Introduction

The Didymellaceae is one of the core families of the Pleosporales in the class Dothideomycetes (Hyde et al. 2013; Hongsanan et al. 2020). Members of this family are mainly plant pathogens (Rouxel & Balesdent 2005; McDonald & Peck 2009; Salam et al. 2011; de Gruyter et al. 2013), and some of them are also opportunistic pathogens of human and animals throughout the world (Boerema et al. 2004; de Hoog et al. 2011). Some species are endophytes of plants or saprobes often found in association with hosts and non-host living organisms (Schulz and Boyle 2005; Tahtamouni et al. 2016).

The family Didymellaceae was proposed by de Gruyter et al. (2009) to accommodate some phoma-like taxa (Boerema et al. 2004), which formed a monophyletic clade containing the type species of the sections Macrospora (Phoma zeae-maydis), Peyronellaea (Phoma glomerata), Phoma (Phoma herbarum), Phyllostictoides (Phoma exigua var. exigua.), and Sclerophomella (Phoma complanata). Phoma-like taxa that grouped phylogenetically in different lineages outside the Didymellaceae, including most members of four other sections Heterospora, Paraphoma, Pilosa, and Plenodomus, were accommodated in distinct families namely Coniothyriaceae, Cucurbitariaceae, Leptosphaeriaceae, Phaeosphaeriaceae, and Pleosporaceae (Aveskamp et al. 2010; de Gruyter et al. 2010, de Gruyter 2012). However, the family Didymellaceae included mostly the species traditionally classified in the genera Ascochyta, Didymella, and Phoma (de Gruyter et al. 2009). In a study by Zhang et al. (2009), the sexual genera Didymella, Leptosphaerulina, Macroventuria, Monascostroma, and Platychora grouped phylogenetically in the Didymellaceae. In another molecular study based on multilocus phylogenetic analysis, Aveskamp et al. (2010) introduced the genus Boeremia to accommodate species closely related to Phoma exigua (Syn. Boeremia exigua). In this study, most species of section Sclerophomella were transferred to Epicoccum and Peyronellaea, while Stagonosporopsis species from section Heterospora were reclassified in existing genera within the Didymellaceae.

A comprehensive molecular study on phoma-like taxa revealed nine new genera in the Didymellaceae including Allophoma, Calophoma, Heterophoma, Neoascochyta, Neodidymelliopsis, Nothophoma, Paraboeremia, Phomatodes, and Xenodidymella (Chen et al. 2015). In this study, Chen et al. (2015) proposed the family Microsphaeropsidaceae to accommodate two species of Microsphaeropsis that grouped together basal to the Didymellaceae, but this family was not accepted in next studies and Microsphaeropsis remained in the Didymellaceae (Hyde et al. 2020; Hou et al. 2020a, b). Recently, several other new genera have been assigned to the Didymellaceae based on molecular studies, i.e. Briansuttonomyces, Chaetasbolisia, Coniothyrium, Cumuliphoma, Didymellocamarosporium, Didysimulans, Dimorphoma, Ectodidymella, Ectophoma, Endophoma, Heracleicola, Juxtiphoma, Longididymella, Macroascochyta, Neocucurbitaria, Neodidymella, Neomicrosphaeropsis, Nothomicrosphaeropsis, Paramicrosphaeropsis, Phaeomycocentrospora, Pleiochaeta, Pseudoascochyta, Remotididymella, Sclerotiophoma, Similiphoma, Vacuiphoma, Vandijckomycella, and Verrucoconiothyrium (Ariyawansa et al. 2015; Crous & Groenewald 2016; Crous et al. 2016; Thambugala et al. 2016; Wijayawardene et al. 2016; Valenzuela-Lopez et al. 2018; Hou et al. 2020a, b; Crous et al. 2021). However, Hou et al. (2020a) did not accept some genera in the Didymellaceae due to lack of genetic and morphological divergence, including Neodidymella, Didymellocamarosporium, Endocoryneum, Heracleicola, and Pseudohendersonia (Ariyawansa et al. 2015; Wijayawardene et al. 2016; Chen et al. 2017).

In recent decades, phylogenetic species recognition using several unlinked DNA loci has resulted in taxonomic changes of most fungal taxa and the finding of new taxa. Accordingly, the number of recognized genera and species in the Didymellaceae is continuously growing. In this study, four novel species of the family Didymellaceae are proposed based on a polyphasic approach, combining morphological and phylogenetic analyses. These species are isolated from plant sources in Iran.

