. viticis in its larger pycnidia [110–450 × 110–350 μm vs. 120–200 × 100–175 μm] and shorter conidiogenous cells (5–8 × 4–6.5 μm vs. 5.5–9 × 3–6 μm). Moreover, Ep. variabile differed from Ep. viticis by producing pycnidia with inconspicuous ostioles and globose conidia (Chen et al. 2017). Although the ex-type culture of Ep. camelliae was sterile, they differed genetically in their rpb2 and tub2 sequences.
Clade 13: Similiphoma Valenz.-Lopez et al., Stud. Mycol. 90: 37. 2017 (2018).
Type species: Similiphoma crystallifera (de Gruyter et al.) Valenz.-Lopez et al.
Clade 14: Sclerotiophoma L.W. Hou, L. Cai & Crous, gen. nov. MycoBank MB833512.
Etymology: Name refers to the producing of pycnosclerotia.
Conidiomata pycnidial, globose, subglobose, scattered, solitary or confluent, semi-immersed or immersed in agar, without distinct ostioles, occasionally with a pore; pycnidial wall pseudoparenchymatous, multi-layered, outer layers slightly pigmented. Pycnosclerotia present. Conidiogenous cells phialidic, hyaline, smooth, ampulliform or bottle-shaped, with minute periclinal thickening. Conidia ellipsoidal to ovoid, smooth- and thin-walled, aseptate, with or without several guttules. Chlamydospores not observed. Sexual morph unknown.
Notes: According to Boerema et al. (2004), most species of Phoma section Sclerophomella have thick-walled pseudoparenchymatous pycnidia in vivo. The type species Phoma complanata of this section has been recombined into Calophoma, and most other species have also been transferred to different genera based on the current phylogenetic analysis, such as Phoma dictamnicola to Heterophoma, and Phoma polemonii to Neodidymelliopsis (Chen et al. 2015). Among the species included in Phoma section Sclerophomella, Phoma versabilis is characterised by the production of pycnidia showing a retarded development of the pycnidial cavity (pycnosclerotia), containing a compact mass of cells that afterwards disintegrates (Boerema et al. 2004). In the present study, phylogenetic analysis showed that this species formed a separate lineage distant from all known genera in Didymellaceae. Therefore a new genus Sclerotiophoma is introduced.
Type species: Sclerotiophoma versabilis (Boerema et al.) L.W. Hou, L. Cai & Crous
Sclerotiophoma versabilis (Boerema et al.) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833530. Fig. 22
Sclerotiophoma versabilis (CBS 876.97). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Conidia. Scale bars: H = 50 μm; I = 10 μm.
Basionym: Phoma versabilis Boerema et al., Persoonia 16: 154. 1996.
Synonym: Ascochyta versabilis (Boerema et al.) Qian Chen & L. Cai, Stud. Mycol. 82: 190. 2015.
Description: Boerema et al. (1996).
Typus: Germany, Westfalen, Oberdresselendorf, on stems of Cardamine impatiens (Brassicaceae), Oct. 1925, A. Ludwig (holotype L 995.229.369).
Materials examined: Denmark, from human toenail, date unknown, D.M. Saunte, culture CBS 124689. The Netherlands, Wageningen, from a stem of Silene sp. (Caryophyllaceae), Jun. 1997, specimen CBS H-24329, culture CBS 876.97 = PD 82/1008.
Notes: This species was introduced by Boerema et al. (1996), on stems of Cardamine impatiens collected in Germany (Boerema et al. 1996). Later, Boerema et al. (2004) described a representative strain for Phoma versabilis (CBS 876.97) which morphologically agreed well with the original description.
In the present study, Phoma versabilis was re-examined based on two isolates (CBS 876.97 and CBS 124689). In our phylogenetic analysis, these two isolates formed an independent lineage which was distant from other genera in Didymellaceae, closely related to the genus Similiphoma and Epicoccum (Fig. 1). Thus, Sclerotiophoma was introduced to accommodate this species. The type species of this genus is characterised by producing pycnosclerotia, which gradually develop into poroid pycnidia.
Clade 15: Didymella Sacc., Michelia 2(no. 6): 57. 1880, emend. Chen et al., Stud. Mycol. 82: 173. 2015.
Synonym: Peyronellaea Goid. ex Togliani, Ann. Sperim. Agrar. II 6: 93. 1952.
Type species: Didymella exigua (Niessl) Sacc.
Didymella acetosellae (A.L. Sm. & Ramsb.) Qian Chen & L. Cai, Stud. Mycol. 82: 173. 2015. Fig. 23.
Didymella acetosellae (CBS 631.76). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–N. Conidiogenous cells. O. Globose conidia. P. Conidia. Scale bars: H–I = 20 μm, J = 10 μm; K–P = 5 μm.
Basionym: Phyllosticta acetosellae A.L. Sm. & Ramsb., Trans. Brit. Mycol. Soc. 4: 173. 1913.
Synonym: Phoma acetosellae (A.L. Sm. & Ramsb.) Aa & Boerema, Persoonia 18: 16. 2002.
Description from ex-epitype (CBS 631.76): Conidiomata pycnidial, semi-immersed or immersed, scattered, mostly solitary, sometimes 2–3 confluent; cover by whitish mycelium, (sub-)globose, pale brown, thin-walled, with hyphal outgrowths, ostiolate, 170–345 × 145–300 μm. Ostioles 1–3(–7), non-papillate. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 3–4 layers, 5.5–26 μm thick, outer cell layers pigmented. Conidiogenous cells phialidic, hyaline, smooth, subglobose, lageniform, ampulliform, slightly papillate, 3.5–11.5 × 3.5–10.5 μm. Conidia variable in shape and size, always two types: cylindrical, ovoid or globose, smooth- and thin-walled, aseptate, the cylindrical conidia 5–9.5(–11) × 2.5–3.5 μm; globose conidia 3.6–5.5 × 3–5, mostly with 2–6 minute, polar guttules. Conidial matrix milky to buff.
Culture characteristics: Colonies on OA reaching 30–35 mm diam after 7 d, margin irregular, aerial mycelium floccose, flat mycelium in the middle, whitish to mouse grey; reverse buff to dark brown. Colonies on MEA reaching 25–30 mm diam after 7 d, margin irregular, covered with felty aerial mycelium, whitish to pale grey; reverse brown to darker brown, with yellow edge. Colonies on PDA reaching 20–25 mm diam after 7 d, margin irregular, covered by felty aerial mycelium, whitish, pale grey near the centre; reverse buff to olivaceous grey. NaOH spot test negative on OA.
Typus: UK, Glangonner, Lanarkshire, on Rumex acetosella (Polygonaceae), 29 Jun. 1912, D.A. Boyd (holotype BM). France, Corrèze, Monteil sur Bois, from leaf spot on Rumex acetosella (Polygonaceae), 21 Jul. 1976, H.A. van der Aa (epitype designated here CBS H-16138, MBT389708, ex-epitype living culture CBS 631.76).
Notes: Phyllosticta acetosellae was originally collected from leaves of Rumex acetosella in the UK, having ellipsoidal to cylindrical conidia measuring 8–10 × 3–4 μm (Smith & Ramsbottom 1913). Based on morphology, De Gruyter et al. (2002) regarded isolate CBS 631.76 to represent Phy. acetosellae, and recombined it into the genus Phoma. Subsequently this species was transferred to Didymella after phylogenetic analysis (Chen et al. 2015). According to De Gruyter et al. (2002), CBS 631.76, isolated from the leaf spot of Rumex acetosella from France, was a good representative of this species as it morphologically agreed well with the original description. We followed this suggestion and designated CBS 631.76 as ex-epitype culture of Phyllosticta acetosellae. In the present study, CBS 631.76 was found to be phylogenetically distant from CBS 179.97 (also received as “Phoma acetosellae”), the latter being re-identified as Did. rumicicola.
Didymella aloeicola L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833514. Fig. 24.
Didymella aloeicola (CBS 562.88). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–N. Conidiogenous cells. O. Conidia. Scale bars: H–I = 20 μm; J = 10 μm; K–O = 5 μm.
Etymology: Name reflects the host genus Aloe, from which this species was isolated.
Description: Conidiomata pycnidial, produced on the agar surface or (semi-)immersed, scattered, mostly solitary, sometimes 2–3 confluent, mostly globose, subglobose or lageniform, brown or darker brown, thin-walled, with hyphal outgrowths, ostiolate, 60–200 × 55–175 μm. Ostioles 1–2, mostly central, not to slightly papillate. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 4–6 layers, 16–19 μm thick, outer cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, ampulliform, doliiform to lageniform, 3.5–5.5 × 3.5–5 μm. Conidia ovoid or oblong, hyaline, smooth- and thin-walled, aseptate, 3.5–4 × 1.5–2 μm, mostly with 1–2 minute, polar guttules. Conidial matrix whitish to buff.
Culture characteristics: Colonies on OA reaching 60–65 mm diam after 7 d at 25 °C, margin regular, aerial mycelium sparse, pale olivaceous grey near the centre, saffron towards periphery; reverse concolourous. Colonies on MEA reaching 60–65 mm diam after 7 d, margin regular, covered with flat aerial mycelium, pale mouse grey to mouse grey; reverse dark brown. Colonies on PDA reaching 75–80 mm diam after 7 d, margin regular, covered by floccose aerial mycelium, vinaceous; reverse concolourous. NaOH spot test negative on OA.
Typus: Italy, Sicily, Aloe sp. (Asphodelaceae), date unknown, F. Seigle-Murandi (holotype CBS H-23683, ex-type living culture CBS 562.88).
Notes: Phylogenetically, Didymella aloeicola formed a distinct lineage from other species in the genus. Morphologically, Did. aloeicola was easily distinguished from its closest neighbour Did. pedeiae in the NaOH reactions (pale reddish discolouration on Did. aloeicola, and no effect on Did. pedeiae) (Aveskamp et al. 2010).
Phoma aloes was originally isolated from dead bark of Aloe dichotoma, the same host genus as for Did. aloeicola, in South Africa (Crous et al. 2013b). However, it clearly differed from Did. aloeicola phylogenetically (Fig. S1). Morphologically, Did. aloeicola produced smaller conidiogenous cells [3.5–5.5 × 3.5–5 μm vs. (7–)8(–9) × (3–)4 μm] and conidia (3.5–4 × 1.5–2 μm vs. 4–7 × 2–2.5 μm) than Phoma aloes.
Didymella anserina (Marchal) Qian Chen & L. Cai, Stud. Mycol. 82: 173. 2015.
Basionym: Phoma anserina Marchal, Champignon Copr. Belg. 6: 11. 1891.
Synonyms: Peyronellaea anserina (Marchal) Aveskamp et al., Stud. Mycol. 65: 31. 2010.
Phoma radicis-callunae R.W. Rayner, Bot. Gaz. 73: 231. 1922.
Phoma suecica J.F.H. Beyma, Antonie van Leeuwenhoek 8: 110. 1942.
Description: De Gruyter & Noordeloos (1992).
Materials examined: Germany, Giessen, Dec. 1979, R. Hadlok, specimen CBS H-16562, culture CBS 253.80; former West-Germany, from plastic, Dec. 1965, H. Kühlwein, culture CBS 397.65; Pfalz, Robinia pseudoacacia (Fabaceae), unknown date and collector, culture CBS 300.36. The Netherlands, Ter Apel, from potato flour, 1983, collector unknown, culture CBS 360.84. UK, from Calluna sp. (Ericaceae), Nov. 1929, R.W. Rayner (ex-type living culture of Phoma radicis-callunae CBS 285.29). Unknown, wood pulp, Jan. 1940, E. Rennerfelt (ex-type living culture of Phoma suecica CBS 167.42).
Notes: Phoma suecica was treated as synonym of Didymella anserina based on a reference isolate CBS 397.65 (Chen et al. 2015). In this study, the ex-type culture of Phoma suecica (CBS 167.42) was examined. In our phylogenetic analyses, it clustered with the representative strain of Did. anserina (CBS 360.84), which agreed with the findings of Chen et al. (2015).
Didymella combreti (Crous) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833516.
Basionym: Peyronellaea combreti Crous, Persoonia 32: 223. 2014.
Description: Crous et al. (2014).
Typus: Zambia, S16°55.766', E27°75.914', on Combretum mossambicensis (Combretaceae), 28 Feb. 2013, M. van der Bank (holotype CBS H-21694, ex-type living culture CPC 22587 = CBS 137982).
Notes: Peyronellaea combreti was originally introduced based on a phylogenetic study of ITS and LSU sequence data (Crous et al. 2014). A more extensive phylogenetic analysis on DNA sequences from four loci ITS, LSU, rpb2 and tub2 in this study revealed that the ex-type isolate (CBS 137982) grouped in the genus Didymella and was distinct from other species (Fig. 1).
Didymella guttulata L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833521. Fig. 25.
Didymella guttulata (CBS 127976). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H–I = 20 μm; J = 10 μm; K–N = 5 μm.
Etymology: Name derived from the guttulate conidia produced by this species.
Description: Conidiomata pycnidial, produced superficially on the agar as well as in the mycelium, solitary, scattered or aggregated, mostly globose, subglobose or flask-shaped, darker brown, with hyphal outgrowths, ostiolate, 120–360 × 105–280 μm. Ostioles single, centric. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 3–5 layers, 23.5–51.5 μm thick, outer 1–3 cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, ampulliform, doliiform or lageniform, 4.5–9 × 4–7.5 μm. Conidia oblong, ovoid or ellipsoidal, smooth- and thin-walled, aseptate, (3.5–)4.5–7(–8.5) × 2.5–4 μm, mostly with two minute, polar guttules, sometimes 3–4 minute guttules. Conidial matrix pale pink.
Culture characteristics: Colonies on OA reaching 60–65 mm diam after 7 d at 25 °C, margin regular, aerial mycelium felty, dark mouse grey to pale mouse grey, margin brownish, abundant production of pycnidia; reverse olivaceous. Colonies on MEA reaching 65–70 mm diam after 7 d, margin regular, covered with moderate aerial mycelium, felty, olivaceous grey, pale grey towards the periphery; reverse olivaceous grey with brown edge. Colonies on PDA reaching 68–72 mm diam after 7 d, margin regular, covered by felty aerial mycelium, olivaceous, mouse grey towards periphery; reverse dark mouse grey. NaOH spot test negative on OA.
Typus: Zimbabwe, near Umtali, from soil in deciduous forest, date and collector unknown (holotype CBS H-23691, ex-type living culture CBS 127976).
Notes: Didymella guttulata was phylogenetically allied to Did. combreti and Did. eucalyptica. Morphologically, it differed from Did. combreti in having larger pycnidia (120–360 × 105–280 μm vs. up to 250 μm diam), smaller conidia [(3.5–)4.5–7(–8.5) × 2.5–4 μm vs. (5–)8–10(–12) × 3.5(–4) μm] and absence of chlamydospores (Crous et al. 2014). Didymella guttulata could be distinguished from Did. eucalyptica by producing larger pycnidia (120–360 × 105–280 μm vs. 120–250 × 80–200 μm) and larger, guttulate conidia [(3.5–)4.5–7(–8.5) × 2.5–4 μm vs. 2.8–4.2 × 1–2 μm] (De Gruyter & Noordeloos 1992).
Didymella indica L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833517. Fig. 26.
Didymella indica (CBS 653.77). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidia. I–J. Section through pycnidium. K–O. Conidiogenous cells. P. Conidia. Scale bars: H = 20 μm; I–J = 10 μm; K–P = 5 μm.
Etymology: Named after the country where this species was collected, India.
Description: Conidiomata pycnidial, produced on the agar surface or semi-immersed, scattered and solitary, obpyriform or subglobose, brown or darker brown, thin-walled, with hyphal outgrowths, ostiolate, 145–220 × 85–160 μm. Ostioles single, central, slightly papillate. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 3–4 layers, 8–22 μm thick, outer three cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, ampulliform to lageniform, 6.5–9 × 6–8 μm. Conidia oblong to ellipsoidal, hyaline, smooth- and thin-walled, aseptate, 5–9 × 3.5–4.5 μm, mostly with minute guttules. Conidial matrix buff.
Culture characteristics: Colonies on OA, 65–70 mm diam after 7 d at 25 °C, margin regular, aerial mycelium felty, whitish, olivaceous towards periphery; reverse pale olivaceous. Colonies on MEA 70–75 mm diam after 7 d, margin regular, covered with medium aerial mycelium, felty, whitish, olivaceous near the centre; reverse buff to pale brownish. Colonies on PDA, 70–75 mm diam after 7 d, margin regular, covered by felty aerial mycelium, buff, but aerial mycelium near margin flat, pale olivaceous; reverse olivaceous black, with buff edge. NaOH spot test negative on OA.
Typus: India, Kurukshetra, host and date unknown, K.R. Aneja (holotype CBS H-24311, ex-type living culture CBS 653.77).
Notes: Didymella indica formed a well-supported and distinct clade on the four-locus phylogenetic tree (Fig. 1), closely related to Did. boeremae and Did. dimorpha. Didymella indica differs genetically from Did. boeremae in 17 bp of the ITS nucleotide sequence, 2 bp of LSU, 10 bp of tub2 and 35 bp of rpb2; and it differs from Did. dimorpha in 6 bp of the ITS nucleotide sequence, 10 bp of tub2 and 36 bp of rpb2, based on alignment of the concatenated four loci deposited in TreeBASE (S25826). Morphologically, Did. indica can be distinguished from Did. dimorpha by producing wider conidia [3.5–4.5 μm vs. (2–)2.5–3(–3.5) μm] in vitro; from Did. boeremae by producing shorter conidia (5–9 μm vs. 8–15 μm). Besides, Did. indica is clearly different from the latter two species by producing obpyriform pycnidia while Did. boeremae and Did. dimorpha produce (sub-)globose to irregular pycnidia.
Didymella mitis L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833519. Fig. 27.
Didymella mitis (CBS 443.72). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–L. Conidiogenous cells. M. Globose conidia. N. Conidia. Scale bars: H = 100 μm; I = 50 μm; J–N = 5 μm.
Etymology: Name derived from the soft pycnidia wall produced by this species, mitis = soft.
Description: Conidiomata pycnidial, produced partly in the agar, solitary, sometimes 2–5 confluent, scattered or aggregated, mostly (sub-)globose, always irregular-shaped with age, buff to pale brown at the beginning, darker brown with age, thin-walled, glabrous, 270–600 × 180–400 μm. Ostioles inconspicuous. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 3–4 layers, 13–25 μm thick, outer cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, (sub-)globose, ampulliform, 5.5–6.5 × 5.5–6 μm. Conidia oblong, ovoid or ellipsoidal, hyaline, smooth- and thin-walled, aseptate, (3.5–)4.5–6.5 × 2.5–3.5 μm, with two minute guttules or eguttulate; globose conidia exist, smooth- and thin-walled, aseptate, 3–4 × 2.5–4 μm. Conidial matrix whitish.
Culture characteristics: Colonies on OA reaching 60–65 mm diam after 7 d at 25 °C, margin regular, aerial mycelium flat, salmon near the centre and olivaceous towards periphery, abundant production of pycnidia; reverse concolourous. Colonies on MEA reaching 55–60 mm diam after 7 d, margin regular, aerial mycelium flat, sparse, grey with brown margin, abundant production of pycnidia, pinkish conidial matrix visible; reverse dark brown with pale brown margin. Colonies on PDA reaching 55–60 mm diam after 7 d, margin regular, covered by sparse floccose aerial mycelium, pale olivaceous; reverse olivaceous black. NaOH spot test negative on OA.