Material and methods

Strains, media, and morphological observations

Three unidentified phoma-like strains, deposited in the Iranian Fungal Culture Collection (IRAN…C) including IRAN 2641C, IRAN 3051C, and IRAN 2929C, were included in our study. Three additional strains were isolated from symptomatic plants in Khuzestan Province during 2019–2020, including IRAN 4375C and SCUA-Ah-W4-2 from leaf spot and blight of Mentha piperita (Lamiaceae) and SCUA-Ah-B from stem canker of Quercus brantii (Fagaceae). Small pieces from areas between asymptomatic and symptomatic tissues were excised and superficially desinfested in 1% sodium hypochlorite for 2–4 min, followed by dipping in sterile water (2 min) and drying on sterile paper. Dried pieces were placed in Petri plates containing potato dextrose agar (PDA, potato extract 200–400 g L−1, sucrose 10 g L−1, agar 12 g L−1, streptomycin sulfate 30 mg L−1) and then plates were incubated at 25 °C in darkness for 7–12 days. Small agar plugs from the edge of emerging colonies were cut out and transferred on fresh PDA plates. The purification of the isolates was carried out by isolating single spores (Babaahmadi et al. 2018; Larki et al. 2019).

Dried cultures of holotypes are preserved at the Fungus Reference Collection (IRAN…F) of Herbarium Ministerii Iranici Agriculturae “IRAN”, Iranian Research Institute of Plant Protection (Tehran). Subcultures of the ex-type strains and others included in this study (Table 1) are preserved at the Iranian Fungal Culture Collection (IRAN…C) of “IRAN” Herbarium and the Collection of Fungal Cultures, Department of Plant Protection, Shahid Chamran University of Ahvaz, Iran (SCUA).

Table 1 Strains used in phylogenetic analyses. The new sequences are designated in bold.
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Morphological study

Microscopic characteristics of the isolates were determined on PDA and oatmeal agar (OA, oatmeal 30–60 g L−1, agar 12 g L−1) after 7–20 days of incubation at 25 °C under a photoperiod of 12 h. Fungal structures (including pycnidia, conidia, and chlamydospores) were mounted in a drop of lactophenol or lactophenol cotton blue on microscopic slides. Pycnidial wall was studied using the microtome sections of 3-μm thickness, prepared with a Leica RM 2235 microtome, and stained with hematoxylin and eosin. Photomicrographs were taken with an OLYMPUS DP12 digital camera fixed on OLYMPUS BX51 microscope. At least, 50 measurements of each fungal structure were made using a Leitz Wetzlar (SM-LUX) Basic Biological Light Microscope at 400× and 1000× magnification. A maximum and minimum range, 95% confidence intervals, means and standard deviations were calculated for the size of the each structure.

DNA extraction, amplification, and sequencing

The isolates were grown on PDA at 25 °C in darkness for 1–2 weeks. Mycelial biomass grown on the surface of each culture was harvested using a sterile glass slide. DNA extraction proceeded as described by Ahmadpour et al. (2017). The internal transcribed spacer regions 1 and 2 including the intervening 5.8S nuclear ribosomal DNA (ITS) and partial nuclear 28S ribosomal DNA (LSU) were amplified using primer pair ITS1 and NL4 (White et al. 1990; O’Donnell 1993), partial β-tubulin (TUB2) with primer pair Btub2Fd and T2 (O’Donnell & Cigelnik 1997; Woudenberg et al. 2009), and RNA polymerase II second largest subunit (RPB2) with primer pair RPB2-5F2 and fRPB2-7cR (Liu et al. 1999; Sung et al. 2007). PCR mixture was prepared with 3 μL of 1× PCR buffer (GenetBio, South Korea), 3 μl of genomic DNA (20 ng), 1.2 μl of each primer (10 μM), 1.2 μl of dNTP mix (2.5 mM of each dNTP), 2.4 μl of MgCl2 (25 mM), 3 units of Prime Taq DNA polymerase (GenetBio, South Korea), and DNase free Milli-Q water up to final volume (30 μl). The temperature regime of PCR in a MJ Mini™ Gradient Thermal Cycler consisted of initial denaturation at 94 °C for 3–5 min; 35 cycles of denaturation at 94 °C for 30 s, primer annealing at 56 °C (ITS-LSU) or 58 °C (TUB2 and RPB2), and primer extension at 72 °C for 40 s, and a final extension at 72 °C for 5 min.

PCR products were analyzed and sequenced as described by Safi et al. (2020). PCR products were stained with a commercial safe stain (Sinaclon, Iran) and electrophoresed in 1% agarose gels in TAE buffer. The amplification fragments with the expected size were purified using the Gel Extraction Kit (Geneall biotechnology, South Korea), and sequenced with forward and reverse primers by Codon Genetics incorporated (Tehran, Iran).

Phylogenetic analyses

Generated sequences were edited using BioEdit v. 7.0.9.0 (Hall 1999) and assembled with DNA Baser Sequence Assembler v4 (2013, Heracle BioSoft, www.DnaBaser.com). Obtained sequences were deposited in GenBank (Table 1) and compared against the NCBI’s GenBank nucleotide database using BLASTn search algorithm to find the closest related species. The ITS, LSU, TUB2, and RPB2 sequences of ex-type or authentic representatives of the genera under study were retrieved from GenBank. All generated and downloaded sequences of each locus were aligned using BioEdit v. 7.0.9.0 and manually repaired. Phylogenetic analyses were performed first for each locus (not shown), and then for the four loci combined (ITS + LSU + TUB2 + RPB2). A multi-locus dataset was generated by concatenation of all single-locus alignments. The unavailable sequences in the concatenated alignment were treated as missing data. Congruency between the four loci was estimated from results obtained in single-locus phylogenetic analyses. Neodidymelliopsis cannabis strain CBS 121.75 was used as the outgroup taxon. Maximum likelihood (ML) analysis was done using raxmlGUI 2.0 beta (Edler et al. 2019), and started with the following options: the general time-reversible (GTR) model with a gamma-distributed rate variation and thorough bootstrapping analysis with 1000 replicates (MLBS). Maximum parsimony (MP) analyses were run in MEGA7 (Tamura et al. 2013) with the heuristic search option and 1000 pseudo-sampling in bootstrapping analysis. The Bayesian analysis (BI) was run using MrBayes v.3.2.6 (Ronquist et al. 2012), with the best evolutional models for each locus estimated by jModelTest 2 (Darriba et al. 2012). Accordingly, GTR +I + G was used for ITS, TUB2, and RPB2 loci and K80 + I for LSU. In BI analysis, the Markov chain Monte Carlo (MCMC) analysis was performed with the following options: four MCMC chains were sampled over 5,000,000 generations, sampling every 1000 generations, the standard deviation below 0.01 and posterior probability values (BPP) were determined after removing the first 25% of trees. New sequences were deposited in GenBank (Table 1) and new species in MycoBank.

Results

DNA analyses and phylogeny

Phylogenetic analysis of combined ITS, LSU, TUB2, and RPB2 loci included 317 sequences from 81 strains, with the outgroup taxon. The concatenated alignment comprised a total length of 1608 nucleotide sites, including gaps (ITS: 407 bp, LSU: 521 bp, TUB2: 243 bp, RPB2: 437 bp). Of those, 1254 were constant (ITS: 341 bp, LSU: 492 bp, TUB2: 147 bp, RPB2: 274 bp), 47 were parsimony uninformative (ITS: 17 bp, LSU: 15 bp, TUB2: 6 bp, RPB2: 9 bp), and 301 were parsimony informative (ITS: 44 bp, LSU: 14 bp, TUB2: 89 bp, RPB2: 154 bp). The topology of single-gene trees was congruent (not shown), allowing to combine the sequences to a concatenated dataset. The ML tree was used to show the phylogenetic relationships of the representative taxa from four genera of the Didymellaceae (Fig. 1). The BI and MP trees had a similar topology to the ML tree in major clades. In the phylogenetic tree (Fig. 1), four major clades representing Ascochyta (MLBS 97%, MPBS 96%, BPP 1.00), Didymella (MLBS 100%, MPBS 99%, BPP 1.00), Xenodidymella (MLBS 97%, MPBS 99%, BPP 1.00), and the generic complex Microsphaeropsis (MLBS 91%, MPBS 81%, BPP 1.00) are supported by bootstrap and posterior probability values. The phylogenetic analyses based on combined ITS, LSU, TUB2, and RPB2 loci showed that the strains under study placed in four independent lineages representing four new species of the Didymellaceae (Fig. 1), i.e. Ascochyta amygdali, Didymella cylindrica, Paramicrosphaeropsis iranica, and Xenodidymella menthae. In addition, the morphological comparison of these strains with closely related species in the Didymellaceae revealed enough differences to regard them as distinct species.

Fig. 1
figure 1

Phylogenetic tree constructed from a maximum likelihood analysis based on the combined ITS, LSU, TUB2 and RPB2 sequences. Bootstrap values obtained in maximum likelihood (MLBS) and maximum parsimony (MPBS) analyses ≥ 50% and Bayesian posterior probability values (BYPP) ≥ 0.95% are shown at the nodes, respectively. The scale bar shows the expected number of changes per site. The tree is rooted to Neodidymelliopsis cannabis strain CBS 121.75. Letter T indicates the ex-type strains.

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Taxonomy

Ascochyta amygdali S.A. Ahmadp., M. Mehrabi-Koushki, Farokhinejad & Asgari, sp. nov. (Fig. 2)

Fig. 2
figure 2

Ascochyta amygdali (IRAN 2641C). a, b Colony on OA after 8 days at 28 °C (top and reverse). c, d Colony on PDA after 8 days at 28 °C (top and reverse). e, f Old colony on OA after 23 days at 28 °C (top and reverse). g–i Pycnidia. j, k Section of pycnidia. l Conidiogenous cells. m Chlamydospores. n Conidia. Scale bars: g–h and j = 200 μm; k–n = 20 μm.

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MycoBank: MB 841334

Holotype: IRAN. Hamadan Province, Nahavand, isolated from Amygdalus scoparia Spach (Rosaceae), Dec. 2016, S. Bagher Abadi (holotype, IRAN 18141F; ex-type cultures, IRAN 2641C = SCUA-Bt25).

Etymology: Name refers to the host genus, Amygdalus, from which the fungus was isolated.