Typus: South Africa, near Otavi, from soil, Oct. 1971, G. Franz (holotype CBS H-16400, ex-type living culture CBS 443.72).
Additional material examined:Namibia, about 18 km west of Mariental, 90 km east of Maltahohe, from surface soil, desert, clay hardpan, under Boscia albitrunca (Capparaceae), date unknown, M. Christensen, culture CBS 126162.
Notes: Isolate CBS 443.72 was initially deposited as “Didymella musae”. In this study, CBS 443.72 together with CBS 126162 clustered in a well-supported clade in the genus Didymella, distant from the representative culture of Did. musae (CBS 463.69; Fig. 1). Phylogenetically, Didymella mitis was closely related to Did. sancta, Did. keratinophila and Did. coffeae-arabicae, but distinguished from the latter three species by producing inconspicuously ostiolate pycnidia. Moreover, Didymella mitis differed from Did. sancta and Did. coffeae-arabicae by producing smaller conidia [(3.5–)4.5–6.5 × 2.5–3.5 μm in Did. mitis, 5–7(–7.5) × 2.5–4(–4.5) μm in Did. sancta, and (4–)4.5–6(–7) × (2.5–)3–4(–4.5) μm in Did. coffeae-arabicae] (Aveskamp et al. 2009a).
Didymella pinodella (L.K. Jones) Qian Chen & L. Cai, Stud. Mycol. 82: 178. 2015.
Basionym: Ascochyta pinodella L.K. Jones, Bull. New York Agric. Exp. Sta., Geneva 547: 10. 1927.
Synonyms: Phoma medicaginis var. pinodella (L.K. Jones) Boerema, Netherlands J. Pl. Pathol. 71: 88. 1965.
Ascochyta sojicola Abramov, Bolezni i Vrediteli Soievykh Bobov na Dal’nem Vostoke 68. 1931.
Phoma trifolii E.M. Johnson & Valleau, Bull. Kentucky Agric. Exp. Sta. 339: 57. 1933.
Phomatosphaeropsis pinicola Ribaldi, Ann. Sperim. Agrar. 7(3): 849. 1953.
Phoma pinodella (L.K. Jones) Morgan-Jones & K.B. Burch, Mycotaxon 29: 485. 1987.
Phoma sojicola (Abramov) Kövics, Gruyter & Aa, Mycol. Res. 103(8): 1066. 1999.
Peyronellaea pinodella (L.K. Jones) Aveskamp et al., Stud. Mycol. 65: 33. 2010.
Description: De Gruyter et al. (2002).
Materials examined: Hungary, from seed of Glycine max (Fabaceae), 2000, Plant Protection Service Wageningen, culture CBS 100580 = PD D/ 054; from G. max, 1997, G. Kövics, culture CBS 567.97 = PD 97/2160. Italy, Torino, from bark and wood of Pinus nigra var. austriaca (Pinaceae), 1953, M. Ribaldi, culture CBS 300.53 (ex-type living culture of Phomatosphaeropsis pinicola). The Netherlands, from a stem of Pisum sativum (Fabaceae), Jul. 1990, M.E. Noordeloos, culture CBS 318.90 = PD 81/729. USA, Minnesota, from Trifolium pratense (Fabaceae), Sep. 1966, culture CBS 531.66; from Trifolium sp. (Fabaceae), May 1934, E.M. Johnson & W.D. Valleau, culture CBS 351.34 = MUCL 18217 = MUCL 9927 (authentic strain of Phoma trifolii).
Notes: Phoma trifolii was originally described by Johnson & Valleau (1933) from Trifolium spp. in America and later treated as a variety Phoma medicaginis var. pinodella based on morphology (Boerema et al. 1965). However, Phoma medicaginis var. pinodella was recently elevated to species status as Didymella pinodella (Chen et al. 2015). Another species, Phomatosphaeropsis pinicola, was originally isolated from Pinus nigra var. austriaca in Italy. In this study, based on the multi-locus phylogenetic analyses, the ex-type strain of Phomatos. pinicola (CBS 300.53) and the authentic strain of Phoma trifolii (CBS 351.34), clustered with the reference culture of Did. pinodella. Therefore, we regarded Phomatos. pinicola and Phoma trifolii as synonyms of Did. pinodella.
Phoma sojicola was first described from Glycine max in Russia. De Gruyter et al. (2002) described CBS 100580 as a representative strain of Phoma sojicola as it was morphologically similar to the original description. Together with CBS 100580, another two isolates CBS 567.97 and CBS 113.53 were also originally identified as Phoma sojicola. However, CBS 100580 and CBS 567.97 (from Glycine max in Hungary), clustered with Did. pinodella in the phylogenetic tree (Fig.1). Therefore, Phoma sojicola was reduced to synonymy with Did. pinodella. Isolate CBS 113.53 clustered in the genus Allophoma, and was re-identified as Al. zantedeschiae.
Didymella pomorum (Thüm.) Qian Chen & L. Cai, Stud. Mycol. 82: 179. 2015.
Basionym: Phoma pomorum Thüm., Fungi Pomicoli: 105. 1879.
Synonyms: Phoma triticina E. Müll., Phytopathol. Z. 19: 413. 1952.
Peyronellaea nigricans Kusnezowa, Novoste Sist. Nizsh. Rast. 8: 191. 1971.
Peyronellaea circinata Kusnezowa, Novoste Sist. Nizsh. Rast. 8: 189. 1971.
Phoma jolyana var. circinata (Kusnezowa) Boerema & Kesteren, Kew Bull. 31: 535. 1977.
Phoma pomorum var. circinata (Kusnezowa) Aveskamp et al., Mycologia 101: 377. 2009.
Peyronellaea pomorum var. circinata (Kusnezowa) Aveskamp et al., Stud. Mycol. 65: 33. 2010.
Phoma cyanea Jooste & Papendorf, Mycotaxon 12: 444. 1981.
Phoma pomorum var. cyanea (Jooste & Papendorf) Aveskamp et al., Mycologia 101: 377. 2009.
Peyronellaea pomorum var. cyanea (Jooste & Papendorf) Aveskamp et al., Stud. Mycol. 65: 32. 2010.
Peyronellaea pomorum var. pomorum (Thüm.) Aveskamp et al., Stud. Mycol. 65: 33. 2010.
Description: Boerema (1993).
Materials examined: Russia, Siberia, Novosibirsk, Hortus Botanicus, from a leaf of Allium nutans (Liliaceae), 1963, T.T. Kuznetsova (holotype of Peyronellaea nigricans CBS H-16399, ex-type living culture CBS 286.76 = ATCC 26242 = IMI 176743 = VKM F-1844). The Netherlands, Wageningen, from Polygonum tataricum (Polygonaceae), Sep. 1966, M.M.J. Dorenbosch, specimen CBS H-16540, culture CBS 539.66 = ATCC 16791 = IMI 122266 = PD 64/914.
Notes: Peyronellaea nigricans was originally isolated from a leaf of Allium nutans in Siberia (Kuznetsova 1971). However, the ex-type culture of Pe. nigricans (CBS 286.76) clustered with the representative strain of Didymella pomorum (CBS 539.66) based on phylogenetic data (Aveskamp et al. 2010). In the present study, this species was re-examined and rpb2 sequences were provided.
Didymella prolaticolla L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833518. Fig. 28.
Didymella prolaticolla (CBS 126182). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K, L. Conidiogenous cells. M. Conidia. Scale bars: H = 50 μm; I = 20 μm, J = 10 μm; K–M = 5 μm.
Etymology: Name refers to the elongated ostiolar necks, prolatus (= extended), collum = neck.
Description: Conidiomata pycnidial, semi-immersed, scattered or aggregated, mostly solitary, sometimes confluent, mostly (sub-)globose or lageniform, darker brown, with hyphal outgrowths, ostiolate, (100–)190–350(–470) × (90–)175–275 μm. Ostioles 1–3, mostly 1–2, developing into elongated necks. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 4–6 layers, 16–51 μm thick, outer three cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, subglobose, ampulliform to lageniform, sometimes with a papilla or a column at top, 5–10 × 4.5–8 μm. Conidia ovoid or ellipsoidal, hyaline, smooth- and thin-walled, aseptate, 5–7 × 2.5–4 μm, eguttulate or with 1–2 minute, polar guttules. Conidial matrix whitish, turning buff or pale brown with age.
Culture characteristics: Colonies on OA reaching 65–70 mm diam after 7 d at 25 °C, margin regular, aerial mycelium sparsely, flat, straw but mouse grey near the central zone; reverse concolourous. Colonies on MEA reaching 55–60 mm diam after 7 d, margin regular, covered with medium aerial mycelium, felty, whitish to buff; reverse orange to brick. Colonies on PDA reaching 80–85 mm diam after 7 d, margin regular, covered by felty aerial mycelium, pale olivaceous, but aerial mycelium near centre floccose, buff; reverse olivaceous. NaOH spot test results in a pale reddish discolouration near the margin of mycelium on OA.
Typus: Namibia, about 18 km west of Mariental, 90 km east of Maltahohe, from surface soil, clay hardpan, under Boscia albitrunca in desert, date unknown, M. Christensen (holotype CBS H-23664, ex-type living culture CBS 126182).
Notes: Phylogenetically, isolate CBS 126182 formed a distinct lineage sister to Didymella gardeniae. Morphologically, CBS 126182 clearly differed from Did. gardeniae by producing larger pycnidia with more ostioles and elongated necks, [(100–)190–350(–470) × (90–)175–275 μm, 1–3 ostioles vs. 50–180 μm diam, single ostiole]. In addition, CBS 126182 produced shorter conidia than Did. gardeniae [5–7 × 2.5–4 μm vs. (3.5–)5–8.5(–10.5) × (1.5–)2–3.5(–4.5) μm]. Therefore, CBS 126182 was introduced as a new species, Did. prolaticolla.
Didymella prosopidis (Crous & A.R. Wood) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833520.
Basionym: Peyronellaea prosopidis Crous & A.R. Wood, Persoonia 31: 207. 2013.
Description: Crous et al. (2013b).
Typus: South Africa, Western Cape Province, Calvinia, associated with a stem disease of Prosopis sp. (Fabaceae), Sep. 2012, A. Wood (holotype CBS H-21425, ex-type living culture CPC 21698 = CBS 136414).
Additional material examined: South Africa, Western Cape Province, Calvinia, associated with a stem disease of Prosopis sp. (Fabaceae), Sep. 2012, A. Wood, culture CPC 21704 = CBS 136550.
Notes: The holotype of Peyronellaea prosopidis was originally isolated from stems of a Prosopis sp. in South Africa (Crous et al. 2013b). Later, the genus Peyronellaea was treated as synonym of Didymella (Chen et al. 2015). Based on the combined four-gene phylogenetic analysis in the present study, Pe. prosopidis clustered in a distinct and well-supported lineage in Didymella. Therefore, a new combination was proposed as Did. prosopidis.
Didymella subglobispora L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833515. Fig. 29.
Didymella subglobispora (CBS 364.91). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H–I = 20 μm; J = 10 μm; K–N = 5 μm.
Etymology: Name refers to the subglobose conidia produced by this species.
Description: Conidiomata pycnidial, immersed or semi-immersed, scattered or aggregated, solitary, mostly globose, flask-shaped, brown to darker brown, thin-walled, glabrous, ostiolate, 100–230 × 95–190 μm. Ostiole single and centric, up to four with age. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 2–4 layers, 7.5–18.5 μm thick, outer cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, subglobose, ampulliform or lageniform, 5–9 × 4–7 μm. Conidia globose, subglobose or broad ellipsoidal, smooth- and thin-walled, aseptate, 2.5–3.5 × 2–3 μm, mostly with one minute-sized guttule. Conidial matrix buff.
Culture characteristics: Colonies on OA reaching 70–75 mm diam after 7 d at 25 °C, margin regular, covered by medium aerial mycelium, flat, pale mouse grey; reverse concolourous. Colonies on MEA reaching 65–70 mm diam after 7 d, margin regular, covered with medium aerial mycelium, flat, mouse grey, some radially furrowed zones near the centre; reverse olivaceous, olivaceous black towards periphery. Colonies on PDA reaching 75–80 mm diam after 7 d, margin regular, covered by felty aerial mycelium, pale mouse grey, dark mouse grey towards periphery; reverse dark mouse grey. NaOH spot test negative on OA.
Typus: Unknown, from fruit of Ananas sativus (Bromeliaceae), Jun. 1991, Plant Protection Service, Wageningen (holotype CBS H-23663, ex-type living culture CBS 364.91 = PD 81/290).
Notes: The cultures CBS 364.91, CBS 365.91 and CBS 363.91 were originally received as Phoma anserina, a species that was described from goose manure in Werbomont, Belgium (Saccardo 1892). Later Boerema et al. (2004) proposed CBS 365.91 and CBS 363.91 as representative cultures for Phoma anserina, and they were subsequently transferred to the genus Didymella as Did. anserina (Chen et al. 2015). In this study, CBS 364.91 was phylogenetically closely related to other isolates of Did. anserina, but formed a separate lineage with 1 bp difference in the LSU nucleotide sequence, 4 bp of tub2 and 11 bp of rpb2, based on alignment of the four sequenced loci. Morphologically, CBS 364.91 was characterised by having globose to subglobose conidia, while Did. anserina had broadly ellipsoidal conidia. Besides, CBS 364.91 was recorded as different from the other two isolates in having less intensely pigmented colonies (De Gruyter & Noordeloos 1992). Therefore, we treated CBS 364.91 as a distinct species.
Didymella subrosea L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833513. Fig. 30.
Didymella subrosea (CBS 733.79). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Globose conidia. O. Conidia. Scale bars: H = 100 μm; I = 20 μm, J, O = 10 μm; K–N = 5 μm.
Etymology: Name reflects the rosy colour of the colony on OA medium.
Description: Conidiomata pycnidial, produced on the surface of agar, mostly aggregated and confluent, seldom solitary, mostly globose or subglobose, whitish at beginning, turning pale brown and irregular shaped with age, especially around the ostioles (darker brown), thick-walled, with white hyphal outgrowths, ostiolate, variable in size, 160–560(–1 080) × 120–425(–630) μm. Ostioles 3–5, sometimes up to 12, slightly papillate or non-papillate. Pycnidial wall pseudoparenchymatous, composed of isodiametric or oblong cells, 4–6 layers, outer cell layers slightly pigmented, 17–50 μm thick. Conidiogenous cells phialidic, hyaline, smooth, (sub-)globose, lageniform or ampulliform, 8.5–15 × 7–12.5 μm. Conidia variable in shape and size, ovoid, fusoid to cylindrical or globose to ellipsoidal, hyaline, smooth- and thin-walled, 0–1-septate, 13–22.5 × 3.5–6 μm when 1-septate, 5–20 × 3–6 μm when aseptate, 3–6 × 2.5–4 μm for globose conidia, mostly with numerous minute guttules scattered or aggregated. Conidial matrix whitish to pink.
Culture characteristics: Colonies on OA reaching 45–50 mm diam after 7 d 25 °C, margin irregular, aerial mycelium sparse, whitish to rosy buff because of the pink conidial matrix, abundant production of pale brown pycnidia; reverse buff. Colonies on MEA reaching 35–40 mm diam after 7 d, margin irregular, covered with medium aerial mycelium, felty, whitish; reverse brownish, yellow towards periphery. Colonies on PDA reaching 45–50 mm diam after 7 d, margin irregular, covered by flat aerial mycelium, pale olivaceous grey to whitish, with some olivaceous black zones; reverse pale brown to dark brown, margin buff. NaOH spot test negative on OA.
Typus: France, Lyon, from Abies alba litter (Pinaceae), date unknown, F. Gourbière (holotype CBS H-9103, ex-type living culture CBS 733.79).
Notes: Didymella subrosea was proposed to accommodate isolate CBS 733.79 collected from litter of Abies alba in France. In the phylogenetic tree, Did. subrosea formed a distinct lineage separate from other species in this genus and most closely related to Did. macrostoma (Fig. 1). Morphologically, Did. subrosea was well distinguished from Did. macrostoma by producing larger pycnidia with more ostioles [160–560(–1080) × 120–425(–630) μm, numbers of ostioles up to 12 vs. 80 × 300 μm, 1–2 ostioles] and larger conidia (13–22.5 × 3.5–6 μm vs. 8.5–14 × 2.5–4 μm; Boerema et al. 2004). Didymella subrosea differed from Phoma abietis-albae (also on Abies) by producing larger conidia (13–22.5 × 3.5–6 μm vs. 5–7 × 1.5–3.5 μm; Allescher 1898).
Didymella variabilis L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833522. Fig. 31.
Didymella variabilis (CBS 254.79). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H = 100 μm; I = 50 μm; J, N = 10 μm; K–M = 5 μm.
Etymology: From Latin variabilis, due to the variable shape of the pycnidia.
Description: Conidiomata pycnidial, immersed or semi-immersed, scattered, mostly solitary, sometimes 2–3 confluent, variable in shape and size, mostly irregularly shaped, glabrous, brown, thin-walled, pale brown gelatinous outside layer, 230–875 × 180–740 μm. Ostiole inconspicuous. Pycnidial wall pseudoparenchymatous, composed of oblong, globose or isodiametric cells, 2–4 layers, 17.5–54.5 μm thick, no pigmented layers or outer cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, globose, ampulliform, 5–9.5 × 5–9 μm. Conidia smooth- and thin-walled, variable in shape and size, 1-septate or aseptate, round-ellipsoidal or oblong with both ends rounded, hyaline and aseptate at the beginning, 5.5–9 × 4–6 μm; turning pale brown and 1-septate with age, 7–12(–14) × 4.5–7 μm, mostly with minute polar guttules; globose conidia present, 3.5–6.5 × 3.5–5.5 μm. Conidial matrix not observed.
Culture characteristics: Colonies on OA reaching 65–70 mm diam after 7 d at 25 °C, margin regular, aerial mycelium sparse, buff but pale olivaceous grey near the central zone; reverse concolourous. Colonies on MEA reaching 55–60 mm diam after 7 d, margin regular, covered with sparse aerial mycelium, brown to dark brown, buff towards periphery; reverse concolourous. Colonies on PDA, 80–85 mm diam after 7 d, margin regular, covered by floccose aerial mycelium, buff to honey; reverse concolourous. NaOH spot test results in a pale reddish discolouration near the margin of mycelium on OA.
Typus: Italy, Bari, from Vitis vinifera (Vitaceae), date unknown, A. Ciccarone (holotype CBS H-23693, ex-type living culture CBS 254.79).
Notes: Phylogenetically Did. variabilis clustered in the genus Didymella, and formed a distinct lineage sister to Did. negriana by having 21 bp differences, mainly in rpb2 and tub2 sequences. Morphologically, Did. variabilis differed from Did. negriana by producing larger pycnidia with inconspicuous ostioles [230–875 × 180–740 μm, ostioles inconspicuous vs. 70–220 um, 1–2(–4) papillate ostioles] and larger conidia [7–12(–14) × 4.5–7 μm vs. 4.5–8.5 (–10.5) × 2–4 μm]. These species could also be distinguished based on their conidial colour and septation: conidia brown and 0–1-septate in Did. variabilis, hyaline and aseptate in Did. negriana.