Colonies on OA reaching 45–46 mm-diam after 8 days of incubation at 24 ± 0.5 °C, and 29–30-mm diam at 28 ± 0.5 °C, circular with undulate margin, initially hyaline to pale peach, with age becoming greyish pale peach, covered by white cottony aerial mycelia; reverse pale brown to brown with paler margin. Colonies on PDA reaching 35–36-mm diam after 8 days of incubation at 24 ± 0.5 °C, and 27–28-mm diam at 28 ± 0.5 °C, circular with regular margin, initially golden olive, with age becoming brown with hyaline margin, covered by floccose aerial mycelia; reverse brown with hyaline margin and light and dark brown rings near the center. Hyphae hyaline, septate, branched, smooth-walled, 2–4.5 μm. Conidiomata pycnidial. Pycnidia abundant, solitary or aggregated, scattered, immersed or semi-immersed in the agar, globose, subglobose, cylindrical or irregular in shape, initially pale brown and with age becoming dark brown, 133.8–263.3(–309.2) × 94.5–224 μm, 95% confidence limits = 192.3–219.4 × 137.2–161.2 μm, (\( \overline{x} \) ± SD = 205.9 ± 44.5 × 149.6 ± 38.3 μm, n = 50). Ostioles distinct, 1–7(9), frequently with 1–2(5) long elongated broad necks which are tri- or multi-furcated, 99.75–379.2(–436.3) μm, 95% confidence limits = 189.1–264.4 μm, (\( \overline{x} \) ± SD = 266.7 ± 97 μm, n = 50). Pycnidial wall pseudoparenchymatous, composed of isodiametric angular cells, 3–4 layered, initially pale brown, with age becoming dark brown. Conidiogenous cellsphialidic, hyaline, smooth, ampulliform to doliiform, 6.3–8.7 × 4.3–7.2 μm, 95% confidence limits = 6.4–7.5 × 4.5–6.3 μm, (\( \overline{x} \) ± SD = 7 ± 0.7 × 5.4 ± 1.1 μm, n = 20). Conidia globose, sub-globose, ellipsoidal, thin and smooth walled, initially hyaline, with age becoming pale brown, aseptate, 3.6–7.6 × 3.2–5.4 μm, 95% confidence limits = 5.4–6 × 4–4.3 μm, (\( \overline{x} \) ± SD = 5.7 ± 0.9 × 4.1 ± 0.5 μm, n = 50). Swollen cells intercalary or terminal, solitary or in chains, globose or subglobose, ellipsoidal, thin-walled, 7.5–11.8 × 4.8–11.3 μm, 95% confidence limits = 8.5–10.2 × 6.4–8.1 μm, (\( \overline{x} \) ± SD = 9.4 ± 1.6 × 7.2 ± 1.5 μm, n = 40). Chlamydospores unicellular or multicellular, globose to subglobose, ellipsoidal or cylindrical, intercalary or terminal, solitary or in spiral chain, smooth, dark brown, 8.2–17.4(–19.2) × 7.1–15.3(–18.5) μm, 95% confidence limits = 11.7–13.1 × 10.3–11.6 μm, (\( \overline{x} \) ± SD = 12.4 ± 2.3 × 10.9 ± 2.1 μm, n = 40), where multicellular irregular in shape (dictyosporous or pseudo-microsclerotioid). Sexual morph not observed.

Notes: Ascochyta amygdali is closely related to A. herbicola (MLBS 72%, MPBS 67%, BPP 0.99), but is easily distinguished from it by lacking cylindrical to subcylindrical conidia (Li et al. 2020). The width and length of conidia in A. amygdali are also different from A. herbicola (4–7 × 1.5–2.6 μm). In addition, some pycnidia in A. amygdali are multiostiolate (1–7(9)-ostiolate), while pycnidia in A. herbicola are without ostiole, dehiscing by an irregular rupture in the apical wall (Li et al. 2020). A comparison of nucleotides of A. amygdali with A. herbicola (CBS 629.97) revealed a difference of 0.8% in the TUB2 region (244 bp). All attempts, including the reduction of annealing temperature and an increased concentration of MgCl2 in PCR mixture, to amplify partial RPB2 region of the new species with mentioned primers failed.

Didymella cylindrica S.A. Ahmadp., M. Mehrabi-Koushki, Farokhinejad & Asgari, sp. nov. (Fig. 3)

Fig. 3
figure 3

Didymella cylindrica (IRAN 3051C). a, b Colony on OA after 8 days at 28 °C (top and reverse). c, d Colony on PDA after 8 days at 28 °C (top and reverse). e, f Old colony on OA after 23 days at 28 °C (top and reverse). g–i Pycnidia. j Section of pycnidia. kSection of pycnidial wall. l Conidiogenous cells. m Conidia. Scale bars: h = 500 μm; i = 200 μm; j = 105 μm; k–m = 20 μm.

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MycoBank: MB 841336

Holotype: IRAN. Mazandaran Province, Savadkuh, Zirab, Sorkh Kolah village, isolated from Pteridium aquilinum (Dennstaedtiaceae), Nov. 2018, A. Javadi Estahbanati (holotype, IRAN 18140F; ex-type cultures, IRAN 3051C = SCUA-Bt 22).

Etymology: Name reflects the cylindrical conidia.

Colonies on OA reaching 18–19-mm diam after 8 days of incubation at 25 ± 0.5 °C, and 15–16-mm diam at 28 ± 0.5 °C, irregular with undulate margin, initially pale brown, becoming brown to dark brown with age, covered by whitish grey aerial mycelium, floccose, reverse dark brown. Colonies on PDA reaching 21–22-mm diam after 8 days of incubation at 25 ± 0.5 °C, and 20–21-mm diam at 28 ± 0.5 °C, circular with regular margin, initially white and gradually pale olivaceous to brownish olivaceous, with age becoming brown with white cottony aerial mycelium, pycnidia scattered as black dots, reverse brownish olivaceous. Hyphae hyaline to pale brown, septate, branched, smooth-walled, 2–4.5 μm. Conidiomata pycnidial. Pycnidia solitary, mainly superficial, sometimes partly immersed in the agar, mostly globose or subglobose to ellipsoid, but also elongate or irregularly-shaped, brown, with hyphal outgrowths, ostiolate, sometimes with 1–3 necks, 112–478.8 × 88.9–427.4 μm, 95% confidence limits = 240.7–285.4 × 166–196.2 μm, (\( \overline{x} \) ± SD = 263 ± 85.5 × 181.1 ± 58 μm, n = 60). Ostioles 1–6(–9), papillate. Pycnidial wall pseudoparenchymatous, variable in thickness, 4–8-layered, composed of isodiametric and oblong cells, brown to dark brown, outer cell layers more pigmented. Conidiogenous cells phialidic, hyaline, smooth, ampulliform to doliiform, 7.5–14 × 2.03–3.7 μm, 95% confidence limits = 8.4–11.4 × 2.1–2.7 μm, (\( \overline{x} \) ± SD = 9.9 ± 2.3 × 2.4 ± 0.5 μm, n = 20). Conidia allantoid to cylindrical, straight or curved, broadly rounded at both ends, sometimes flatted in basal end, hyaline, smooth-and thin-walled, eguttulate or with several guttules, 0–3-euseptate, mostly 2–3-celled, 13.4–30.5 × 2.5–5.9 μm, 95% confidence limits = 19.5–21.3 ×3.4–3.7 μm, (\( \overline{x} \) ± SD = 20.4 ± 3.7 × 3.5 ± 0.6 μm, n = 80). Chlamydospores absent. Sexual morph not observed.

Notes: Didymella cylindrica is phylogenetically closely related to D. subrosea (MLBS 100%, MPBS 100%, BPP 1.00), but can easily be distinguished by smaller pycnidia (112–478.8 × 88.9–427.4 μm vs. 160–560 × 120–425 μm) and longer cylindrical conidia (13.4–30.5 × 2.5–5.9 μm vs. 5–22.5 × 3–6 μm) (Hou et al. 2020a). Besides this, mature pycnidia in D. cylindrica sometimes develop 1–3 necks, but D. subrosea lacks any neck (Hou et al. 2020a). Single-nucleotide polymorphisms (SNP) analysis of three gene regions showed D. cylindrica and D. subrosea having one base pair difference (0.24%) across 416 nucleotides of the ITS region, five different base pairs (2%) across 244 nucleotides of the TUB2 region, and a difference of seven base pairs (1.6%) across 440 nucleotides of the RPB2 region.

Paramicrosphaeropsis iranica S.A. Ahmadp., M. Mehrabi-Koushki, Farokhinejad & Asgari, sp. nov. (Fig. 4)

Fig. 4
figure 4

Paramicrosphaeropsis iranica (IRAN 2929C). a, b Colony on OA after 23 days at 28 °C (top and reverse). c, d Colony on PDA after 23 days at 28 °C (top and reverse). f–i Pycnidia. j Chlamydospores. k Conidiogenous cells. l Conidia. Scale bars: f–h = 200 μm; i = 50 μm; j–l = 20 μm.

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MycoBank: MB 841337

Holotype: IRAN. Kohgiluyeh and Boyer-Ahmad Province, Boyer-Ahmad, isolated from Quercus brantii, Nov. 2016, M. Ghobad-Nejhad (holotype, IRAN 18142F; ex-type cultures, IRAN 2929C = SCUA-Bt26).

Etymology: Refers to Iran, the country where the specimens were collected.