Multiple phoma-like species were found in association with Vitis vinifera, e.g. Phoma flaccida, Phoma vitis and Phoma viniferae. Phoma flaccida had been reallocated to Macrophoma. The new species Didymella variabilis clearly differed from Phoma vitis and Phoma viniferae by its larger conidia (3.5–14 × 3.5–7 μm vs. 3–3.5 × 1–2 μm in Phoma vitis and 7 × 4 μm in Phoma viniferae) (Bonorden, 1864, Saccardo, 1884, 500).
Clade 16: Paraboeremia Qian Chen & L. Cai, Stud. Mycol. 82: 183. 2015.
Type species: Paraboeremia selaginellae (Sacc.) Qian Chen & L. Cai
Paraboeremia putaminum (Speg.) Qian Chen & L. Cai, Stud. Mycol. 82: 184. 2015.
Basionym: Phoma putaminum Speg., Atti Soc. Crittog. Ital. 3: 66. 1881.
Synonyms: Aposphaeria putaminum (Speg.) Sacc., Syll. Fung. 3: 177. 1884.
Coniothyrium putaminum (Speg.) Kuntze, Revis. Gen. Pl. 3 (3): 459. 1898.
Phoma dunorum Ten Houten, Kiemplziekt. Conif. [Thesis Univ. Utrecht]: 88. 1939. Nom. inval.
Description: De Gruyter & Noordeloos (1992).
Materials examined:Denmark, from the rhizosphere of Malus sylvestris (Rosaceae), Feb. 1969, E. Sønderhousen, culture CBS 130.69 = CECT 20054 = IMI 331916. The Netherlands, from a branch of Ulmus sp. (Ulmaceae), Jun. 1991, G.H. Boerema, culture CBS 372.91 = PD 75/960; Gelderland Province, Wageningen, from Zygocactus sp. (Cactaceae), unknown date, M.M.J. Dorenbosch, culture CBS 538.66 = PD 62/93; North Holland Province, Bakkum, Pinus nigra var. austriaca (Pinaceae) seedling root, on dune soil, unknown date, J.G. ten Houten, culture CBS 299.39 (ex-type living culture of Phoma dunorum).
Notes: Phoma dunorum was originally described from Pinus nigra var. austriaca (Ten Houten 1939) and later treated as facultative or heterotypic synonym of Phoma putaminum (currently: Paraboeremia putaminum) based on morphological characteristics (De Gruyter & Noordeloos 1992). According to the phylogenetic analysis in this study (Fig. 1), the ex-type strain of Phoma dunorum (CBS 299.39) clustered with the representative strains of Parab. putaminum (CBS 372.91 and CBS 373.91), confirming the treatment of De Gruyter & Noordeloos (1992). Didymella aeria is genetically similar with Parab. putaminum based on ITS, LSU, tub sequences, taxonomy of which await to be confirmed with more genes (Fig. S1).
Clade 17: Cumuliphoma Valenz.-Lopez et al., Stud. Mycol. 90: 38. 2017(2018).
Type species: Cumuliphoma omnivirens (Aveskamp et al.) Valenz.-Lopez et al.
Clade 18: Macroventuria Aa, Persoonia 6: 359. 1971.
Type species: Macroventuria anomochaeta Aa
Macroventuria angustispora L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833523. Fig. 32.
Macroventuria angustispora (CBS 502.72). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G–H. Pseudothecia. I. Section through pseudothecia. J–K. Asci. L. Ascospores. Scale bars: G–J = 20 μm; K–L = 10 μm.
Etymology: Name reflects the narrowing cell of the ascospores produced by this species.
Description: Ascomata pseudothecial, superficial on the agar or immersed, solitary and scattered, uniloculate, (sub-)globose, with a prominent papilla, membranous, dark brown, 95–180 × 85–165 μm. Ostioles inconspicuous. Pseudothecial wall black, 2–4 layers, 15.5–45 μm, outer wall comprising two layers of dark brown, thick-walled cells, textura angularis. Setae apical on pseudothecia, pale brown, with sub-hyaline tips. Pseudoparaphyses absent. Asci 59.5–91 × 18–30.5 μm, 8-spored, biseriate, bitunicate, fissitunicate, saccate, cylindrical or obpyriform. Ascospores 2-celled, often constricted at the septum, the upper one slightly larger than the lower one, often with one cell narrowing at the middle, 23–28 × 10.5–13 μm, hyaline, smooth-walled, surrounded by distinct mucilaginous sheath. Chlamydospores not observed.
Culture characteristics: Colonies on OA, 40–45 mm diam after 7 d 25 °C, margin entire, regular, aerial mycelium sparse, flat, olivaceous black; reverse leaden grey. Colonies on MEA 30–35 mm diam after 7 d, margin irregular, aerial mycelium sparse, flat, whitish near the centre, lavender grey with an orange edge, some radially furrowed zones near the centre; reverse concolourous. Colonies on PDA, 30–35 mm diam after 7 d, margin irregular, covered by flat mycelium, olivaceous black to olivaceous, with a buff edge; reverse concolourous. NaOH spot test negative on OA.
Typus: South Africa, Cape Province, from trunks of Medicago sativa (Fabaceae), Jun. 1972, W.F.O. Marasas (holotype CBS H-14193, ex-type living culture CBS 502.72).
Notes: CBS 502.72 was initially received as Macroventuria anomochaeta. However, this isolate formed a distinct lineage in the genus Macroventuria and can be distinguished from the ex-type strain of Ma. anomochaeta (CBS 525.71) by producing much larger asci (59.5–91 × 18–30.5 μm vs. 60–75 × 16–21 μm) and wider ascospores (23–28 × 10.5–13 μm vs. 20–27 × 9–11 μm). Therefore, Ma. angustispora was introduced as a new species, based on isolate CBS 502.72.
Macroventuria terrestris L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833524. Fig. 33.
Macroventuria terrestris (CBS 127771). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Perithecia forming on OA. H. Pseudothecia. I. Section through pseudothecium. J. Section of pseudothecial wall. K–L. Asci. M. Ascospores. Scale bars: H = 50 μm; I, K = 20 μm, J, L–M = 10 μm.
Etymology: The species name refers to soil, the substrate from which this fungus was isolated.
Description: Pseudothecia superficial on or semi-immersed in the agar, solitary and scattered, uniloculate, subglobose to ellipsoidal, membranous, dark brown to black, 135–295 × 95–285 μm. Ostioles inconspicuous, central. Setae pale brown/olivaceous, with sub-hyaline tip, cylindrical or tapering to the rounded or somewhat pointed tip. Pseudothecial wall black, 17.5–43.5 μm, 4–5 layers, outer wall comprising 1–3 layers of pigmented, thin-walled cells, of textura angularis. Pseudoparaphyses absent. Asci 102–134 × 20.5–29 μm, 8-spored, uniseriate, bitunicate, fissitunicate, saccate, cylindrical to club-shaped. Ascospores 2-celled, often constricted at the septum, the upper cell slightly larger than the lower one, 20–29.5 × 11.5–13 μm, hyaline, smooth- and thin-walled, surrounded by distinct mucilaginous sheath. Chlamydospores not observed.
Culture characteristics: Colonies on OA, 25–35 mm diam after 7 d, margin irregular, covered by flat aerial mycelium, buff to darker olivaceous; reverse concolourous. Colonies on MEA 55–60 mm diam after 7 d, margin regular, aerial mycelium flat, pale olivaceous grey, with orange margin; reverse yellow to brown. Colonies on PDA, 15–20 mm diam after 7 d, margin irregular, covered by felty, olivaceous aerial mycelium, with thin buff margin; reverse vinaceous buff to olivaceous. NaOH spot test results in a pale brown discolouration on OA.
Typus: USA, Utah, Canyonlands National Park, from soil, 1997, M. Christensen (holotype CBS H-23670, ex-type living culture CBS 127771).
Notes: Isolate CBS 127771 was received as Macroventuria wentii. However, it was phylogenetically distinct from the ex-type strain of Ma. wentii (CBS 526.71). Isolate CBS 127771 also differed morphologically from Ma. wentii by its ellipsoidal pseudothecia (globose or piriform in Ma. wentii). Furthermore, CBS 127771 produced larger pseudothecia than Ma. wentii (134–295 × 95–285 μm vs. 135–180 × 105–160 μm).
Clade 19:Juxtiphoma Valenz.-Lopez et al., Stud. Mycol. 90: 40. 2017(2018).
Notes: Juxtiphoma was introduced by Valenzuela-Lopez et al. (2018) to accommodate Phoma eupyrena reported on stems of Solanum tuberosum (geographic origin not cited; Boerema et al.2004). It formed a well-supported monophyletic clade, and is separated from the other genera of Didymellaceae in the phylogenetic tree (Valenzuela-Lopez et al. 2018). However, when this genus was introduced, Endophoma elongata that was isolated from soil of a bat cave in Canada, was not included in the phylogenetic analysis. In the present study, Endophoma elongata clusters within the Juxtiphoma clade. However, they are clearly different morphologically: E. elongata is characterised by producing two kinds of pycnidia, spherical conidiomata and branched, non-ostiolate, cylindrical conidiomata, both of which always produced conidia endogenously. In contrast, species of Juxtiphoma only produce globose pycnidia with normal phoma-like conidia. Therefore, these two genera appear to be distinct.
Type species: Juxtiphoma eupyrena (Sacc.) Valenz.-Lopez et al.
Juxtiphoma kolkmaniarum Hern.-Restr. et al., MycoKeys 65: 76. 2020.
Description: Hou et al. (2020).
Typus: The Netherlands, Ophemert, isolated from garden soil, Mar. 2017, L. & M. Kolkman (holotype CBS H-24214, ex-type culture CBS 146005 = JW 185006).
Additional materials examined: Canada, Alberta, Whitehorse Wildland Provincial Park, Boulder Run in Cadomin Cave, soil in bat cave, 13 Jun. 2010, A. Hudgins, (holotype of Endophoma elongata UAMH 11216). Germany, Kiel-Kitzeberg, from wheat field soil, unknown collector and collect date, specimen CBS H-16216, culture CBS 527.66.
Notes: Juxtiphoma kolkmaniarum was isolated from Dutch garden soil and was named after the sample collectors (Hou et al. 2020). Endophoma elongata was isolated from soil of a bat cave in Canada, but the DNA sequences proved to be identical to J. kolkmaniarum (based on ITS, LSU and tub2 sequences; Tsuneda et al. 2011). However, E. elongata lacks rpb2 sequence data for analysis. Although the sequence data show that they might be synonyms, their morphological characters are very different.
Clade 20: Nothophoma Qian Chen & L. Cai, Stud. Mycol. 82: 212. 2015.
Conidiomata pycnidial, globose to elongated, or irregular, superficial on or immersed into the agar, solitary or confluent, ostiolate, sometimes with a short neck. Pycnidial wall pseudoparenchymatous, 2–9-layered, outer wall pigmented. Conidiogenous cells phialidic, hyaline, smooth, ampulliform, doliiform or globose, sometimes flask-shaped. Conidia variable in shape, hyaline but incidentally brown, smooth- and thin-walled, (0–)1(–2)-septate, i.e. ovoid, oblong to ellipsoidal, eguttulate or guttulate (Chen et al. 2015).
Type species: Nothophoma infossa (Ellis & Everh.) Qian Chen & L. Cai
Notes: Two newly added species of Nothophoma, N. infuscata and N. acaciae produce 0–1-septate conidia, and N. eucalyptigena produces conidia with (0–)1(–2)-septate, therefore, the generic circumscription of Nothophoma was emended according to the morphological features of the new member introduced in this genus.
Nothophoma acaciae (Crous) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833533.
Basionym: Didymella acaciae Crous, Persoonia 39: 407. 2017.
Description: Crous et al. (2017b).
Typus: Australia, New South Wales, Merimbula, on leaves of Acacia melanoxylon (Fabaceae), 28 Nov. 2016, P.W. Crous (holotype CBS H-23295, ex-type living culture CPC 32504 = CBS 143404).
Notes: This species, originally published as Didymella acaciae (Crous et al. 2017b), was mentioned as being phylogenetically closely related to several genera, including Didymella, Paraboeremia, Peyronellaea and Verrucoconiothyrium, containing several species which clustered in the genus Nothophoma in the present study. Phylogenetically, Didymella acaciae grouped in a sister lineage to No. prosopidis (Fig. 1). Therefore, a new combination was introduced as No. acaciae. Morphologically, No. acaciae is characterised by producing 1-septate conidia, which is rare in the genus Nothophoma.
Nothophoma arachidis-hypogaeae (V.G. Rao) Qian Chen & L. Cai, Stud. Mycol. 82: 213. 2015. Fig. 34.
Nothophoma arachidis-hypogaeae (CBS 125.93). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G–H. Pycnidia forming on OA. I. Conidia. J–K. Pycnidia. L–M. Conidiogenous cells. Scale bars: I, L–M = 5 μm; J–K = 20 μm.
Basionym: Phyllosticta arachidis-hypogaeae V.G. Rao, Sydowia 16: 275. 1962.
Synonyms: Phoma arachidis-hypogaeae (V.G. Rao) Aa & Boerema, Persoonia 15: 388. 1993.
Description from ex-epitype (CBS 125.93): Conidiomata pycnidial, semi-immersed or superficial on the agar surface, mostly aggregated, solitary or confluent, mostly (sub-)globose or flask-shaped, pale brown, dark brown near the ostioles, glabrous, with hypha around the ostioles, 95–260 × 80–190 μm. Ostioles single, central, papillate or elongated to a neck, up to 70 μm. Pycnidial wall pseudoparenchymatous, composed of oblong, isodiametric or globose cells, 3–5 layers, 18–23 μm thick, outer two cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, (sub-)globose, ampulliform or lageniform, 5.5–8.5 × 4.5–8 μm. Conidia oblong, smooth- and thin-walled, hyaline, aseptate, 4–7 × 2–3 μm, with two minute, polar guttules. Conidial matrix whitish to buff.
Culture characteristics: Colonies on OA, 65–70 mm diam after 7 d at 25 °C, margin regular, aerial mycelium flat, buff to dark brown; reverse pale brown. Colonies on MEA 60–65 mm diam after 7 d, margin regular, densely covered with floccose aerial mycelium, buff; reverse luteous to dark brown. Colonies on PDA, 70–75 mm diam after 7 d, margin regular, densely covered by floccose aerial mycelium, buff, rosy buff towards periphery; reverse dark brown. NaOH spot test negative on OA.
Typus: India, Poona, from leaves of Arachis hypogaea (Fabaceae), Sep. 1962, V. Rao (holotype M.A.C.S. No. 134). India, Madras, from a leaf of Arachis hypogaea (Fabaceae), dep. Jan. 1993, J. de Gruyter (epitype designated here CBS H-24315, MBT389731, ex-epitype living culture CBS 125.93 = PD 77/1029).
Notes: Phyllosticta arachidis-hypogaeae was originally described from leaves of Arachis hypogaea in Poona, India (Rao 1962), with conidia measuring 5.25–6.9 × 2.2–3.57 μm. The morphology of the specimen selected in this study, isolated from the same host and country as the holotype, agreed well with the original description (conidia 4–7 × 2–3 μm), and thus CBS H-24315 was chosen as epitype, with CBS 125.93 as ex-epitype culture.
Nothophoma brennandiae Hern.-Restr. et al., Mycokeys 65: 77. 2020. Fig. 35.
Nothophoma brennandiae (CBS 125539). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N–O. Conidia. Scale bars: H = 50 μm; I = 20 μm, J = 10 μm; K–O = 5 μm.
Description from culture CBS 125539: Conidiomata pycnidial, semi-immersed or superficial on the agar surface, mostly aggregated and five or more confluent, some scattered and solitary, mostly cylindrical, club-shaped, flask-shaped or subglobose, dark brown, thick-walled, with hyphal outgrowths, especially around the central ostioles, 210–400 × 150–300 μm. Ostioles 1–2, slightly papillate. Pycnidial wall pseudoparenchymatous, composed of oblong, isodiametric or globose cells, 4–5 layers, 11.5–38.5 μm thick, outer three cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, ampulliform, lageniform or doliiform, 3.5–8.5 × 3–6.5 μm. Conidia oblong to ovoid, smooth- and thin-walled, hyaline, turning brown with age, aseptate, 4.5–8.5 × 3–3.5 μm, with small polar guttules. Conidial matrix dark brown or black.
Culture characteristics: Colonies on OA, 55–60 mm diam after 7 d at 25 °C, margin regular, aerial mycelium flat, pale mouse grey; reverse olivaceous. Colonies on MEA 45–50 mm diam after 7 d, margin regular, covered with sparse aerial mycelium, flat, mouse grey with brown edge, black in the centre because of the black conidial exudate, with pale mouse grey sterile section; reverse concolourous. Colonies on PDA, 55–60 mm diam after 7 d, margin regular, covered by floccose aerial mycelium, olivaceous to pale olivaceous grey, with some pale brown area; reverse olivaceous black with olivaceous edge. NaOH spot test results in a brown discolouration near the margin of mycelium on OA.
Typus: The Netherlands, Limburg Province, Ell, isolated from garden soil, Mar. 2017, K. Brennand (holotype CBS H-24103, ex-type living culture CBS 145912 = JW 53011).
Additional materials examined: Canada, Regina, Municipality of Sherwood, Saskatchewan, house dust, unknown date and collector, culture CBS 140540; ibid., cultures CBS 140541, CBS 140542. Greece, Northern Greece, Prefecture of Imathia, from shoot of Amygdalus sp. (Rosaceae; canker), 6 Dec. 2008, T. Thomidis, culture CBS 125539. Italy, Apulia, from Olea europaea (Oleaceae), 6 Dec. 2008, S. Frisullo, culture CBS 262.95; from branches of Ostrya carpinifolia (Betulaceae; hop hornbeam), unknown date and collector, culture CBS 125540. The Netherlands, North Holland province, Amsterdam, garden soil, Mar. 2017, J. van Dijk, JW 1066.
Notes: Three isolates from plant material in two southern European countries clustered with the ex-type strain of Nothophoma brennandiae (CBS 145912; Fig. 1), which was isolated from Dutch garden soil (Hou et al. 2020), revealing the widespread distribution of No. brennandiae.
Nothophoma eucalyptigena (Crous) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833534.
Basionym: Verrucoconiothyrium eucalyptigenum Crous, Persoonia 38: 299. 2017.
Description: Crous et al. (2017a).
Typus: Australia, Western Australia, Perth, King’s Park Botanic Gardens, on leaf litter of Eucalyptus sp. (Myrtaceae), 27 Sep. 2015, P.W. Crous (holotype CBS H-23098, ex-type living culture CPC 29000 = CBS 142535).
Notes: This species was introduced as Verrucoconiothyrium eucalyptigenum based on the megablast result of ITS and LSU sequences (Crous et al. 2017a). The multi-locus phylogenetic analyses indicated, however, that this species grouped within the genus Nothophoma, and formed a distinct lineage closely related to No. infuscata (Fig. 1), the novel species collected from dying leaves of Acacia longifolia (Fabaceae) and described in the present study. Morphologically, both species produced brown and septate conidia (see No. infuscata for discussion).
Nothophoma infuscata L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833535. Fig. 36.
Nothophoma infuscata (CBS 121931). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidia. I. Section through pycnidium. J. Section of pycnidial wall. K–N. Conidiogenous cells. O. Conidia. Scale bars: H = 50 μm; I = 20 μm; J = 10 μm; K–O = 5 μm.
Etymology: Epithet derived from the brown conidial matrix produced by this species.
Description:Conidiomata pycnidial, mostly immersed in the agar, sometimes semi-immersed, scattered, and arranged in radiate lines, solitary or 2–3 confluent, (sub-)globose, brown to dark brown, thin-walled, with abundant hyphal outgrowths, especially around the ostioles, 115–335 × 95–280 μm. Ostioles single, mostly in the centre, papillate or not papillate, sometimes elongated into a short neck, up to 50 μm. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 4–6 layers, 10.5–32 μm thick, outer 1–2 cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, subglobose, ampulliform or lageniform, 7–11 × 5.5–10.5 μm. Conidia ovoid, ellipsoidal, oblong with round end or cylindrical, hyaline, smooth- and thin-walled, brown, 0–1-septate, (4.5–)6–11 × 3.5–5.5 μm, mostly with 5–6 minute scattered guttules. Conidial matrix dark brown.
Culture characteristics: Colonies on OA reaching 30–35 mm diam after 7 d at 25 °C, margin regular, without aerial mycelium, olivaceous but with some rosy buff sterile sections, production of pycnidia abundant; reverse concolourous. Colonies on MEA reaching 30–35 mm diam after 7 d, margin regular, covered with medium aerial mycelium, felty, pale olivaceous grey, salmon towards periphery, abundant production of pycnidia; reverse dark brown with orange edge. Colonies on PDA reaching 35–40 mm diam after 7 d, margin regular, covered by flat aerial mycelium, sparse, rosy buff to buff, production of pycnidia abundant; reverse concolourous. NaOH spot test results in a pale reddish discolouration near the margin of mycelium.
Typus: New Zealand, Auckland, Waterview, Traherne Island, from dying leaves of Acacia longifolia (Fabaceae), 16 May 2007, H. Pearson (holotype CBS H-23674, ex-type living culture CBS 121931).
Notes: Nothophoma infuscata was isolated from the same host as Phoma acaciae (Acacia longifolia, Italy). However, its conidia differ from the original description of Phoma acaciae [(4.5–)6–11 × 3.5–5.5 μm vs. 7–8 × 2–3 μm; Saccardo 1884]. It is genetically distinct from other species in the genus Nothophoma, forming a sister lineage to No. eucalyptigena (Fig. 1). Morphologically, No. eucalyptigena has smaller conidiogenous cells (6–8 × 5–7 μm vs. 7–11 × 5.5–10.5 μm) and larger conidia than No. infuscata [(8–)9–13(–15) × (4–)5(–6) μm vs. (4.5–)6–11 × 3.5–5.5 μm; Crous et al. 2017a].
Nothophoma nullicana L.W. Hou, L. Cai & Crous, nom. nov. MycoBank MB834021.
Basionym: Verrucoconiothyrium acaciae Crous, Persoonia 38: 403. 2017.
Etymology: Name refers to Nullica State Forest, Australia, where this fungus was collected.
Description: Crous et al. (2017b).
Typus: Australia, New South Wales, Nullica State Forest, on leaves of Acacia falciformis (Fabaceae), 29 Nov. 2016, P.W. Crous (holotype CBS H-23292, culture ex-type CPC 32330 = CBS 143448).
Notes: Verrucoconiothyrium acaciae was described from leaves of Acacia falciformis (Fabaceae), the same host genus with Didymella acaciae, which was treated as a new combination, Nothophoma acaciae, in the present study. Phylogenetically, the ex-type strain of Verrucoconiothyrium acaciae (CBS 143448) also clusters within Nothophoma and shares a close phylogenetic affinity with N. acaciae and N. prosopidis, but forms a clearly separate lineage that is distinct from the latter two species. However, the epithet is already occupied, and therefore, a new name is proposed for this species as Nothophoma nullicana. Nothophoma nullicana can be easily distinguished from N. acaciae in producing brown, aseptate and shorter conidia measuring (6.5–)8–9(–10) × (3–)3.5(–4) μm, while N. acaciae produces hyaline, 1-septate conidia measuring (16–)18–21(–25) × (3–)3.5(–4) μm. Nothophoma nullicana differs from N. prosopidis by producing conidiomata with smaller ostioles (10–15 μm diam. vs. 80 μm diam.) and narrower conidia [(6.5–)8–9(–10) × (3–)3.5(–4) μm, fusoid-ellipsoid vs. (7–)8–9(–10) × (4–)5–6(–7) μm, ellipsoid to globose] (Crous et al. 2013b).
Nothophoma prosopidis (Crous & A.R. Wood) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833536.
Basionym: Coniothyrium prosopidis Crous & A.R. Wood, Persoonia 31: 207. 2013.
Synonym: Verrucoconiothyrium prosopidis (Crous & A.R. Wood) Crous, Persoonia 38: 299. 2017.
Description: Crous et al. (2013b).
Typus: South Africa, Northern Cape Province, Calvinia, associated with a bark disease on Prosopis sp. (Fabaceae), Sep. 2012, A. Wood (holotype CBS H-21424, ex-type living culture CPC 21699 = CBS 136415).
Additional material examined: South Africa, Northern Cape Province, Calvinia, associated with a bark disease on Prosopis sp. (Fabaceae), Sep. 2012, A. Wood, culture CPC 21701; ibid., culture CPC 21705.
Notes: This species was initially introduced as Coniothyrium prosopidis (Crous et al. 2013b) and later recombined in Verrucoconiothyrium as Ve. prosopidis (Crous et al. 2017a). In the present study, Ve. prosopidis grouped in the genus Nothophoma and formed a distinct lineage closely related to No. acaciae, thus a new combination, No. prosopidis, was introduced here.
Clade 21: Vacuiphoma Valenz.-Lopez et al., Stud. Mycol. 90: 40. 2017 (2018).
Type species: Vacuiphoma bulgarica (Aveskamp et al.) Valenz.-Lopez et al.
Vacuiphoma ferulae (Pat.) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833531. Fig. 37.
Vacuiphoma ferulae (CBS 353.71). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H–I = 50 μm; J, L–N = 10 μm; K = 5 μm.
Basionym: Ascochyta ferulae Pat., Énum. Champ. Tunisie (Paris) 17. 1892.
Description from ex-neotype (CBS 353.71): Conidiomata pycnidial, semi-immersed, immersed or produced on aerial mycelium, mostly scattered, solitary, sometimes 2–3 confluent, (sub-)globose or flask-shaped, pale brown, glabrous, ostiolate, 130–380 × 90–245 μm. Ostioles single, central, slightly papillate or non-papillate. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 2–5 layers, 9–26 μm thick, outer 1–3 cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, subglobose, lageniform, ampulliform to doliiform, 7.5–16.5 × 5.5–11.5 μm. Conidia oblong with rounded apices, smooth- and thin-walled, hyaline, 0–1-septate and constricted at the septum, 6.5–17 × 3.5–6.5 μm, eguttulate or with minute scattered guttules. Conidial matrix slightly pink.
Culture characteristics: Colonies on OA, 75–80 mm diam after 7 d 25 °C, margin irregular, aerial mycelium woolly, whitish; reverse buff. Colonies on MEA 75–80 mm diam after 7 d, margin regular, aerial mycelium woolly, whitish to pale grey; reverse dark brick. Colonies on PDA, 75–80 mm diam after 7 d, margin regular, covered by floccose aerial mycelium, buff to olivaceous grey; reverse olivaceous grey to olivaceous. NaOH spot test: a pale red discolouration on OA.
Typus: Italy, Sardegna, Monte Toddeito, from dead stem of Ferula communis (Apiaceae), 24 Aug. 1970, W. Gams (neotype designated here CBS H-23660, MBT389727, ex-neotype living culture CBS 353.71).
Notes: Ascochyta ferulae was originally described from a Ferulae sp. in Monastir, Tunisia (Patouillard 1892). However, no type material could be located (FH, PC, B), and it is thus considered lost. Culture CBS 353.71 was received as As. ferulae, the morphological characters of which agrees well with the description of As. ferulae from Saccardo & Sydow (1899). Isolate CBS 353.71 was therefore designated as ex-neotype culture for As. ferulae.
Vacuiphoma laurina (Thüm.) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833532. Fig. 38.
Vacuiphoma laurina (CBS 119636). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H–I = 20 μm; J = 10 μm; K–N = 5 μm.
Basionym: Phoma laurina Thüm., Boll. Soc. Adriat. Sci. Nat. Trieste. 3 (2): 452. 1877.
Description from ex-neotype (CBS 119636): Conidiomata pycnidial, abundant, semi-immersed or immersed, mostly aggregated, solitary, sometimes 2–4 confluent, (sub-)globose or flask-shaped, brown, with sparse hyphal outgrowths, ostiolate, 105–435(–505) × 90–300(–425) μm. Ostioles 1–4(–5), up to 10 with age, slightly papillate or elongated into a short collared neck. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 4–5 layers, 12–32 μm thick, outer 2–3 cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, ampulliform, doliiform or lageniform, 5–8 × 4.5–7.5 μm. Conidia oblong with rounded apices, smooth- and thin-walled, hyaline, aseptate, 4.5–7.5 × 2–3 μm, eguttulate or with 2–4 minute, polar guttules. Conidial matrix white, hyaline.
Culture characteristics: Colonies on OA reaching 55–60 mm diam after 7 d at 25 °C, margin regular, aerial mycelium flat, pale mouse grey near the centre, with a buff circle, pale olivaceous towards periphery; reverse concolourous. Colonies on MEA reaching 55–60 mm diam after 7 d, margin regular, aerial mycelium flat, concentric circles of different colours, mouse grey near the centre, greyish in the middle, salmon towards periphery; reverse dark brown, orange towards periphery. Colonies on PDA reaching 60–65 mm diam after 7 d, margin regular, covered by floccose aerial mycelium, pale olivaceous; reverse olivaceous black with buff edge. NaOH spot test results in a pale reddish discolouration, with pale olivaceous near the centre on OA.
Typus: Turkey, Western Anatolia, from Laurus nobilis (Lauraceae), date unknown, M.E. Göre (neotype designated here CBS H-23686, MBT389728, ex-neotype living culture CBS 119636).
Notes: Phoma laurina was originally described from Laurus nobilis in Gorizia, Italy, with ellipsoidal conidia measuring 4–6 × 1.5–2 μm (Von Schlechtendal, 1824, Saccardo, 1884, Bolle and von Thümen, 1877). However, the type specimen of Phoma laurina could not be traced. In the present study, isolate CBS 119636 (Laurus nobilis, Turkey) morphologically agrees well with the original description of Phoma laurina, having cylindrical to oblong conidia measuring 4.5–7.5 × 2–3 μm. We therefore designated CBS 119636 as ex-neotype culture of Phoma laurina. Phylogenetically, CBS 119636 grouped in the genus Vacuiphoma, and thus a new combination was introduced.
Clade 22: Microsphaeropsis Höhn., Hedwigia 59: 267. 1917.
Type species: Microsphaeropsis olivacea (Bonord.) Höhn.
Notes: The genus Microsphaeropsis was introduced by Von Höhnel (1917) and was originally placed in Montagnulaceae to accommodate species with pigmented, phoma-like conidia. Later a new family Microsphaeropsidaceae was introduced to accommodate this genus (Chen et al. 2015). However, in the present study, all remaining isolates accommodated as “Microsphaeropsis spp.” in the CBS culture collection were sequenced. The phylogenetic analysis based on the increased number of isolates and expanded dataset revealed that Microsphaeropsis species reside inside the Didymellaceae, being closely related to Neomicrosphaeropsis and Paramicrosphaeropsis. Moreover, all three genera are morphologically comparable in initially producing hyaline conidia that turn brownish with age. Therefore, the family Microsphaeropsidaceae was reduced to synonymy, and the genus Microsphaeropsis was again placed in Didymellaceae.
Microsphaeropsis fusca L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833537. Fig. 39.
Microsphaeropsis fusca (CBS 116670). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H–I. Pycnidia. J. Section through pycnidium. K–N. Conidiogenous cells. O. Conidia. Scale bars: H–J = 50 μm; K–O = 5 μm.
Etymology: Name refers to the dark brown pycnidia produced by this species.
Conidiomata pycnidial, semi-immersed in the agar, solitary, scattered, sometimes confluent, (sub-)globose or irregular-shaped, dark brown to black, with hyphal outgrowths, 210–335 × 190–260 μm. Ostioles inconspicuous. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 3–4 layers, with outer 3 layers pigmented, 16.5–30 μm thick. Conidiogenous cell phialidic, hyaline, smooth, ampulliform, lageniform or doliiform, 7–14 × 4.5–10.5 μm. Conidia variable in shape and size, (sub-)globose, oblong, ellipsoidal or oval, hyaline at beginning, pale brown with age, smooth- and thin-walled, aseptate, 5–10.5 × 3.5–6.5 μm, guttulate.
Culture characteristics: Colonies on OA, 40–45 mm diam after 7 d at 25 °C, margin regular, covered by sparsely floccose aerial mycelium, buff to cinnamon; reverse concolourous. Colonies on MEA 35–40 mm diam after 7 d, margin regular, covered by woolly aerial mycelium, white to cinnamon; reverse orange to sienna. Colonies on PDA, 35–40 mm diam after 7 d, margin regular, covered by woolly aerial mycelium, whitish, buff toward periphery; reverse buff, pale orange near the centre. NaOH spot test negative on OA.
Typus: The Netherlands, from twig lesions of Sarothamnus scoparius (Fabaceae), 23 Aug. 2004, G. Verkley & M. Starink (holotype CBS-H 24339, ex-type living culture CBS 116670).
Additional materials examined: The Netherlands, from twig lesions of Sarothamnus scoparius (Fabaceae), 23 Aug. 2004, G. Verkley & M. Starink, culture CBS 116669. Germany, from Sarothamnus scoparius (Fabaceae), 21 Dec. 2013, R.K. Schumacher, culture CBS 139603.
Notes: Microsphaeropsis fusca was isolated from twig lesions of Sarothamnus scoparius in the Netherlands, and formed a well-supported sister clade with Mi. proteae (Fig. 1). It differed phenotypically from Mi. proteae by its larger conidiogenous cell (7–14 × 4.5–10.5 μm vs. 3–8 × 3–4 μm) and larger conidia (5–10.5 × 3.5–6.5 μm vs. 3–4 × 2–2.5 μm in vitro; Swart et al. 1998, Crous et al. 2011).
Microsphaeropsis olivacea (Bonord.) Höhn., Hedwigia 59: 267. 1917.
Basionym: Coniothyrium olivaceum Bonord., Jahrb. Nassauischen Vereins Naturk. 23–24: 377. 1869.
Description: Chen et al. (2015).
Typus: Austria, on stem of Hedera helix (holotype BPI 797151, ex herb. Fuckel, ex herb. Boiss).
Materials examined: France, Lorraine, Cronartium ribicola (Cronartiaceae), May 1976, M. Morelet, CBS 320.76; Nancy, from needle of Pinus laricio (Pinaceae), unknown date, M. Morelet, CBS 233.77; Germany, Baden-Württemberg, from wood of Picea abies (Pinaceae), unknown date, S. Schönhar, CBS 336.78. The Netherlands, Baarn, Groeneveld, Tremella foliacea (Tremellaceae), 2 Mar. 1972, J. Stalpers, CBS 608.72. Switzerland, Egerkingen, Pinus sylvestris (Pinaceae), unknown date and collector, CBS 617.83.
Notes: Microsphaeropsis olivacea was originally collected from stems of Hedera helix in Austria. In this study, additional isolates cluster with the reference strain of Microsphaeropsis olivacea (CBS 233.77), but were collected from different hosts. Therefore, further studies are needed to resolve its typification and to clarify its phylogeny.
Microsphaeropsis taxicola L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB835482. Fig. 40.
Microsphaeropsis taxicola (CBS 442.83). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J–K. Conidiogenous cells. L. Conidia. M–O. Chlamydospores. Scale bars: H = 100 μm; I = 50 μm; J–O = 10 μm.
Etymology: Named after the host genus Taxus, from which this species was isolated.
Description: Conidiomata pycnidial, (semi-)immersed in the agar, solitary and aggregate, (sub-)globose, dark brown or black, with white hyphal outgrowths, especially around the ostioles, ostiolate, 275–545 × 245–435 μm. Ostioles 1–3, slightly papillate. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 3–4 layers, with outer 3 layers pigmented, 10–18 μm thick. Conidiogenous cell phialidic, hyaline, smooth, globose, ampulliform, lageniform, 5.5–10.5 × 4–8 μm. Conidia oblong, hyaline at beginning, pale brown with age, smooth- and thin-walled, aseptate, 3.5–6 × 2.5–3.5 μm, 1–2 guttulate, minute. Chlamydospores uni- or multicellular, unicellular intercalary, guttulate, thick-walled, pale brown to brown, 6–10 μm diam, multicellular irregular dictyo/phragmosporous, somewhat botryoid and in combination with unicellular chlamydospores, dark greenbrown, 16–32 × 15–25 μm. Conidial matrix brown.
Culture characteristics: Colonies on OA, 30–35 mm diam after 7 d at 25 °C, margin regular, covered by sparsely floccose aerial mycelium, with sepia, white, cinnamon and buff concentric rings; reverse concolourous. Colonies on MEA 25–30 mm diam after 7 d, margin irregular, covered by felty aerial mycelium, buff at centre, whitish at periphery; reverse dark brick, orange towards periphery. Colonies on PDA, 30–35 mm diam after 7 d, margin regular, covered by felty aerial mycelium, pale luteous, luteous to whitish towards periphery, with vinaceous pigment on the agar; reverse brown vinaceous, saffron towards periphery. NaOH spot test negative on OA.
Typus: The Netherlands, Baarn, Taxus baccata (Taxaceae), unknown date and collector, (holotype CBS H-24379, ex-type living culture CBS 442.83).
Additional materials examined: The Netherlands, Baaer, from wood of Rhus typhina (Anacardiaceae), Jul. 1980, W. Gams, culture CBS 469.80; Kortenhoef, Opuntia cladodes (Cactaceae, cultivated, several years ago imported from Spain), May 1992, J. Daams, culture CBS 427.92.
Notes: Three isolates collected from different hosts in the Netherlands and formed an isolated clade with moderate support values, showing a close phylogenetic affinity to Microsphaeropsis olivacea and Mi. viridis. A new species is therefore proposed here as Mi. taxicola. The morphological differences between Mi. taxicola and Mi. viridis are discussed under Mi. viridis. Microsphaeropsis taxicola can be clearly distinguished from Mi. olivacea by producing aseptate conidia, while the latter produces 0–1-septate conidia.
Microsphaeropsis viridis L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB835483. Fig. 41.
Microsphaeropsis viridis (CBS 432.71). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H–I. Pycnidia. J. Section through pycnidium. K–M. Conidiogenous cells. N. Conidia. Scale bars: H–J = 50 μm; K–N = 5 μm.
Etymology: Name refers to the dark green discolouration on the agar by application of NaOH.