Colonies on OA reaching 28–29-mm diam after 8 days of incubation at 24 ± 0.5 °C, and 21–22-mm diam at 28 ± 0.5 °C. Circular with regular margin, initially white, finally coral buff with hyaline margin, floccose, sparse aerial mycelium bearing white pycnidia that cannot be seen with the naked eye; reverse pale brown to pink with hyaline margin. Colonies on PDA reaching 25–26-mm diam after 8 days of incubation at 24 ± 0.5 °C, and 16–17-mm diam at 28 ± 0.5 °C, circular to slightly irregular with entire margin, white, with age becoming pale buff to buff towards the centre of the colony, floccose; reverse similar. Hyphae hyaline, septate, branched, smooth-walled, 1.8–4.7 μm. Conidiomata pycnidial. Pycnidia solitary, sometimes confluent in small groups, scattered, mostly superficial on the medium or in aerial mycelium, but also semi-immersed in the agar, globose or subglobose to ellipsoidal, hyaline to pale brown, highly thick-walled, sometimes with a very small lumen, producing a few conidia or lacking conidia, occasionally with a beak-shaped neck, 79–314.4 × 62–220.7 μm, 95% confidence limits = 154.12–197.7 × 117.6–144.5 μm, (\( \overline{x} \) ± SD = 175.9 ± 68.2 × 131 ± 42.1 μm, n = 40). Pycnidial wall pseudoparenchymatous, variable in thickness, 10–25-layered, composed of isodiametric cells, initially hyaline, approximately after two mount outer cell layers turn brown to dark brown pigmented. Conidiogenous cells phialidic, hyaline, smooth, ampulliform to doliiform, 7.8–10.3 × 4–6.5 μm, 95% confidence limits = 7.7–9.5 × 4.3–5.3 μm, (\( \overline{x} \) ± SD = 8.6 ± 1.06 × 4.7 ± 0.7 μm, n = 20). Conidia subglobose to ellipsoidal or irregular, smooth, pale brown to brown, aseptate, 4–7.7 × 3–6.3 μm, 95% confidence limits = 6–6.5 × 4–4.4 μm, (\( \overline{x} \) ± SD = 6.3 ± 0.8 × 4.2 ± 0.7 μm, n = 50). Swollen cells intercalary or terminal, solitary or in chains, subglobose to ellipsoidal or irregular in shape, thin-walled, 6.3–14.3 × 5.5–13.8 μm, 95% confidence limits = 8.9–11.5 × 7.2–9.6 μm, (\( \overline{x} \) ± SD = 10.2 ± 2.4 × 8.2 ± 2.2 μm, n = 50). Chlamydospores variable and irregular in shape, mostly subglobose to ellipsoid, single or in chains, unicellular or multicellular, intercalary or terminal, smooth, brown to dark brown, 8.4–14.1 × 7.7–12 μm, 95% confidence limits = 10.3–11.7 × 8.6–9.6 μm, (\( \overline{x} \) ± SD = 11 ± 1.5 × 9.1 ± 1 μm, n = 50); where multicellular irregular in shape (dictyosporous or pseudo-microsclerotioid). Sexual morph not observed.

Additional specimen examined: IRAN. Khuzestan Province; Andimeshk, Mangareh village, from stem canker of Quercus brantii, Mar. 2020, S.A. Ahmadpour (SCUA-Ah-B).

Notes: Paramicrosphaeropsis iranica is morphologically different from the previously described species P. ellipsoidea in having smaller pycnidia (79–314.4 × 62–220.7 μm vs. 150–490 × 110–440 μm) and shorter conidia (4–7.7 × 3–6.3 μm vs. 5–11 × 3.5–6.5 μm). In addition, pycnidia in the new species, P. iranica, are very thick-walled (10–25 layers) producing only few conidia or even lacking conidia, while pycnidia in P. ellipsoidea are thin-walled (4–5 layers) with a large number of conidia (Hou et al. 2020a). The SNP analysis of three regions showed P. iranica and P. ellipsoidea having 3 base pair difference (0.7%) across 416 nucleotides of the ITS region, 3 different base pairs (1.2%) across 244 nucleotides of the TUB2 region, and a difference of 9 base pairs (2%) across 440 nucleotides of the RPB2 region.

Xenodidymella menthae S.A. Ahmadp., M. Mehrabi-Koushki, Farokhinejad & Asgari, sp. nov. (Fig. 5)

Fig. 5
figure 5

Xenodidymella menthae (IRAN 4375C). a, b Colony on OA after 8 days at 28 °C (top and reverse). c, d Colony on PDA after 8 days at 28 °C (top and reverse). e, f Old colony on OA after 23 days at 28 °C (top and reverse). g Lesions on leaves of Mentha piperita. h–j Pycnidia. k Section of pycnidia. l Section of pycnidial wall. m Chlamydospores. n Conidiogenous cells. o Conidia. Scale bars: h–i = 500 μm; j–k = 105 μm; l = 20 μm; m = 50 μm; n–o = 20 μm

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MycoBank: MB 841338

Holotype: IRAN. Khuzestan Province; Shavur, isolated from leaf spot and blight of Mentha piperita, Dec. 2019, S.A. Ahmadpour (holotype, IRAN 18138F; ex-type cultures, IRAN 4375C = SCUA-Ah-W4).

Etymology: Name refers to the host genus Mentha from which it was isolated.