Description: Conidiomata pycnidial, (semi-)immersed in the agar, solitary, aggregate, (sub-)globose or irregular-shaped, pale brown, with hyphal outgrowths, especially around the ostioles, ostiolate, 150–395 × 135–285 μm. Ostioles 0–2, slightly papillate. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 3–4 layers, with outer 2–3 layers pigmented, 17–40 μm thick. Conidiogenous cell phialidic, hyaline, smooth, ampulliform, lageniform or doliiform, 5–8 × 4.5–7 μm. Conidia variable in shape and size, oblong, ellipsoidal, hyaline at beginning, pale brown with age, smooth- and thin-walled, aseptate, 5.5–8.5 × 2.5–3.5 μm, guttulate. Conidial matrix hyaline to pale brown.
Culture characteristics: Colonies on OA, 35–40 mm diam after 7 d at 25 °C, margin regular, covered by sparsely floccose aerial mycelium, with white, pale brown and buff concentric rings, pycnidia aggregate and formed in the middle rings; reverse concolourous. Colonies on MEA 30–35 mm diam after 7 d, margin regular, covered by felty aerial mycelium, with buff, pale grey, brick and whitish concentric rings; reverse with umber, orange, ochreous rings. Colonies on PDA, 30–35 mm diam after 7 d, margin regular, covered by felty aerial mycelium, whitish, with isabelline areas, cinnamon at periphery; reverse with sepia, fawn, and saffron rings. Application of NaOH results in a dark green discolouration of the agar.
Typus: The Netherlands, Valkenswaard, from dead twig and pod of Sarothamnus sp. (Fabaceae), 21 Feb. 1971, H.A. van der Aa (holotype CBS H-10870, ex-type living culture CBS 432.71).
Additional materials examined: France, Meurthe et Moselle, from leaf of Populus tremula (Salicaceae), M. Morelet, 17 Aug. 1972, culture CBS 763.73; Haute Vienne, Pseudotsuga menziesii (Pinaceae), M. Morelet, 21 Mar. 1972, culture CBS 762.73. Germany, University of Freiburg, Nov. 1980, G. Schüler, Abies alba (Pinaceae), culture CBS 639.80. The Netherlands, Baarn, Valkenswaard, garden Peperstraat 6, from leaf spots of Berberis sp. (Berberidaceae), cult. H.A. van der Aa, 23 Mar. 1969, culture CBS 354.69.
Notes: Microsphaeropsis viridis is represented by five isolates from different hosts. It forms a well-supported clade and is closely related with Mi. olivacea and Mi. taxicola (Fig. 1). In our phylogenetic study, Mi. viridis is distinct from the references strain of Mi. olivacea (CBS 233.77) in 17 bp of rpb2 and 3 bp of tub2 based on alignment of the concatenated four loci deposited in TreeBASE (S25826); from Mi. taxicola differing in 2 bp of ITS and 13 bp of rpb2. This new species differs from Mi. taxicola by its longer conidia (5.5–8.5 × 2.5–3.5 μm vs. 3.5–6 × 2.5–3.5 μm); from Mi. olivacea by its aseptate conidia (Chen et al. 2015). Microsphaeropsis viridis could also be distinguished from the other two species by producing pycnidia with 0–2 ostioles, while pycnidia of Mi. olivacea has single ostiole and Mi. taxicola has 1–3 ostioles. Besides, the application on NaOH results in a dark green discolouration of the agar of Mi. viridis, with Mi. taxicola having a negative reaction in NaOH, and that of Mi. olivacea is not recorded.
Clade 23: Neomicrosphaeropsis Thambug. et al., Fungal Diversity 82: 261. 2016.
Type species: Neomicrosphaeropsis italica Thambug. et al., Fungal Diversity 82: 264. 2016.
Clade 24: Paramicrosphaeropsis L.W. Hou, L. Cai & Crous, gen. nov. MycoBank MB833538.
Etymology: From Greek παρα-, beside, referring to the close morphological and phylogenetic relationship with the genus Microsphaeropsis.
Conidiomata pycnidial, mostly semi-immersed, abundant, scattered or aggregated, mostly solitary, sometimes confluent, globose to subglobose, pale brown and the pycnidial wall hyaline at first, dark brown with age. Pycnidial wall pseudoparenchymatous, thin, multi-layered. Conidiogenous cells phialidic, hyaline, smooth, subglobose, ampulliform to lageniform. Conidia variable in shape, broad ellipsoidal, oblong or ovoid, thin-walled, smooth, hyaline at beginning, brown with age, aseptate, mostly guttulate. Sexual morph unknown.
Notes: Paramicrosphaeropsis is introduced as a new genus belonging to Didymellaceae based on the multi-locus phylogenetic analysis and morphological characters. This genus is phylogenetically close to Neomicrosphaeropsis and Microsphaeropsis, and distant from all other known genera in Didymellaceae with high statistical support (100 % MLBS/1.0 BPP, Fig. 1). Morphologically, Paramicrosphaeropsis could be distinguished from other genera in this family by producing pycnidia with an extremely thin and hyaline pycnidial wall (almost hyaline when the conidia have exuded).
Type species: Paramicrosphaeropsis ellipsoidea L.W. Hou, L. Cai & Crous
Paramicrosphaeropsis ellipsoidea L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833539. Fig. 42.
Paramicrosphaeropsis ellipsoidea (CBS 194.97). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–N. Conidiogenous cells. O. Conidia. Scale bars: H–I = 20 μm; J, O = 10 μm; K–N = 5 μm.
Etymology: Name refers to the ellipsoidal conidia of this species.
Description: Conidiomata pycnidial, semi-immersed, abundant, scattered or aggregated, mostly solitary, sometimes 2–5 confluent, globose to subglobose, pale brown to dark brown, with hyphal outgrowths, (100–)150–490 × (80–)110–440 μm. Ostiole inconspicuous. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, thin, hyaline at beginning, dark brown with age, 4–5 layers, 10–30 μm thick, outer 1–2 cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, subglobose, ampulliform or doliiform, 7–11 × 5.5–10 μm. Conidia variable in shape, mostly broad ellipsoidal, some oblong or ovoid, straight or slightly curved, smooth- and thin-walled, hyaline, later becoming brown with age, aseptate, 5–11 × 3.5–6.5 μm, with numerous minute guttules. Conidial matrix brownish.
Culture characteristics: Colonies on OA reaching 10–15 mm diam after 7 d at 25 °C, margin irregular, aerial mycelium flat, olivaceous, some zone formed pycnidia without aerial mycelium and in buff colour; reverse dark olivaceous with buff edge. Colonies on MEA 10–15 mm diam after 7 d, margin regular, aerial mycelium flat to floccose, pink to white, with some iron grey zone; reverse pale brown with buff edge, with some iron grey zone. Colonies on PDA reaching 10–15 mm diam after 7 d, margin regular, covered by flat aerial mycelium, rosy buff to pink, some brown pycnidia visible near the centre; reverse concolourous. NaOH spot test: a pale red-brown discolouration on OA.
Typus: Spain, from Quercus ilex (Fagaceae), 1996, R.F. Castañeda (holotype CBS H-23671, ex-type living culture CBS 194.97).
Additional material examined: Spain, from decayed twig of Quercus ilex (Fagaceae), 20 Jul. 1996, R.F. Castañeda, culture CBS 197.97.
Notes: Isolates CBS 194.97 and CBS 197.97 were previously identified as “Microsphaeropsis sp.”, and characterised by producing brown conidia. However, based on the phylogenetic analysis (Fig. 1), these two isolates formed a distinct clade basal to Neomicrosphaeropsis and Microsphaeropsis. Therefore, we accommodated CBS 197.97 and CBS 194.97 as a new species in a novel genus, Paramicrosphaeropsis. Morphologically, Paramicrosphaeropsis differs from Neomicrosphaeropsis in its subglobose, ampulliform or doliiform conidiogenous cells, while Neomicrosphaeropsis produces subcylindrical conidiogenous cells.
Clade 25: Calophoma Qian Chen & L. Cai, Stud. Mycol. 82: 191. 2015.
Type species: Calophoma clematidina (Thüm.) Qian Chen & L. Cai
Calophoma parvula L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833540. Fig. 43.
Calophoma parvula (CBS 620.68). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H–I. Pycnidia. J. Section through pycnidium. K–L. Conidiogenous cells. M. Chlamydospore. N. Conidia. Scale bars: H–I = 50 μm; J = 20 μm; K–L, N = 5 μm; M = 10 μm.
Etymology: Latin parvula (= very small), refers to the size of the pycnidia produced by this species.
Description: Conidiomata pycnidial, immersed, scattered and solitary, globose to subglobose, with slightly papilla at the top, pale brown and thin-walled, glabrous, ostiolate, 65–155 × 60–140 μm. Ostiole single. Pycnidial wall pseudoparenchymatous, composed of oblong or isodiametric cells, 2–3 layers, 8–37 μm thick, outer 1–2 cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, subglobose, ampulliform or lageniform, 5.5–9.5 × 3.5–8 μm. Conidia oblong with both ends rounded or broad ellipsoidal, smooth- and thin-walled, hyaline, aseptate, 4–7 × 2–3.5 μm, with two medium-sized polar guttules. Chlamydospores multi-celled, dark brown, smooth- and thick-walled, singly, globose to ellipsoidal, 18–30 × 18–25 μm.
Culture characteristics: Colonies on OA reaching 60–65 mm diam after 7 d at 25 °C, margin irregular, aerial mycelium flat, pale olivaceous to olivaceous black, saffron near the centre; reverse dark olivaceous. Colonies on MEA 55–60 mm diam after 7 d, margin regular, aerial mycelium woolly, white to buff; reverse pale brown with buff edge, some with a black zone. Colonies on PDA reaching 55–60 mm diam after 7 d, margin regular, covered by felty aerial mycelium, whitish, pale olivaceous towards periphery; reverse saffron to pale brown, buff near the centre. NaOH spot test: a pale red-brown discolouration on OA.
Typus: The Netherlands, Baarn, Buitenzorg, from leaf spots of Aegopodium podagraria (Umbelliferae), 7 Jul. 1968, H.A. van der Aa (holotype CBS H-16196, ex-type living culture CBS 620.68).
Notes: Isolate CBS 620.68 was originally identified as “Calophoma complanata”, but is distinct from Ca. complanata (CBS 268.92) both in morphology and phylogeny (Fig. 1). Isolate CBS 620.68 was characterised by smaller pycnidia (65–155 × 62–140 μm), while Ca. complanata produced larger pycnidia (300–400 μm) (Boerema & de Gruyter 1998). Moreover, CBS 620.68 was well distinguished from Ca. complanata in producing smaller and aseptate conidia (aseptate, 4–7 × 2–3.5 μm vs. 0–1-septate, 5–9 × 2–3.5 μm; Boerema & de Gruyter 1998). We consequently described CBS 620.68 as a new species, Ca. parvula.
Calophoma vincetoxici (De Not.) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833541. Fig. 44.
Calophoma vincetoxici (CBS 186.55). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section of pycnidial wall. J. Section through pycnidium. K–L. Conidiogenous cells. M. Conidia. Scale bars: H = 50 μm; J = 20 μm; I, K–M = 10 μm.
Basionym: Amphisphaeria vincetoxici De Not., Sf. Ital. 72. 1863.
Synonyms: Didymella vincetoxici (De Not.) Sacc., Syll. Fung. (Abellini) 1: 552. 1882.
Cercidospora vincetoxici (De Not.) Kuntze, Revis. Gen. Pl. 3(3): 454. 1898.
Description from ex-epitype culture (CBS 186.55): Conidiomata pycnidial, semi-immersed or immersed in the agar, mostly solitary, sometimes aggregated, globose, subglobose or flask-shaped, glabrous or with some hyphal outgrowths, ostiolate, (70–)220–380 × (70–)190–380 μm. Ostiole single, non-papillate or slightly papillate, sometimes elongated to a short neck. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 2–4 layers, 10.5–30 μm thick, outer wall 1–3-layered, pigmented. Conidiogenous cells phialidic, hyaline, smooth, (sub-)globose, ampulliform or flask-shaped, (7.5–)9.5–17.5 × (6.5–)8.5–13.5 μm. Conidia variable in shape and size, subcylindrical with obtuse ends, oblong or allantoid, smooth- and thin-walled, hyaline, 0–1-septate, (9–)14–22.5 × 4.5–7 μm, with small guttules per cell. Conidial matrix pale salmon.
Culture characteristics: Colonies on OA, 25–35 mm diam after 7 d at 25 °C, margin entire, regular, densely covered by olivaceous buff and floccose aerial mycelium, greyish pink or greyish brown near the margin; reverse olivaceous, reddish brown, greyish brown near the margin. Colonies on MEA 25–30 mm diam after 7 d, margin regular, aerial mycelium floccose, white, with a buff concentric ring; reverse orange in outer ring, darkening towards the centre of the colony, brick to darker brown. Colonies on PDA, 35–40 mm diam after 7 d, margin regular, covered by floccose, white mycelium, with flat and pale reddish brown near the margin; reverse buff in outer ring, darkening towards the centre of the colony, honey, brick to darker brown. NaOH spot test: a pale greenish discolouration on OA.
Typus: Japan (lectotype designated here of Amphisphaeria vincetoxici, Sferiacei Italici, Centuria 1–Fascicolo 1: illustration on page 72, 1863, G. De Notaris, MBT389739). Switzerland, Gabi am Simplon, from Vincetoxicum officinale (Apocynaceae), 30 Oct. 1953, E. Müller (epitype designated here CBS H-24309, MBT389812, ex-epitype living culture CBS 186.55).
Additional material examined: Switzerland, St. Gallen, Speergebiet, Durchschlägi, from Ononis sp. (Fabaceae), 30 Oct. 1953, E. Müller, culture CBS 185.55.
Notes: Didymella vincetoxici (basionym: Amphisphaeria vincetoxici) was originally isolated from Cynanchi vincetoxici (currently: Vincetoxicum officinale) in Italy (De Notaris, 1863, Saccardo, 1882). However, the original description did not refer to a holotype specimen, nor could we locate one in the present study. Isolate CBS 186.55 was originally received as Did. vincetoxici and was isolated from the same host plant (Vin. officinale) in Europe. Thus, we proposed CBS 186.55 as ex-epitype culture. The morphology of the epitype (CBS H-24309), which we designated here, agrees with the description of Did. vincetoxici by Saccardo (1882). In our study, these isolates clustered in the genus Calophoma and resided in an independent clade, forming a distinct sister group with Ca. complanata (Fig. 1).
Clade 26: Pseudoascochyta Valenz.-Lopez et al., Persoonia 37: 255. 2016.
Type species: Pseudoascochyta pratensis Valenz.-Lopez et al.
Clade 27: Briansuttonomyces Crous, Fungal Biology 120: 1412. 2016.
Type species: Briansuttonomyces eucalypti Crous
Clade 28: Ascochyta Lib. emend. Qian Chen & L. Cai, Stud. Mycol. 82: 185. 2015.
Type species: Ascochyta pisi Lib.
Ascochyta astragalina (Rehm ex Sacc.) L.W. Hou, L. Cai & Crous, stat. et. comb. nov. MycoBank MB834116.
Basionym: Didymella bryoniae var. astragalina Rehm ex Sacc., Syll. fung. (Abellini) 1: 557. 1882.
Synonym: Didymella astragalina (Rehm ex Sacc.) Corbaz, Phytopath. Z. 28: 395. 1957.
Description: Corbaz (1957).
Material examined: Sweden, Uppland, on Lathyrus vernus (Fabaceae), May 1987, K. & L. Holm, specimen CBS H-24335, culture CBS 113797 = UPSC 2222.
Notes: Isolate CBS 113797 was originally identified as Didymella astragalina, and was from the same host family (Fabaceae) in Europe (Sweden) as the original description (Astragalus sp. in Germany; Saccardo, 1882, Corbaz 1957). Phylogenetically, it clustered in the genus Ascochyta in the present study. However, because the type material could not be traced, and isolate CBS 113797 remains sterile, further studies are needed to resolve its typification.
Ascochyta clinopodiicola D. Pem et al., Mycosphere 9: 288. 2018. Fig. 45.
Ascochyta clinopodiicola (CBS 123526). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H = 100 μm; I = 50 μm; J, N = 10 μm; K–M = 5 μm.
Description from culture CBS 123526: Conidiomata pycnidial, (semi-)immersed, abundant, mostly scattered, few aggregated, solitary, becoming 2–3 confluent with age, globose to subglobose, dark brown, with hyphal outgrowths, especially around the ostioles, 180–475 × 175–410 μm. Ostioles 2–8, slightly papillate. Pycnidial wall pseudoparenchymatous, composed of oblong, globose or isodiametric cells, 4–7-layered, 14–36.5 μm thick, outer 1–2(–4) cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, globose, ampulliform to doliiform, slightly papillate, 5.5–8 × 4–7.5 μm. Conidia oblong with rounded apices, smooth- and thin-walled, hyaline, straight or slightly curved, aseptate, 4.5–7.5 × 2–2.5 μm, with 2–6 small guttules. Conidial matrix whitish.
Culture characteristics: Colonies on OA reaching 45–50 mm diam after 7 d at 25 °C, margin regular, aerial mycelium flat, hazel with honey sterile zones; reverse concolourous. Colonies on MEA reaching 50–55 mm diam after 7 d, margin regular, aerial mycelium floccose, whitish with some grey section; reverse buff to orange, with some brown sections. Colonies on PDA reaching 50–55 mm diam after 7 d, margin regular, covered by felty aerial mycelium, whitish to pale olivaceous grey; reverse buff to dark brown. NaOH spot test: a brownish discolouration on mycelium of OA.
Typus: Italy, Province of Forlì-Cesena, Spinello-Santa Sofia, on dead aerial stem of Clinopodium nepeta (Lamiaceae), 18 May 2017, E. Camporesi, (holotype MFLU 17-1034, ex-type living culture MFLUCC 18-0344).
Additional materials examined: USA, Washington, from Pisum sativum (Fabaceae), 2008, M.I. Chilvers, culture CBS 123526; ibid., culture CBS 123527; Utah, Canyonlands National Park, from soil, 1997, M. Christensen, culture CBS 127776.
Notes: Ascochyta clinopodiicola was originally described from a sexual morph collected on dead stems of Clinopodium nepeta in Italy (Hyde et al. 2018). In this study, three isolates from Pisum sativum were revealed to be genetically identical to the ex-type isolate of As. clinopodiicola (MFLUCC 18-0344) and the morphological characters of the asexual morph were also described (based on CBS 123526).
Ascochyta koolunga (J.A. Davidson et al.) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833543.
Basionym: Phoma koolunga J.A. Davidson et al., Mycologia 101: 124. 2009.
Synonym: Ascochyta boeremae L.W. Hou et al., Stud. Mycol. 87: 126. 2017.
Description: Davidson et al. (2009).
Typus: Australia, Minnipa, Meridionalis, Nova Hollandia, on Pisum sativum (Fabaceae), 26 Oct. 2004, J. Davidson (holotype DAR 78535).
Materials examined: Australia, from a leaf of P. sativum, dep. Sep. 1984, G.H. Boerema (holotype of A. boeremae, CBS H-23017, ex-type living culture CBS 372.84 = PD 80/1246); from a leaf of P. sativum, dep. Sep. 1984, G.H. Boerema, specimen CBS H-9078, culture CBS 373.84 = PD 80/1247.
Notes: Phoma koolunga was isolated from field peas (Pisum sativum) causing Ascochyta blight in South Australia (Davidson et al. 2009). This species was identified based on morphological characters and sequences of the ITS region compared with those of the accepted pathogens causing Ascochyta blight of field peas. In our phylogenetic analyses, the holotype of Phoma koolunga (DAR 78535) clustered in a well-supported clade with the ex-type strain of As. boeremae (CBS 372.84; Chen et al. 2017) which was isolated from same host species (Pisum sativum) without any difference in the ITS and rpb2 sequences. Therefore, As. boeremae was synonymised under Phoma koolunga. A new combination was also proposed for this species as As. koolunga.
Ascochyta pilosella L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833542. Fig. 46.
Ascochyta pilosella (CBS 583.97). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H–I = 50 μm; J, M–N = 10 μm; K–L = 5 μm.
Etymology: Name reflects the pycnidia bearing hairs (= pilus in Latin) produced by this species.
Description: Conidiomata pycnidial, semi-immersed or immersed, abundant, mostly scattered and solitary, sometimes 2–3 aggregated, (sub-)globose or flask-shaped, thick-walled, pale brown, becoming dark brown with age, with hyphal outgrowths, ostiolate, (185–)300–650(–800) × (180–)250–550(–700) μm. Ostioles 1–6(–12), non-papillate or slightly papillate. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 4–12 layers, 35–65 μm thick, outer 2–5 cell layers slightly pigmented. Conidiogenous cells phialidic, hyaline, smooth, subglobose, ampulliform or lageniform, 5–10.5 × 4–8.5 μm. Conidia cylindrical or oblong with rounded apices, smooth- and thin-walled, hyaline, aseptate, 5.5–9 × 2.5–3.5 μm, mostly with two polar guttules, sometimes 2–3 minute guttules at both end. Conidial matrix brownish.
Culture characteristics: Colonies on OA reaching 45–50 mm diam after 7 d at 25 °C, margin regular, aerial mycelium floccose, dark brown near the centre, and flat, buff towards the periphery; reverse concolourous. Colonies on MEA reaching 50–55 mm diam after 7 d, margin regular, aerial mycelium woolly, white with a buff circle, pinkish towards the periphery; reverse pale brown to orange. Colonies on PDA, 50–55 mm diam after 7 d, margin regular, covered by floccose aerial mycelium, buff, aerial mycelium flat towards the periphery; reverse brown to yellow, buff towards periphery. NaOH spot test results in a brownish discolouration on OA.
Typus: Canada, British Columbia, south of Whitler, North-side of Cheakamus River, from leaf spot on Clintonia uniflora (Liliaceae), 18 Aug. 1994, V. Mel’nik (holotype CBS H-23672, ex-type living culture CBS 583.97).
Notes: Isolate CBS 583.97 formed a basal lineage in Ascochyta which was phylogenetically closely related to As. astragalina, As. herbicola and As. rosae (Fig. 1). Although As. astragalina is sterile, isolate CBS 583.97 is well separated from it based on their sequence differences: 1 bp of ITS sequences, 30 bp in rpb2 and 13 bp in tub2 . Besides, isolate CBS 583.97 could be well distinguished from As. herbicola in the number of pycnidial ostioles (1–12 ostioles, non-papillate or slightly papillate vs. 1–2 ostioles, with long elongated neck; De Gruyter et al. 1998); and from As. rosae in the size of its conidia (5.5–9 × 2.5–3.5 μm vs. 3.2–4.4 × 1.2–1.8 μm; Tibpromma et al. 2017). Thus, a new species is proposed here as As. pilosella based on CBS 583.97.
Ascochyta viciae-villosae Ondřej, Biológia (Bratislava) 23: 815. 1968.
Description: Ondřej (1968).
Material examined: Czechoslovakia, on leaf of Vicia villosa (Fabaceae), unknown date and collector, culture CBS 255.92 = CCM F-244.
Note: Isolate CBS 255.92 was from the same host and location with the original description of Ascochyta viciae-villosae (on leaves of Vicia villosa, former Czechoslovakia; Ondřej 1968). However, CBS 255.92 remained sterile in all culture media tested in this study. Therefore, further studies and more suitable cultures are needed to resolve its typification and clarify its phylogeny.
Clade 29: Phomatodes Qian Chen & L. Cai, Stud. Mycol. 82: 191. 2015.
Type species: Phomatodes aubrietiae (Moesz) Qian Chen & L. Cai
Phomatodes nebulosa (Pers.) Qian Chen & L. Cai, Stud. Mycol. 82: 191. 2015.
Basionym: Sphaeria nebulosa Pers., Observ. Disp. Mycol. 2: 69. 1800.
Synonym: Phoma nebulosa (Pers.) Berk., Outl. Brit. Fung. (London): 314. 1860.
Materials examined: Poland, near Gryfice, on Thlaspi arvense (Cruciferae), Dec. 1997, J. Marcinkowska, culture CBS 100191. The Netherlands, from a stem of Mercurialis perennis (Euphorbiaceae), Jan. 1993, J. de Gruyter, culture CBS 117.93 = PD 83/90.
Phomatodes pilosa L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833544. Fig. 47.
Phomatodes pilosa (CBS 628.68). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H = 100 μm; I = 20 μm; J = 10 μm; K–N = 5 μm.
Etymology: Name refers to the piliferous ostioles of the pycnidia produced by this species.
Description: Conidiomata pycnidial, mostly semi-immersed or immersed, abundant, scattered or aggregated, solitary or confluent, (sub-)globose, brown to dark brown, glabrous, with hypha around the ostioles, 210–530 × 200–470 μm. Ostioles mostly single, central, up to 15 with age, slightly papillate or elongated into to a short neck. Pycnidial wall pseudoparenchymatous, composed of isodiametric or oblong cells, 4–8 layers, 14.5–33.5 μm thick, outer 3–4 cell layers pigmented. Conidiogenous cells phialidic, hyaline, smooth, lageniform or ampulliform, 5–7.5 × 3.5–7.5 μm. Conidia oblong with rounded apices, smooth- and thin-walled, hyaline, aseptate, 4.5–7 × 2–3.5 μm, mostly with two polar guttules, sometimes 1–4 minute-sized guttules. Conidial matrix whitish.
Culture characteristics: Colonies on OA reaching 45–50 mm diam after 7 d at 25 °C, margin regular, without aerial mycelium, olivaceous, production of pycnidia abundant, formed in the centre as well as in several radial lines near the margin; reverse dark olivaceous with a grey margin. Colonies on MEA reaching 45–50 mm diam after 7 d, margin regular, aerial mycelium flat to floccose, black at the centre and pale olivaceous grey towards the periphery, some radially furrowed zones from the centre to the edge; reverse pale brown to dark brown. Colonies on PDA reaching 45–50 mm diam after 7 d, margin regular, covered by floccose aerial mycelium, olivaceous grey, with olivaceous edge; reverse olivaceous black. NaOH test results in a brownish discolouration on OA.
Typus: The Netherlands, Wageningen, from stem of Humulus lupulus (Cannabaceae), Sep. 1968, Plant Protection Service, Wageningen (holotype CBS H-23680, ex-type living culture CBS 628.68 = PD 67/884).
Notes: Phylogenetically, Phomatodes pilosa was allied to the ex-type strain of Phomat. aubrietiae (CBS 627.97; Fig. 1). However, it differed from the latter in the number of pycnidial ostioles and in conidial size. Phomatodes aubrietiae produced smaller pycnidia measuring 110–255(–290) × 90–215(–245) μm with a single ostiole, and longer conidia measuring 6–8.5 × 2.5–3 μm (Chen et al. 2015). Phomatodes pilosa produced larger pycnidia, 210–530 × 200–470 μm, with more ostioles (up to 15), but smaller conidia, 4.5–7 × 2–3.5 μm. Furthermore, conidia produced by Phoma humuli-japonici (isolated from Humulus japonicus) were much larger than those of Phomat. pilosa (10–13 × 3.5–4 μm vs. 4.5–7 × 2–3.5 μm; Roumeguère 1891).
Clade 30: Phoma Sacc., Michelia 2: 4. 1880. emend. Q. Chen & L. Cai, Stud. Mycol. 82: 194. 2015.
Type species: Phoma herbarum Westend.
Phoma herbarum Westend., Bull. Acad. Roy. Sci. Belgique, Cl. Sci. 19: 118. 1852, emend. Qian Chen & L. Cai, Stud. Mycol. 82: 195. 2015.
Synonyms: Phoma lignicola Rennerf., Svensk Skogsvårdsförening Tidskr. 35(1): 60. 1937.
Phoma cruris-hominis Punith., Nova Hedwigia 31: 135. 1979.
Atradidymella muscivora M.L. Davey & Currah, Amer. J. Bot. 96: 1283. 2009.
Phoma muscivora M.L. Davey & Currah, Amer. J. Bot. 96: 1283. 2009.
Description: Chen et al. (2015).
Materials examined:Sweden, from wood pulp, unknown date, E. Rennerfelt (ex-type living culture of Phoma lignicola, culture CBS 276.37 = MUCL 9920). The Netherlands, Flevoland Province, Emmeloord, from the stem of Rosa multiflora var. cathayensis (Rosaceae), Dec. 1975, G.H. Boerema, culture CBS 615.75 = PD 73/665 = IMI 199779; South Holland Province, Naaldwijk, from a stem base of Nerium sp. (Apocynaceae), Sep. 1991, J. de Gruyter, culture CBS 502.91 = PD 82/276; Emmeloord, from a leaf of Delphinium sp., deposited in CBS Feb. 1996, CBS 134.96 = PD 84/676. UK, from a leg of woman, Apr. 1977, Y.M. Clayton (holotype of Phoma cruris-hominis IMI 213845, ex-type living culture CBS 377.92 = IMI 213845); near Dumfries, from die-back of Picea excelsa (Pinaceae), Oct. 1937, T.R. Peace, culture CBS 274.37. USA, Michigan, Wolf Lake, from gametophytes of Polytrichum juniperinum (Polytrichaceae) growing on the base of an uprooted Picea mariana (Pinacea) tree, 2008, M.L. Davey (ex-type living culture of Atradidymella muscivora UAMH 10909 = CBS 127589 = Pj8-D); Maryland, from fruit Malus sylvestris, unknown date, M.A. Smith, specimen CBS H-7608, culture CBS 567.63 = ATCC 15053 = MUCL 9889. USSR, unknown substrates, unknown date and collector, culture CBS 105.30.
Notes: Phoma lignicola was originally isolated from wood pulp in Sweden (Rennerfelt 1937). In our phylogenetic tree, the ex-type strain of Phoma lignicola (CBS 276.37) clustered with the representative strain of Phoma herbarum (CBS 615.75; Fig. 1), and therefore Phoma lignicola is reduced to synonymy here.
Clade 31: Leptosphaerulina McAlpine, Fungus diseases of stone-fruit trees in Australia: 103. 1902.
Type species: Leptosphaerulina australis McAlpine
Leptosphaerulina briosiana (Pollacci) J.H. Graham & Luttr., Phytopathology 51: 685. 1961.
Basionym: Pleosphaerulina briosiana Pollacci, Atti Ist. Bot. R. Univ. Pavia, 2 Sér. 7: 51. 1902.
Synonym: Pseudoplea briosiana (Pollacci) Höhn., Ann. Mycol. 16 (1–2): 162. 1918.
Description: Graham & Luttrell (1961).
Typus: Italy, Utinum, from leaves of Medicaginis sativae (Fabaceae), lectotype designated here of Pleosphaerulina briosiana, Sopra una nuova malattia dell'erba medica (Pleosphaerulina briosiana Pollacci). Atti dell’Istituto Botanico della Università e Laboratorio Crittogamico di Pavia: illustration on page 51, 1902, Pollacci G, MBT390299). The Netherlands, from leaf Medicago sativa (Fabaceae), date unknown, Plant Protection Service, Wageningen (epitype designated here CBS H-24328, MBT390093, ex-epitype living culture CBS 533.66).
Additional materials examined: USA, Minnesota, St. Paul, from leaf Medicago sativa (Fabaceae), Feb. 1966, L. Sundheim, culture CBS 156.66; Minnesota, University of farm, St. Paul, from leaf M. sativa, Feb. 1954, R.R. Nelson, culture CBS 301.54; Georgia, from M. sativa, 23 Mar. 1954, E.S. Luttrell, culture CBS 214.55; host unknown, Jul. 1974, K.L. O’Donnell, culture CBS 441.74.
Notes: According to the original literature, Pleosphaerulina briosiana was described from Medicago sativa collected in Italy (Pollacci 1902), and later recombined into Leptosphaerulina as Le. briosiana based on morphological characters (Graham & Luttrell 1961). Unfortunately, no type was indicated. In this study, four cultures received as Le. briosiana, which were isolated from the same host as Le. briosiana in the USA were examined. Based on the phylogenetic analysis, these four isolates clustered together with isolate CBS 533.66, which was received as Ascochyta medicaginicola but was phylogenetically distant from the ex-type strain of As. medicaginicola (CBS 112.53; Fig. 1). Because CBS 533.66 was collected from the same host and continent as the holotype of Le. briosiana, it was designated as ex-epitype culture.
Leptosphaerulina chartarum Cec. Roux, Trans. Brit. Mycol. Soc. 86: 320. 1986.
[not sexual morph of Pithomyces chartarum (Berk. & M.A. Curtis) M.B. Ellis, Mycol. Pap. 76: 13 (1960)]
Typus: South Africa, Cape Province, Middelburg, from Galenia procumbens (Aizoaceae), 1981, coll. A. Barnhoorn, dep. C. Roux (holotype PREM 47900, ex-type living culture CBS 329.86).
Notes: Leptosphaerulina chartarum was originally described as the sexual morph of Pithomyces chartarum (Roux 1986). However, phylogenetic analyses revealed that the ex-type strain (CBS 329.86) of Le. chartarum did not belong to the Pi. chartarum clade, nor was it related to any of the Pithomyces lineages within Montagnulaceae (Da Cunha et al. 2014). In the present study, Le. chartarum grouped with other Leptosphaerulina species and together clustered within Didymellaceae (Fig. 1), which further revealed that Le. chartarum is not the sexual morph of Pi. chartarum.
Leptosphaerulina gaeumannii (E. Müll.) Wehm., World Monograph of the Genus Pleospora and its Segregates: 322. 1961.
Basionym: Pleospora gaeumannii E. Müll., Ber. Schweiz. Bot. Ges. 61: 165. 1951.
Synonym: Pseudoplea gaeumannii (E. Müll.) Wehm., Mycologia 47(2): 164. 1955.
Typus: Switzerland, Zürich, lawn, Aug. 1950, E. Müller, specimen CBS H-24338, ex-type living culture of Pleospora gaeumannii CBS 311.51.
Additional material examined:The Netherlands, Baarn, Pekingtuin, soil, J.A. Stalpers, Sep 1969, culture CBS 939.69.
Notes: Pleospora gaeumannii was originally described from dead stems of Poaceae in Switzerland (Müller 1951), and later placed in Leptosphaerulina based on morphology (Wehmeyer 1961). In the present study, the ex-type strain of Le. gaeumannii (CBS 311.51) formed a distinct lineage within Leptosphaerulina. Unfortunately, this isolate remained sterile in all culture media tested in this study.
Leptosphaerulina obtusispora L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833545. Fig. 48.
Leptosphaerulina obtusispora (CBS 569.94). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pseudothecia forming on OA. H. Pseudothecium. I. Section through pseudothecium. J. Section of pseudothecial wall. K. Asci and ascospores. L. Ascospores. Scale bars: H–I, K = 20 μm; J, L = 10 μm.
Etymology: Name refers to the ascospores with obtuse apices.
Description: Ascomata pseudothecial, superficial, or semi-immersed, solitary or clustered, scattered, uniloculate, globose, subglobose or pyriform, membranous, brown to dark brown, 100–310 × 80–285 μm. Ostioles circular, central, papillate. Pseudothecial wall black, 3–4 layers, 18–42 μm thick, outer wall comprising 1–2 layers of brown to dark brown cells, of textura angularis. Pseudoparaphyses not observed. Asci 84–130.5 × 41.5–64 μm, 8-spored, bitunicate, fissitunicate, saccate, obpyriform or ovoid. Ascospores 31–43.5 × 13–16.5 μm, irregularly tri-seriate, cylindrical or ellipsoidal, hyaline, muriform, with 4–5 transverse septa, and 3–4 longitudinal septa, apex obtuse, base broadly obtuse to subobtuse, slightly constricted at the septum, smooth-walled, surrounded by distinct mucilaginous sheath. Chlamydospores not observed.
Culture characteristics: Colonies on OA reaching 45–50 mm diam after 7 d at 25 °C, margin entire, regular, covered by flat aerial mycelium, olivaceous to olivaceous black; reverse buff to black. Colonies on MEA reaching 45–50 mm diam after 7 d, margin regular, covered by flat aerial mycelium, some radially furrowed zones near the centre, concentric circles of different colours, centre buff, greyish, orange, pale olivaceous towards the periphery; reverse dark mouse grey, brick to orange edge, with pale brown radial lines from centre. Colonies on PDA reaching 30–35 mm diam after 7 d, margin regular, covered by flat aerial mycelium, pale olivaceous grey, buff to olivaceous toward periphery; reverse black with buff edge. NaOH spot test negative on OA.
Typus: The Netherlands, South Holland Province, Boskoop, tree nursery, from leaf spot of Lonicera periclymenum (Caprifoliaceae), 5 Oct. 1994, Plant Protection Service, Wageningen (holotype CBS H-23669, ex-type living culture CBS 569.94).
Additional material examined:The Netherlands, Utrecht Province, Baarn, CBS garden, lawn, Apr. 1958, J.A. von Arx, culture CBS 234.58.
Notes: Leptosphaerulina argentinensis was originally described from Datura stramonium (Solanaceae) collected in Argentina, with ascospores measuring 30–35 × 12–16 μm (Spegazzini 1909, Graham & Luttrell 1961). Isolate CBS 569.94 was originally received as Le. argentinensis, but differed morphologically by producing larger pseudothecia, asci and ascospores (pseudothecia: 100–310 × 80–285 μm vs. 100–120 μm, asci: 84–130.5 × 41.5–64 μm vs. 80–90 × 40–45 μm, ascospores: 31–43.5 × 13–16.5 μm vs. 30–35 × 12–16 μm). Besides, CBS 569.94 could be easily differentiated from its most closely related species Le. saccharicola in ascospore morphology. Isolate CBS 569.94 produced ascospores with 4–5 transverse septa, and 3–4 longitudinal septa, while Le. saccharicola produced ascospores with 4 transverse septa, and 0–2 longitudinal septa. Furthermore, CBS 569.94 could be distinguished from the latter by producing larger ascomata (100–310 × 80–285 μm vs. 70–110 μm × 100–140 μm) and larger ascospores [31–43.5 × 13–16.5 μm vs. (25–)27–32(–35.5) × (9–)10–11.5(–12) μm]. Thus, we proposed a new species to accommodate this isolate.
Leptosphaerulina sisyrinchiicola L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833837. Fig. 49.
Leptosphaerulina sisyrinchiicola (CBS 121688). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pseudothecia forming on OA. H–I. Pseudothecia. J. Section through pseudothecium. K. Asci. L. Ascospores. Scale bars: H–I = 50 μm; J, K = 20 μm; L = 10 μm.