Colonies on OA reaching 52–53-mm diam after 8 days of incubation at 25 ± 0.5 °C, and 11–12-mm diam at 28 ± 0.5 °C, circular with regular margin, initially white, with age becoming dark grey at the centre of colony, Covered by white floccose aerial mycelia, pycnidia abundant and scattered as dark brown to dark spherical dots at the centre of the colony; reverse dark grey with hyaline margin. Colonies on PDA reaching 45–46-mm diam after 8 days of incubation at 25 ± 0.5 °C, and 16–17-mm diam at 28 ± 0.5 °C, circular with white regular margin, initially white, with age becoming olivaceous with white margin, darker toward the centre of the colony, pycnidia covering the whole centre or developing in dark concentric rings on or partly in the agar; reverse greenish brown to dark brown with white margin, smoky grey to bitumen-like black near the centre. Hyphae hyaline, septate, branched, smooth-walled, 1.8–5 μm. Conidiomata pycnidial. Pycnidia abundant, solitary or aggregated, superficial or semi-immersed, rarely in aerial mycelium or submerged, mostly globose to subglobose or ellipsoidal, but also flask-shaped or irregular, ostiolate, sometimes with a short neck, brown to dark brown, glabrous or with some hyphal outgrowths, (106.4–)123.8–304.8 × 102.2–264.1 μm, 95% confidence limits = 185.8–215.4 × 154.5–176.2 μm, (\( \overline{x} \) ± SD = 200.6 ± 49.7 × 165 ± 36.5 μm, n = 50). Ostioles 0–1, slightly papillate or non-papillate. Pycnidial wall pseudoparenchymatous, 2–5-layered, composed of isodiametric and oblong cells, dark brown to brown, outer cell layers more pigmented. Conidiogenous cells phialidic, hyaline, smooth, globose to flask-shaped or doliiform, 3.2–5.9(–8) × 2.2–3.5(–5.8) μm, 95% confidence limits = 3.3–6 × 2.04–3.7 μm, (\( \overline{x} \) ± SD = 4.6 ± 1.6 × 2.9 ± 1 μm, n = 50). Conidia smooth, thin-walled, oblong to cylindrical, straight or slightly curved, aseptate, rounded at both ends, with two polar and some small guttules, hyaline, 5–8.4 × 1.5–2.7 μm, 95% confidence limits = 6.3–6.7 ×2.2–2.3 μm, (\( \overline{x} \) ± SD = 6.5 ± 0.7 × 2.2 ± 0.3 μm, n = 60). Swollen cells (pseudochlamydospores) intercalary or terminal, mostly in chains, globose, subglobose to oblong, darker than the hyphae, thin-walled, 9.4–17.9 × 6.7–15.3 μm, 95% confidence limits = 11.5–13.1 × 9.9–11 μm, (\( \overline{x} \) ± SD = 12.3 ± 2.2 × 10.4 ± 1.6 μm, n = 50). Chlamydospores unicellular or multicellular, solitary or in chains, intercalary or terminal, globose to subglobose, smooth, brown to dark brown, 7.9–18.2 × 7.2–16.4 μm, 95% confidence limits = 12.1–13.6 × 9.3–10.6 μm, (\( \overline{x} \) ± SD = 12.9 ± 2.4 × 10 ± 2.1 μm, n = 50); where multicellular irregular in shape (dictyosporous or pseudo-microsclerotioid). Sexual morph not observed.

Additional specimen examined: IRAN. Khuzestan Province; Hamidiyeh, from leaf spot and blight of Mentha piperita, Dec. 2019, S.A. Ahmadpour (SCUA-Ah-W4-2).

Notes: Xenodidymella menthae is phylogenetically closely related to X. catariae (MLBS 89%, MPBS 97%, BPP 1.00). The SNP analysis of three regions showed that X. menthae and X. catariae had a 2-base pair difference (0.5%) across 416 nucleotides of the ITS region, 10 different base pairs (4.1%) across 244 nucleotides of the TUB2 region, and a difference of 13 base pairs (6%) across 203 nucleotides of the RPB2 region. Morphologically, X. menthae produces smaller aseptate conidia in vitro, while some conidia formed in the cultures of X. catariae are uniseptate and longer (5–8.4 × 1.5–2.7 μm vs. 9.5–14.5 × 2.5–5 μm) (de Gruyter et al. 2002). Some pycnidia in X. menthae have a short neck, but X. catariae has yet been reported to form a distinct neck (de Gruyter et al. 2002).