Etymology: Named after the host genus Sisyrinchium, from which this species was isolated.
Description: Ascomata pseudothecial, superficial or semi-immersed in agar, solitary or clustered, scattered, uniloculate, globose or subglobose, brown to dark brown, 175–325 × 175–260 μm. Ostioles central, papillate, with mycelium around the ostioles. Pseudothecial wall black, 4–5 layers, 14.5–43 μm, outer wall comprising 2–3 layers of dark brown cells, of textura angularis. Pseudoparaphyses not observed. Asci 70–113.5 × 56–73 μm, 8-spored, bitunicate, fissitunicate, saccate, subglobose, obpyriform or ovoid. Ascospores (28–)33–50.5 × (10.75–)11.5–18 μm, irregularly tri-seriate, oval to cylindrical or ellipsoidal, hyaline, muriform, with 4–5 transverse septa, and 2 longitudinal septa, apex obtuse, base broadly obtuse to subobtuse, usually widest at the third cell, slightly constricted at the septum, smooth-walled, surrounded by distinct mucilaginous sheath. Chlamydospores not observed.
Culture characteristics: Colonies on OA reaching 45–50 mm diam after 7 d at 25 °C, margin entire, regular, covered by flat aerial mycelium, olivaceous to olivaceous black; reverse concolourous. Colonies on MEA reaching 55–60 mm diam after 7 d, margin regular, covered by floccose aerial mycelium, some radially furrowed zones near the centre, vinaceous buff to buff; reverse isabelline, yellow toward periphery. Colonies on PDA reaching 45–50 mm diam after 7 d, margin regular, covered by flat aerial mycelium, vinaceous buff to hazel, buff toward periphery; reverse black with buff edge. NaOH spot test negative on OA.
Typus: New Zealand, Auckland, Mt. Albert, Harbutt Street, Phyllis Reserve, from leaf spot on Sisyrinchium sp. (Iridaceae), 10 Dec. 2006, C.F. Hill (holotype CBS H-24330, ex-type culture CBS 121688).
Notes: Isolate CBS 121688 was received as Leptosphaerulina argentinensis, but collected from a different host and locality than the holotype of Le. argentinensis (Graham & Luttrell 1961). Morphologically, CBS 121688 clearly differed from Le. argentinensis by producing larger ascospores (28–50.5 × 11–18 μm vs. 30–35 × 12–16 μm). Based on our phylogenetic analysis, CBS 121688 formed a separate lineage sister to Le. arachidicola (Fig. 1), but differed from the latter by producing larger asci and ascospores (asci: 70–113.5 × 56–73 μm vs. 56–71.75 × 29.75–31.5 μm, ascospores: 28–50.5 × 11–18 μm vs. 25–33.25 × 11.25–16.25 μm). Consequently, we proposed isolate CBS 121688 as a new species, Le. sisyrinchiicola.
Clade 32: Neodidymelliopsis Qian Chen & L. Cai, Stud. Mycol. 82: 207. 2015.
Type species: Neodidymelliopsis cannabis (G. Winter) Qian Chen & L. Cai
Neodidymelliopsis tiliae L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB834685. Fig. 50.
Neodidymelliopsis tiliae (CBS 519.95). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section of pycnidial wall. J. Section through pycnidium. K–M Conidiogenous cells. N. Conidia. Scale bars: H = 100 μm; I = 10 μm; J = 50 μm; K–N = 5 μm.
Etymology: Name reflects the host genus Tilia, from which this species was isolated.
Description: Conidiomata pycnidial, superficial on or semi-immersed in the agar, solitary and aggregated, sometimes confluent, (sub-)globose, brown to dark brown, with white hyphal outgrowths, especially at young age, ostiolate, 290–420 × 190–325 μm. Ostioles 1–3, slightly papillate. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 5–6 layers, 23.5–55.5 μm thick, with outer 2–3-layers pigmented. Conidiogenous cells phialidic, hyaline, smooth, subglobose, ampulliform or doliiform, (5–)6–9.5 × 3.5–8 μm. Conidia oblong, smooth- and thin-walled, hyaline, aseptate, 5–9 × 2.5–3.5 μm, guttulate. Conidial matrix pale salmon.
Culture characteristics: Colonies on OA, 45–50 mm diam after 7 d at 25 °C, margin regular, covered by floccose aerial mycelium, dark grey with olivaceous black cycles near the middle and margin; reverse grey to olivaceous. Colonies on MEA 45–50 mm diam after 7 d, margin regular, covered by woolly aerial mycelium, grey; reverse olivaceous black. Colonies on PDA, 50–55 mm diam after 7 d, margin regular, covered by floccose aerial mycelium, greyish olivaceous to olivaceous; reverse olivaceous black to olivaceous. NaOH spot test negative on OA.
Typus: Italy, Perugia, from branch of Tilia sp. (Tiliaceae), date unknown, T. Schiappa, (holotype CBS H-23684, ex-type culture CBS 519.95).
Additional material examined: Italy, Viterbo, Ocriculum, from leaf of Celtis australis (Cannabaceae) with sooty mould, 26 Oct. 2014, W. Gams, culture CBS 139719.
Notes: Based on the phylogenetic analyses in this study, Neod. tiliae clustered with Neod. moricola, which was described by Hyde et al. (2017) from a dead branch of Morus alba in Russia, with conidia measuring 3.5–6 × 1–3.5 μm (Fig. 1). Neodidymelliopsis tiliae were morphologically similar to Neod. moricola in conidial shape, but differed in producing larger conidia (5–9 × 2.5–3.5 μm vs. 3.5–6 × 1–3.5 μm) and smaller conidiogenous cells [(5–)6–9.5 × 3.5–8 μm vs. 7.5–11.5 × 2.5–5 μm].
Clade 33: Xenodidymella Qian Chen & L. Cai, Stud. Mycol. 82: 205. 2015.
Type species: Xenodidymella applanata (Niessl) Qian Chen & L. Cai
Xenodidymella glycyrrhizicola L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833546. Fig. 51.
Xenodidymella glycyrrhizicola (CBS 684.97). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G–H. Pycnidia forming on OA. I. Section through pycnidium. J. Section of pycnidial wall. K–N. Conidiogenous cells. O. Conidia. Scale bars: I = 50 μm; J–O = 10 μm.
Etymology: Named after the host genus Glycyrrhiza, from which this species was isolated.
Description: Conidiomata pycnidial, superficial on or immersed in the agar, scattered or aggregated, solitary, sometimes 2 or 3 confluent, globose or subglobose, irregular-shaped with age, brown, becoming dark brown or black with age, thick-walled, glabrous or with some hyphal outgrowths, ostiolate, 270–465 × 215–340 μm. Ostioles 2–3, papillate or elongated to a short neck. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 4–6 layers, 20.5–73 μm thick, with outer 2–4 layers pigmented. Conidiogenous cells phialidic, hyaline, smooth, subglobose, ampulliform or doliiform, 6.5–10.5 × 5.5–10.5 μm. Conidia variable in shape and size, oblong, broadly ellipsoidal or subglobose, smooth- and thin-walled, hyaline at beginning, pale brown with age, aseptate, 4.5–9.5 × 3.5–5.5 μm, guttulate. Conidial matrix brown.
Culture characteristics: Colonies on OA reaching 45–50 mm diam after 7 d at 25 °C, margin regular, covered by flat aerial mycelium, felty, pale olivaceous or saffron, abundant production of black pycnidia in circles, especially near the centre; reverse olivaceous, reddish brown, greyish brown near the margin. Colonies on MEA reaching 35–45 mm diam after 7 d, margin irregular, aerial mycelium felty, saffron to olivaceous, black in the middle because of the abundant pycnidia aggregated in several circles, especially near the centre; reverse orange to dark brown, orange near the centre. Colonies on PDA reaching 40–45 mm diam after 7 d, margin irregular, covered by floccose and felty aerial mycelium, pale olivaceous to whitish, aerial mycelium in buff colour near the centre; reverse buff to pale olivaceous, with some black area. NaOH spot test negative on OA.
Typus: New Zealand, Hamilton, from leaf spot on Glycyrrhiza lepidota (Fabaceae), date unknown, J. Follet (holotype CBS H-23673, ex-type living culture CBS 684.97).
Additional material examined: Iran, Hamedan, Aran village, from Glycyrrhiza sp. (Fabaceae), 28 May 2009, P. Razaghi, culture CBS 141234.
Notes: Xenodidymella glycyrrhizicola was represented by two cultures isolated from Glycyrrhiza spp. in New Zealand and Iran, which formed a distinct clade, clearly separated from other species (Fig. 1). Morphologically, it is characterised by producing pale brown conidia, while conidia of other species in Xenodidymella were hyaline. This is the first report of a Didymellaceae species infecting Glycyrrhiza plants.
Clade 34: Dimorphoma L.W. Hou, L. Cai & Crous, gen. nov. MycoBank MB833547.
Etymology: The genus name refers to the dimorphic conidia of the type species.
Conidiomata pycnidial, solitary, (sub-)globose, glabrous or covered with hyphal outgrowths. Ostioles single, papillate. Pycnidial wall pseudoparenchymatous, multi-layered. Conidiogenous cells phialidic, hyaline, simple, smooth, variable in appearance, flask-shaped, oblong or isodiametric. Conidia two types, both originating from the same pycnidium. Conidia of type 1: (sub-)globose, smooth- and thin-walled, hyaline, aseptate, guttulate. Conidia of type 2: cylindrical to ellipsoidal, thin-walled, smooth, hyaline, aseptate, mainly eguttulate. Chlamydospores ubiquitously present in the agar, unicellular, globose, in long chains, greenish pigmented. Sexual morph unknown.
Notes: Although phoma-like species are known from various substrates, the number of rock-inhabiting isolates is relatively low. The type species of Dimorphoma was described from stone and characterised by producing pycnidia with an extremely thin pycnidial wall, being almost hyaline when the conidia have exuded (Aveskamp et al. 2010). Besides, conidia of the type species are of two types (globose conidia and cylindrical to ellipsoidal conidia), both originating from the same pycnidia. Phylogenetically, this species is also clearly separate from all known genera of Didymellaceae (Fig. 1), and therefore a new genus is introduced as Dimorphoma.
Type species: Dimorphoma saxea (Aveskamp et al.) L.W. Hou, L. Cai & Crous
Dimorphoma saxea (Aveskamp et al.) L.W. Hou, L. Cai & Crous, comb. nov. MycoBank MB833548.
Basionym: Phoma saxea Aveskamp et al., Stud. Mycol. 65: 23. 2010.
Synonym: Xenodidymella saxea (Aveskamp et al.) Valenz.-Lopez et al., Stud. Mycol. 90: 44. 2017 (2018).
Description: Aveskamp et al. (2010).
Typus: Germany, Oldenburg, from corroded Mediterranean marble, Jun. 1992, J. Kuroczkin (holotype CBS H-20240, ex-type living culture CBS 419.92).
Additional material examined:Germany, Oldenburg, from limestone, 1987, J. Kuroczkin, culture CBS 298.89.
Notes: The description of Phoma saxea was based on morphology and molecular phylogenetic analyses of CBS 419.92 and CBS 298.89 isolated from stone (Aveskamp et al. 2010). Later Valenzuela-Lopez et al. (2018) recombined this species into Xenodidymella. However, in our phylogenetic analyses, this species formed an independent lineage clearly distinct from Xenodidymella and other genera in Didymellaceae (Fig. 1). This result was in consensus with that of Aveskamp et al. (2010), and also agreed well with the statement of Valenzuela-Lopez et al. (2018) that this species could represent a distinct genus. Morphologically, this species produced two types conidia, hence the name, Dimorphoma.
Clade 35: Neoascochyta Qian Chen & L. Cai, Stud. Mycol. 82: 198. 2015.
Type species: Neoascochyta exitialis (Morini) Qian Chen & L. Cai
Neoascochyta humicola L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB834683.
Etymology: Name derived from the substrate this species was collected from, soil.
Description: Culture sterile. Neoascochyta humicola differs from its closest phylogenetic neighbour Neoa. fusiformis (type strain: CBS 876.72) by unique fixed alleles in three loci based on alignments of the concatenated four loci deposited in TreeBASE (S25826): ITS position: 445(T, insertion); rpb2 positions: 1 460(T), 1 466(C), 1 577(G), 1 598(A), 1 691(G), 1 727(C), 1 742(T), 1 820(C), 1 823(T), 1 835(T), 1 925(G); tub2 positions: 2 138(G), 2 143(T), 2 152(C).
Typus: USA, Kansas, near Manhattan, from soil, unknown date and collector (holotype CBS H-24343, ex-type living culture CBS 127323).
Notes: Phylogenetically, isolate CBS 127323 formed a distinct lineage sister to Neoa. fusiformis, another new species introduced in the present study (Fig. 1), but remained sterile in culture. Considering that it is phylogenetically distinct, a new species, Neoa. humicola, was introduced here to accommodate it.
Neoascochyta fusiformis L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833549. Fig. 52.
Neoascochyta fusiformis (CBS 876.72). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K. Conidiogenous cells. L. Conidia. Scale bars: H = 50 μm; I = 20 μm; J–L = 10 μm.
Etymology: “fusiformis” referring to the fusiform ascospores of this fungus.
Description: Conidiomata pycnidial, superficial on or semi-immersed in the agar, solitary and aggregated, sometimes confluent, globose, dark brown, later becoming irregular and black with age, thick-walled, with dense greyish green hyphal outgrowths, 230–600 × 220–440 μm. Ostioles single, slightly papillate or elongated to a long neck. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 4–6 layers, with outer 2–3 layers pigmented, 21.5–45 μm thick. Conidiogenous cell phialidic, hyaline, smooth, ampulliform or lageniform, 7.5–11 × 6–10 μm. Conidia variable in shape and size, oblong, fusiform or bacilliform, hyaline, smooth- and thin-walled, 1-septate or aseptate, (16–)17.5–24(–25.5) × 4.5–6.5 μm, guttulate. Conidial matrix whitish cream or pale salmon.
Culture characteristics: Colonies on OA, 70–75 mm diam after 7 d 25 °C, margin regular, densely covered by floccose aerial mycelium, greyish olivaceous; reverse concolourous. Colonies on MEA 50–52 mm diam after 7 d, margin irregular, dendritic, covered by floccose aerial mycelium, white in the outer ring and changing to grey olivaceous to fawn; reverse greenish black with brick margin. Colonies on PDA, 70–75 mm diam after 7 d, margin regular, covered by floccose aerial mycelium, greyish olivaceous, with flat and greenish black flat mycelium near the margin; reverse black olivaceous. NaOH spot test: a dull green discolouration on OA.
Typus: South Africa, North West Province, Potchefstroom, from wheat straw (Poaceae), Oct. 1972, M.C. Papendorf (holotype CBS H-8974, ex-type living culture CBS 876.72).
Notes: Diplodina brachypodii was described from a decaying leaf of Brachypodium silvaticum (Poaceae) collected in Germany (conidiomata 100–150 μm diam, conidia 1-septate, 2–3 guttulate, 16–22 × 5–6 μm). Although isolate CBS 876.72 was originally identified as Dip. brachypodii, it produced much larger conidiomata and larger conidia [conidiomata: 230–600 × 220–440 μm, conidia: (16–)17.5–24(–25.5) × 4.5–6.5 μm]. Furthermore, CBS 876.72 was isolated from wheat straw in South Africa. We therefore regarded CBS 876.72 as distinct, and described it as a new species.
Neoascochyta longispora L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB834684. Fig. 53.
Neoascochyta longispora (CBS 113420). A–B. Colony on OA (front and reverse). C–D. Colony on MEA (front and reverse). E–F. Colony on PDA (front and reverse). G. Pycnidia forming on OA. H. Pycnidium. I. Section through pycnidium. J. Section of pycnidial wall. K–M. Conidiogenous cells. N. Conidia. Scale bars: H–I = 20 μm; J, N = 10 μm; K–M = 5 μm.
Etymology: Name reflects the longer (= Longi, in Latin) conidia characteristic of this species.
Description: Conidiomata pycnidial, semi-immersed, solitary or aggregated, globose or subglobose, dark brown to black, buff to pale brown on the upper part on the agar, later becoming confluent and irregular-shaped, thick-walled, glabrous, 125–295 × 110–245 μm. Ostioles inconspicuous. Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 3–5 layers, with outer 2–3 layers pigmented, 15–20 μm thick. Conidiogenous cell phialidic, hyaline, smooth, ampulliform or lageniform, 6.5–9.5 × 6.5–9 μm. Conidia variable in size, oblong or bacilliform, hyaline, smooth- and thin-walled, 1-septate or aseptate, slightly constricted at the septum, 12.5–19 × 3.5–5 μm, guttulate. Conidial matrix unobserved.
Culture characteristics: Colonies on OA, 70–75 mm diam after 7 d 25 °C, margin regular, covered by flat aerial mycelium, vinaceous buff to pale mouse grey; reverse concolourous. Colonies on MEA 50–55 mm diam after 7 d, margin regular, covered by floccose aerial mycelium, buff to rosy buff; reverse rosy buff to greyish sepia. Colonies on PDA, 70–75 mm diam after 7 d, margin regular, covered by floccose aerial mycelium, buff to vinaceous buff; reverse mouse grey, with olivaceous margin. NaOH spot test negative on OA.
Typus: The Netherlands, Zuid-Holland, De Zilk, from Cerastium semidecandrum (Caryophyllaceae), 4 Jun. 2003, H.V.D. Goes (holotype CBS H-24344, ex-type living culture CBS 113420).
Notes: Neoascochyta longispora was isolated from Cerastium semidecandrum, and forms a distinct lineage close to Neoa. fusiformis and Neoa. humicola. However, Neoa. longispora differs from Neoa. fusiformis in the size and shape of pycnidia: pycnidia of Neoa. longispora are globose or subglobose, measuring 125–295 × 110–245 μm, while those of Neoa. fusiformis are mostly globose, measuring 230–600 × 220–440 μm. Furthermore, Neoa. longispora is also distinguishable from the latter in having smaller conidiogenous cells and conidia: Neoa. longispora produces conidiogenous cell measuring 6.5–9.5 × 6.5–9 μm and conidia measuring 12.5–19 × 3.5–5 μm, while Neoa. fusiformis produces conidiogenous cells measuring 7.5–11 × 6–10 μm and conidia measuring (16–)17.5–24(–25.5) × 4.5–6.5 μm. Although Neoa. humicola is sterile, Neoa. longispora can be clearly distinguished based on sequence differences in four genes.
Neoascochyta mortariensis L.W. Hou, L. Cai & Crous, sp. nov. MycoBank MB833550.
Etymology: Epithet derived from the location where it was collected, Mortara, Italy.
Description: Culture sterile. Neoascochyta mortariensis differs from its closest phylogenetic neighbour Neoa. tardicrescens (type strain: CBS 689.97) by unique fixed alleles in three loci based on alignments of the concatenated four loci deposited in TreeBASE (S25826): ITS position: 68(T), 330(C), 351(A), 408(A), 410(C), 437(C); rpb2 positions: 1 471(G), 1 537(C), 1 549(A), 1 564(C), 1 591(T), 1 594(C), 1 603(T), 1 612(C), 1 616(A), 1 621(C), 1 657(C), 1 663(G), 1 723(G), 1 744(C), 1 765(A), 1 777(T), 1 780(T), 1 786(C), 1 795(G), 1 816(C), 1 837(G), 1 840(G), 1 846(C), 1 879(G), 1 882(G), 1 894(A), 1 906(C), 1 915(C), 1 916(A), 1 930(G), 1 942(G), 1 951(C), 1 954(A), 1 966(T), 1 969(T), 1 975(T), 1 993(G), 1 996(T), 1 999(C), 2 002(G), 2 008(G), 2 014(C); tub2 positions: 2 060(C), 2 069(T), 2 083(A), 2 084(C), 2 086(G), 2 087(A), 2 106(A), 2 112(C), 2 122(A), 2 145(T), 2 229(T), 2 282(G), 2 420(T).
Culture characteristics: Colonies on OA reaching 55–60 mm diam after 7 d 25 °C, margin irregular, aerial mycelium felty, pale mouse grey, brown to dark brown towards periphery; reverse dark mouse grey. Colonies on MEA reaching 75–80 mm diam after 7 d, margin regular, aerial mycelium woolly, whitish pale grey near the centre; reverse dark brown with orange edge. Colonies on PDA reaching 80 mm diam after 7 d, margin regular, covered by dense felty aerial mycelium, pale grey; reverse dark mouse grey. NaOH spot test a pale red-brown discolouration on OA.
Typus: Italy, Centro Richerche sul riso, Mortara, from Oryza sativa (Poaceae), Aug. 1981, E.N.R. (holotype CBS H-11964, ex-type living culture CBS 516.81).
Notes: Isolate CBS 516.81 was received as Didymella graminicola. However, it was phylogenetically distant from the authentic culture of Did. graminicola (currently: Neoascochyta graminicola). In the present study, this isolate formed a distinct lineage in Neoascochyta with 64 bp differences in four loci from its closest neighbour, Neoa. tardicrescens (Fig. 1). Thus, we described it here as a new species, Neoa. mortariensis.
Discussion
The taxonomy of Didymellaceae has undergone significant changes in recent years, especially with extensive application of molecular data to resolve the phylogenetic relationships for taxa with similar morphological characteristics (Aveskamp et al., 2010, Chen et al., 2015, Valenzuela-Lopez et al., 2018). The currently recognised phylogenetic relationships of Didymellaceae were mainly based on the ex-type strains and specimens examined by Boerema and his co-workers. However, hitherto only around 300 of the approximately 5 000 described phoma-like species have been studied using multi-locus molecular data (Aveskamp et al., 2010, Chen et al., 2015, Chen et al., 2017). A large number of species await to be re-examined, including numerous phoma-like species accepted by Boerema et al. (2004) and Aveskamp et al. (2010) such as Phoma nemophilae, Phoma eupatorii, Phoma commelinicola, Didymella macropodii, Ascochyta ferulae, and species introduced based on limited sequence data before the establishment of an extended multi-locus backbone phylogeny of Didymellaceae. Although many species may end up being reduced to synonymy, the total number of Phoma taxa remains largely unknown. The present study provides an extended phylogenetic backbone and natural classification system of Didymellaceae based on the hitherto largest number of cultures with a broad distribution, diverse substrates and geographic origins.
Generic assessment
The number of genera in Didymellaceae has significantly increased in recent years (Aveskamp et al., 2010, Chen et al., 2015). Several additional genera have been introduced (Chen et al. 2017), such as Briansuttonomyces, Neomicrosphaeropsis and Pseudoascochyta from plant materials (Crous and Groenewald, 2016, Crous et al., 2016, Thambugala et al., 2016). Six new genera, namely Cumuliphoma, Ectophoma, Juxtiphoma, Remotididymella, Similiphoma and Vacuiphoma, established based on clinical specimens and several reference species of Phoma from prior studies were also confirmed (Aveskamp et al., 2010, Valenzuela-Lopez et al., 2018). Some genera were reduced to synonymy with others in Didymellaceae, e.g. Heracleicola, or not accepted due to lack of genetic and morphological divergence, e.g. Neodidymella, Didymellocamarosporium, Endocoryneum, and Pseudohendersonia (Ariyawansa et al., 2015a, Wijayawardene et al., 2016, Chen et al., 2017). Furthermore, the taxonomic placement of Didysimulans (Tibpromma et al. 2017) remains unconfirmed, as it clustered together with the genus Vacuiphoma based on the multi-locus phylogenetic analysis in the current study (Supplementary Fig. S1). Species of Didysimulans are similar to those of Didymellaceae by producing a peridium and cylindrical asci with 1-septate ascospores; however, it is characterised by its chlamydospores with hyaline to yellow-brown mycelia, arranged in spiral chains. Currently, species of Didysimulans are only known from their sexual morph, while Vacuiphoma is only known for its asexual morph. Despite Didysimulans being phylogenetically closely related to Vacuiphoma, its relatively low phylogenetic support renders it questionable. Thus, more loci of taxa belonging to this genus need to be sequenced to resolve their taxonomic placement within the Didymellaceae. However, if Didysimulans and Vacuiphoma are shown to be congeneric, then Didysimulans would have preference as it was published on 4 May 2017 and Vacuiphoma on 21 November 2017.
The genus Platychora was established by Petrak (1925) and is considered as a member of Venturiaceae. Recently, a representative strain of the type species Platychora ulmi (CBS 361.52) was sequenced for SSU and LSU, revealing a close relationship with Didymella cucurbitacearum, the taxonomic status of which awaits to be confirmed (Winton et al. 2007). Platychora was therefore relocated to the Didymellaceae (Zhang et al., 2009, Chen et al., 2015, Shen et al., 2020). In the present study, CBS 361.52 clustered with the representative strain of Phoma herbarum (CBS 615.75) (Fig. S1), having identical SSU and LSU sequences. Therefore, these two genera appear to be synonyms. Another genus Endophoma was established to accommodate an endoconidial taxon which was isolated from bat-cave soil. The phylogenetic analysis revealed that it clustered with Phoma eupyrena (currently Juxtiphoma eupyrena; Tsuneda et al. 2011, Valenzuela-Lopez et al. 2018). However, Endophoma differs from the latter in having dimorphic conidiomata in culture, i.e. spherical, ostiolate pycnidium-like conidiomata, and cylindrical, setose and closed conidiomata, both of which produced conidia exclusively endogenously. In the present study, sequences of the ex-type strain (UAMH 11216) of Endophoma elongata were revealed to be identical to Juxtiphoma kolkmaniarum (CBS 146005) (CBS 527.66) (Fig. 1 and Supplementary Fig. S1). However, considering the limited sampling of these two genera and the incomplete sequence datasets, the taxonomic placement of Platychora and Endophoma remains unresolved and will be treated once more loci have been sequenced.
Didymellaceous taxa with a hyphomycetous synasexual morph
Epicoccum is the only genus producing a hyphomycetous synasexual morph in Didymellaceae, which is characterised by darkly pigmented multi-septate conidia (dictyochlamydospores) formed on sporodochia. The taxonomy of Epicoccum has long been a topic of intense debate. The present study revealed a highly-supported monophyletic lineage, based on which a natural classification system of Epicoccum is presented. The multi-locus phylogenetic tree indicated that species clustering in the basal part of Epicoccum were mostly plant-associated, producing hyaline, single-celled conidia in pycnidia (coelomycetous asexual morph, except for Epi. sorghinum). However, those taxa that diverged later were mostly isolated from soil or other substrates (Schol-Schwarz, 1959, Punithalingam et al., 1972, Pieckenstain et al. 2001, Hashem and Ali, 2004, De Cal et al., 2009) and tend to produce pigmented, verruculose dictyoconidia (hyphomycetous synasexual morph). The currently available data strongly suggest that the general evolutionary direction in the genus Epicoccum is from producing a coelomycetous asexual morph to those with a hyphomycetous synasexual morph. This evolutionary trend is understandable as production of epicoccoid conidia from mycelia and sporodochia are probably more nutrient/energy efficient. Besides, the thick-walled and multicellular conidia were reported to successfully survive under extreme temperatures, drought, and UV radiation (Ellis, 1971, Ellis, 1976, Ellis and Ellis, 1997, Grishkan and Nevo, 2010), making it more resistant to the complex soil environment. Further genome and comparative analyses might explain this interesting phenomenon.
In accordance with previous studies, the present study supports 36 well-defined genera in Didymellaceae based on comprehensive morphological and phylogenetic data, including seven new genera introduced in the present study. However, four additional genera, Didysimulans, Endophoma, Platychora and Vacuiphoma, as well as four genera mentioned in Chen et al. (2017), Neodidymella, Didymellocamarosporium, Endocoryneum, and Pseudohendersonia remain unresolved.
Species diversity in Didymellaceae
The 947 isolates belonging to 301 taxa investigated in this study represent the largest sample of Didymellaceae studied to date in a single Didymellaceae phylogenetic study. The majority of these isolates occurred on plants from 287 genera in 121 plant families, either as pathogens, endophytes or saprobes. However, in contrast to the accurate and detailed studies of species on plant material, information is still limited on the distribution and species diversity from other substrates. Several studies have revealed that Didymellaceae species are able to adapt to extreme environmental conditions and grow in exposed habitats such as air, soil, water, limestone in caves (Chen et al. 2017), and inorganic materials including asbestos, cement and paint (Aveskamp et al. 2008), or saline-alkali soils, deep-sea sediments and oligotrophic environments. In the present study, 230 isolates from different substrates other than plant materials were also examined. In total 78 isolates originated from the diverse soil environments including desert soil, saline soil, and soil near a glacier, representing 46 species in 20 genera. Among them, seven new species were described, namely Didymella mitis, D. prolaticolla, D. guttulata, Epicoccum brahmansense, Macroventuria terrestris, Remotididymella brunnea and R. humicola. Besides, species from Allophoma, Cumuliphoma, Ectophoma, Leptosphaerulina, Neoascochyta and Neodidymelliopsis were reported from soil for the first time. Similarly, 12 new species and one new genus were recently described from Dutch garden soils by Hou et al. (2020), which revealed that species from many genera in Didymellaceae could be found in soil environments, representing a species-rich and largely unexplored habitat for Didymellaceae. In addition, a total of 152 isolates representing 72 species in 20 genera, mostly Didymella and Epicoccum, were isolated from other substrates such as house dust (21 strains), air (20 isolates), humans (21 isolates), rocks (five isolates), water (five isolates) and others (decayed canvas, juice, paint, plastic, potato flour, etc.; 80 isolates). Among them two new species were described from coral, i.e. Allophoma anatii and Epicoccum dickmanii, demonstrating their cosmopolitan and wide distribution. This study indicates that diverse undetected natural and artificial substrates/habitats harbour large numbers of novel Didymellaceae. Given their ubiquitous nature, the potential applications in industry, and the economic importance, additional taxonomic and ecological investigations of this group would be useful in understanding their biology and environmental significance, as well as their role in agriculture.
Our study examined isolates originating from seven continents and 92 countries. Although this is the hitherto broadest sampling of Didymellaceae investigated to date, most isolates were from Europe, South Africa and America. Continents such as Africa, Asia, Oceania and Antarctica therefore remain largely unexplored. This fact stresses the need to enhance global community-wide efforts to investigate species diversity of Didymellaceae.
Loci for species and genus resolution in Didymellaceae
Different combinations of genomic loci have been used to resolve the phylogenetic relationships of species in Didymellaceae, and to solve problems related to rapid identification of quarantine species during inspections. The combination of ITS-LSU or ITS-LSU-tub2 has been consistently used in many studies of Didymellaceae, and these combinations are also widely used in other families of Dothideomycetes (Phookamsak et al., 2014, Ertz et al., 2015, Ariyawansa et al., 2015b). However, both combinations failed to provide sufficiently clear species boundaries in most genera, as many genera were found to be polyphyletic. Chen et al. (2015) were the first to utilise the rpb2 locus in phylogenetic analyses of phoma-like taxa, revealing a largely improved phylogeny of Didymellaceae. In the present study we followed Chen et al. (2015) and Valenzuela-Lopez et al. (2018) and used ITS, LSU, tub2 and rpb2 to investigate the unidentified strains in the CBS collection and to complement the backbone phylogenetic tree provided by Chen et al. (2015) and Valenzuela-Lopez et al. (2018). A combination of tub2 and rpb2 loci (TreeBASE S25826) and even the rpb2 locus alone (Supplementary Fig. S2) provided a phylogenetic tree similar to those obtained with all four loci. This combination also proved to be highly suitable for the delineation of genera and species. Boeremia is the only genus for which none of the loci performed well on their own; species in this genus are therefore best resolved based on a combined phylogeny rather than that from any single locus. According to the study of Berner et al. (2015), the combination of five loci, namely actin, beta-tubulin, calmodulin, translation elongation factor 1-alpha, and ITS, is recommended to identify species in Boeremia.
Several markers have previously been proposed and applied as standard loci for DNA barcodes in Didymellaceae, including ITS (Druzhinina et al. 2005), actin (ACT, Aveskamp et al. 2009b), and cytochrome c oxidase subunit I (COI, Seifert 2008). ITS is known to be too conserved to distinguish species or generic level relationships in Didymellaceae. ACT, in contrast, was too diverse to be aligned (Aveskamp et al. 2010). Furthermore, the COI analysis did not reveal taxon-specific conserved SNPs and was not robust enough (Aveskamp et al., 2009b, Aveskamp et al., 2010), therefore all three loci were abandoned for use as barcodes. The partial beta-tubulin gene (tub2) was selected as DNA barcode for some groups of fungi in Dothideomycetes, such as Septoria and allied genera (Verkley et al. 2013). Based on the phylogenetic analysis in the present study, tub2 was also an option as a secondary barcode for Didymellaceae. In Didymellaceae, the tub2 are easily to amplify, therefore the success rate of PCR amplification is always high. In the present study, 97.25 % of isolates have available tub2 sequences. Besides, the reference sequence dataset of tub2 is relatively complete (96 %) in GenBank and Q-bank, enabling BLAST identifications. In addition, most species in Didymellaceae could be distinguished. However, the tub2 locus does have problems associated with it, as the phylogeny using tub2 did not satisfactorily delimitate taxa at generic level, especially in Allophoma, Calophoma, Epicoccum, and Remotididymella. Furthermore, considering that the tub2 sequence is rather short (300–350 bp), the number of informative sites for distinguishing closely related taxa are limited. Therefore, for Didymellaceae, tub2 is more suitable for a quick identification at species level. Compared to tub2, the rpb2 gene proved more advantageous as a DNA barcode. In the present study, rpb2 was confirmed to reliably distinguish 35 genera in Didymellaceae, and to provide a good resolution at both species and generic level, distinguishing 279 species. However, it failed for seven species with rpb2 sequences and 24 isolates belonging to 15 species failed to amplify for this gene. In contrast to tub2, rpb2 works well for generic identification, and it only failed to resolve the genus Calophoma in the present study. The topology of the rpb2 tree is highly similar to that from the combined four-locus tree (Chen et al. 2015; present study). However, in previous studies the success rate for the amplification of rpb2 was unsatisfactory, as only 62 % of strains were successfully amplified for rpb2 in Chen et al. (2015). In the present study, a modified PCR protocol with BSA increased the success rate to 95 %, thus solving the final hurdle of using rpb2 as the secondary barcode in Didymellaceae, especially at generic level. However, tub2 could be used as an alternative barcode for species identification, especially for isolates where PCR amplification of rpb2 prove to be problematic.
The utilisation of the rpb2 barcode for Didymellaceae has high value for rapid, accurate and precise identification for species in a family encompassing numerous plant pathogenic species. In addition, the rpb2 phylogeny has the advantage of being highly congruent with the four-locus tree (95 %), recognising 35 among the 36 genera in this family; at the species level, it distinguished the 279 species studied, and was easily amplified with the improved protocol. Sequences were also easily aligned throughout the family.
Conclusions
In the present study we provided a relatively complete sequence dataset of Didymellaceae consisting of four loci (ITS, LSU, rpb2, tub2; Table S1), aiming to resolve the delimitation of novelties at the species, genus and family levels. Our results resolved 36 well-supported monophyletic clades, representing 36 genera of which seven are described as new. Furthermore, a total of 40 new species and 21 new combinations were also introduced. Six epitypifications and six neotypifications were performed, and four basionyms, namely Epicoccum mezzettii (Goidànich 1937), Epi. oryzae (Ito & Iwadare 1934), Epi. purpurascens and Toruloidea tobaica (Von Szilvinyi 1936) were resurrected based on morphological and phylogenetic evidence of the ex-type or reference strains. Although the genera Didysimulans, Endophoma, Platychora and Vacuiphoma were revealed to belong to Didymellaceae, their taxonomic status remains unresolved.
Acknowledgements
This study was financially supported by the National Natural Science Foundation of China (31725001 and 31600023), China. Lingwei Hou acknowledges the projects from CAS (153211KYSB2016002) and National Key R&D Program of China (2016YFF0203201) for supporting her visit to the Westerdijk Institute (WI). Oded Yarden acknowledges support from ISF (888/19), Israel.
We are grateful to Chen Qian for contributing two new species and her valuable scientific help. We are thankful to Mieke Starink, Margarita Hernández-Restrepo, Feithla Al Abdul Salam for providing technical assistance to Lingwei Hou during her visit to WI, and to Arien van Iperen for depositing the isolates and specimens in the culture collection and fungarium. We are very grateful to the reviewers whose suggestions helped improve this manuscript.
Footnotes
Peer review under responsibility of Westerdijk Fungal Biodiversity Institute.
Appendix ASupplementary data to this article can be found online at https://doi.org/10.1016/j.simyco.2020.05.001.
Appendix A. Supplementary data
The following are the Supplementary data to this article:
Maximum likelihood tree constructed based on concatenated alignment of LSU, ITS, rpb2 and tub2 sequences of 1 127 strains belonging to 346 taxa (including all 947 Didymellaceae strains examined in this study and 180 references strains with partial available sequences from previous studies) representing genera of Didymellaceae. Bootstrap support values above 50 % are shown at the nodes. Strains with special status are indicated with a symbol after the accession number (R: representative; T: ex-type). The scale bar represents the expected number of changes per site. The tree is rooted to Coniothyrium palmarum CBS 400.71 and Neocucurbitaria aquatica CBS 297.74.
Maximum likelihood tree constructed using the rpb2 sequences of 573 isolates from genera of Didymellaceae. Bootstrap support values above 50 % are shown at the nodes. Strains with special status are indicated with a symbol after the accession number (R: representative; T: ex-type). The scale bar represents the expected number of changes per site. The tree is rooted to Coniothyrium palmarum culture CBS 400.71, Neocucurbitaria aquatica culture CBS 297.74 and Pleiochaeta setosa cultures CBS 496.63 and CBS 118.25.
Isolates used in Fig. 1 and Fig. S1, and their GenBank accession numbers. Strains showed in Fig. 1 and newly generated sequences are indicated in bold.
Isolates that were identified as not belonging to genera in Didymellaceae. Predicted generic names and families are listed.
References
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Funding
Funders who supported this work.
Chinese Academy of Sciences (1)
Grant ID: 153211KYSB2016002
Israel Science Foundation (1)
Grant ID: 888/19
National Key Research and Development Program of China (1)
Grant ID: 2016YFF0203201
National Natural Science Foundation of China (2)
Grant ID: 31600023
Grant ID: 31725001