Discussion

Morphological features alone are insufficient to accurately identify fungal species (Taylor et al. 2000; Hebert et al. 2003; Seifert et al. 2007). As other phoma-like taxa, genera in the family Didymellaceae are currently well-delimited based on molecular data (de Gruyter et al. 2009, 2010, de Gruyter 2012; Aveskamp et al. 2010; Chen et al. 2015; Valenzuela-Lopez et al. 2018; Hou et al. 2020a, b). Although the ITS region is the common barcode for fungi (Schoch et al. 2012), other non-linked DNA loci are often essential for the proper identification and delineation of genera and species in the Didymellaceae. The combined ITS, LSU, TUB2, and RPB2 regions have been regarded as the best genomic regions for species delineation in this family (de Gruyter et al. 2009, 2010, de Gruyter 2012; Aveskamp et al. 2010; Chen et al. 2015; Valenzuela-Lopez et al. 2018; Hou et al. 2020a, b). In this study, the phylogenetic analyses of the concatenated four-locus dataset (ITS, LSU, TUB2, and RPB2) resolved the strains under study into four novel species, namely, Ascochyta amygdali, Didymella cylindrica, Paramicrosphaeropsis iranica and Xenodidymella menthae. The trees resulted from phylogenetic analysis in this study showed the same overall topology as those obtained in previous studies (Chen et al. 2015, 2017; Valenzuela-Lopez et al. 2018; Hou et al. 2020a, b).

Ascochyta amygdali is described here from Amygdalus scoparia. So far, no species of Ascochyta has been reported from this host (Farr and Rossman 2021). Ascochyta herbicola (syn. Phoma herbicola), the most closely related species to A. amygdali, was primarily described by Wehmeyer (1946) from old dead stems of Veronica dissecta in the USA. Later, it was reported as a saprophytic fungus from lake water, soil, and various herbaceous plants (Farr and Rossman 2021). The genus Ascochyta was introduced by Libert in 1830, with the description of A. pisi as type species (Boerema & Bollen 1975). The generic circumscription of Ascochyta was emended by Chen et al. (2015) to incorporate the morphological features of sexual morphs. This genus is characterized by glabrous, ostiolate, pycnidial conidiomata with annellidic or phialidic conidiogenous cells and mostly uniseptate, hyaline to slightly coloured conidia. The sexual morph is characterized by ostiolate pseudothecial ascomata containing 8-spored, bitunicate asci, hyaline, thin-walled, septate, filamentous pseudoparaphyses and usually smooth, uniseptate ascospores (Chen et al. 2015).

Didymella cylindrica is described here from Pteridium aquilinum. Up to now, three other species of Didymella have been reported from this host plant including Didymella hyphenis, Didymella lophospora and Didymella prominula (Farr and Rossman 2021). None of these species has yet been phylogenetically confirmed in the genus Didymella. Didymella subrosea, the phylogenetically closest species to D. cylindrica, was originally described from litter of Abies alba (Pinaceae) in France (Hou et al. 2020a). Didymella was established by Saccardo in 1880, with the type species Didymella exigua (Holm 1975). The generic circumscription of Didymella was emended to incorporate the morphological characteristics of newly added genera and species (Chen et al. 2015). The emended genus is characterized by ostiolate or poroid, pycnidial conidiomata with hyaline, smooth, phialidic conidiogenous cells and generally aseptate, guttulate, smooth and thin-walled conidia (Chen et al. 2015). The sexual morph is known by ostiolate pseudothecial ascomata with cylindrical to clavate or saccate, bitunicate asci and uniseptate ascospores (Chen et al. 2015).

Paramicrosphaeropsis iranica is here described from Quercus brantii. So far, several phoma-like taxa belonging to the Didymellaceae have been reported from Quercus spp., including Chaetasbolisia argentina on Q. ilicis, Nothophoma quercina on Quercus sp., P. ellipsoidea on Q. ilex, Querciphoma carteri on Q. alba and Qu. minuta on Q. robur, Q. palustris and Q. suber (Boerema et al. 2004; Chen et al. 2015; Hou et al. 2020a, b). However, these species are morphologically and phylogenetically different from P. iranica.

Xenodidymella menthae is described here from Mentha piperita. To date, no species of Xenodidymella or other phoma-like taxa have been reported from this host, except Boeremia strasseri (Zimowska et al. 2018; Farr and Rossman 2021). These two species are morphologically and phylogenetically distinct (Boerema et al. 2004; Aveskamp et al. 2010). Xenodidymella catariae is the most closely related species of X. menthae. This species (syn. Phoma nepeticola) was primarily introduced by Cooke and Ellis (1877) from Nepeta catenaria (Lamiaceae) in the USA. The genus Xenodidymella was established by Chen et al. (2015) to accommodate several phoma-like taxa, including X. applanata (syn. Didymella applanata), X. asphodeli (syn. D. asphodeli), X. catariae (syn. D. catariae) and X. humicola (syn. Phoma humicola). Xenodidymella is characterized by ostiolate, pycnidial conidiomata with hyaline, smooth, phialidic conidiogenous cells and usually aseptate conidia (Chen et al. 2015). The sexual morph is known by ostiolate or poroid, pseudothecial acomata with cylindrical to subclavate asci and hyaline, uniseptate ascospores (Chen et al. 2015).

The growing number of new fungal species described from non-Iranian samples indicates a need for a DNA-based taxonomic reassessment of the strains deposited in fungal collections of Iran. This also indicates a need to reassess the previous host-species records in Iran.