A revision of the family Cucurbitariaceae with additional new taxa from forest trees in Iran

doi:10.21203/rs.3.rs-3593289/v1

The family Cucurbitariaceae is rich in species diversity, and has a wide host range and geographic distribution. In this study, we identified 12 Cucurbitariaceae isolates which were obtained from disease symptoms in two forest trees in Khuzestan province, Iran. In addition, this family is reassessed using phylogenetic analyses based on DNA sequences from five nuclear regions (ITS, LSU, tub2, tef1a and rpb2). The phylogenetic analyses showed that the present isolates represent one new genus, Nothocucurbitaria, and two new species, Nothocucurbitaria izehica and Parafenestella quercicola which are described and illustrated. Furthermore, the genus Allocucurbitaria is emended to accommodate species of Seltsamia, that grouped with the type species of Allocucurbitaria. Parafenestella pittospori and A. prunicola are recombined into the genera Neocucurbitaria and Nothocucurbitaria, respectively. Comparative analysis of single-locus trees revealed that the tub2 and tef1a can distinguish most genera and species in Cucurbitariaceae, while the ITS and LSU phylogenies show low resolution at both generic and species level. The best single locus marker, rpb2, was able to distinguish all generic and most species lineages in Cucurbitariaceae.

Crataegus

new taxa

pycnidia

phylogenetic analyses

Quercus

Cucurbitariaceae represent an important family in Pleosporales (Dothideomycetes) (Wijayawardene et al. 2020). Most members of Cucurbitariaceae are however saprobic, mainly found on the wood, bark and leaves of shrubs and trees, or in the soil at the base of woody plants (Hyde et al. 2013; Wanasinghe et al. 2017). Some members of Cucurbitariaceae are reported to be endophytes (Jaklitsch et al. 2018; Hill et al. 2023), or plant pathogens, including Rhytidiella moriformis Zalasky causing perennial rough-bark of Populus balsamifera L. (Doilom et al. 2013) and Parafenestella pittospori Crous causing leaf spot of Pittosporum tenuifolium Variegata (Crous et al. 2019b). Others are fungicolous, parasitising genera such as Cytospora Ehrenb. (Jaklitsch and Voglmayr 2020), or opportunistic pathogens of animals and humans, mostly colonising skin and nails (Wanasinghe et al. 2017). Members of this family are mostly widespread in temperate and Mediterranean regions, and are rarely found in the tropical climates (Jaklitsch et al. 2018; Wijayawardene et al. 2020).

The Cucurbitariaceae was established by Winter (1885) to accommodate some genera of pyrenomycetes which produce non-stromatic ascomata in erumpent clusters beneath the host periderm or superficially on wood (Barr 1990), including genera such as Cucurbitaria Gray et al., Nitschkia G.H. Otth ex P. Karst. and Otthia Nitschke ex Fuckel. This family is typified by the genus Cucurbitaria and the type species C. berberidis (Pers.) Gray. Cucurbitariaceae are commonly recognized by having immersed or superficial clustered ascomata with scattered ostioles and hyphal outgrowths, and having cylindrical to oblong bitunicate asci containing ellipsoid, fusoid or oblong muriformly septate ascospores (Jaklitsch et al. 2018, 2020). The taxonomy of Cucurbitariaceae has received considerable attention (Arx and Müller 1975; Barr 1987; Aveskamp et al. 2010; de Gruyter et al. 2010; de Gruyter 2012; Doilom et al. 2013; Wanasinghe et al. 2017; Jaklitsch et al. 2018; Valenzuela-Lopez et al. 2018; Jaklitsch and Voglmayr 2020; Su et al. 2022). Arx and Müller (1975) reduced the family Cucurbitariaceae to the genus Cucurbitaria, and placed it in the Pleosporaceae. Barr (1987), however, recognized several genera in the family, including Cucurbitaria, Cucurbidothis Petr., Otthia, Rhytidiella Zalasky and Syncarpella Theiss. & Syd. However, these classifications were based on morphology only, which led to different interpretations and an incorrect taxonomic placement of taxa within this family (Doilom et al. 2013; Jaklitsch et al. 2018; Jaklitsch and Voglmayr 2020). The taxonomic concept of Cucurbitariaceae was subsequently re-defined based on molecular phylogenetic studies combined with morphology (Aveskamp et al. 2010; de Gruyter et al. 2010; de Gruyter 2012; Doilom et al. 2013). Accordingly, the genera Cucurbidothis, Cucurbitaria, Pyrenochaeta De Not., Pyrenochaetopsis Gruyter et al., Rhytidiella and Syncarpella were accommodated in the family (Doilom et al. 2013). In further studies, species of the genera Cucurbidothis, Pyrenochaeta, and Pyrenochaetopsis were excluded from Cucurbitariaceae and transferred to other families, including Didymosphaeriaceae, Pyrenochaetopsidaceae and Teichosporaceae in Pleosporales (Jaklitsch et al. 2016; Valenzuela-Lopez et al. 2018; Wijayawardene et al. 2020). Recently, several new genera were assigned to the Cucurbitariaceae based on molecular phylogenetic studies, i.e. Allocucurbitaria Valenz.-Lopez et al., Astragalicola Jaklitsch & Voglmayr, Cucitella Jaklitsch & Voglmayr, Fenestella Tul. & C. Tul., Neocucurbitaria Wanas. et al., Paracucurbitaria Valenz.-Lopez et al., Parafenestella Jaklitsch & Voglmayr, Protofenestella Jaklitsch & Voglmayr, Seltsamia Jaklitsch & Voglmayr, and Synfenestella Jaklitsch & Voglmayr (Wanasinghe et al. 2017; Jaklitsch et al. 2018; Valenzuela-Lopez et al. 2018; Wijayawardene et al. 2020).

The aims of the present study were thus to: 1) identify Cucurbitariaceae fungi associated with disease symptoms Crataegus and Quercus plantations using a polyphasic approach based on morphological characters and molecular data and 2) delimit the phylogenetic lineages within Cucurbitariaceae and improve the taxonomic position of the genera and species in this family.

Sample collection and fungal isolation

The specimens were collected from various forest areas in Izeh (Khuzestan Province, Iran) during 2021–2022. Symptomatic trees included Crataegus sp. (stem cankers) and Quercus brantii Lindl. (leaf spots). These specimens were visually inspected to check for the presence of fungal sexual structures under a dissecting microscope (Wild Heerburg MDG17, Switzerland), and then used for fungal isolation. Diseased samples were washed with sterile distilled water and small pieces from the border of infected and healthy tissues were excised. Plant pieces were superficially disinfected in 1–2% sodium hypochlorite for 1–3 min and then washed in sterile water, followed by drying on sterile filter paper and placing on semi-selective potato dextrose agar medium (PDA, potato extract 200–400 gL^− 1, sucrose 10 gL^− 1, agar 12 gL^− 1, streptomycin sulfate 30 mgL^− 1) in Petri plates. Plates were incubated at 25°C in darkness for 5–10 days and examined daily to observe the emerging colonies. Small agar plugs from the edge of dissimilar fungal colonies were transferred to fresh PDA plates for further isolation and purification. A hyphal-tip isolation technique was performed to obtain pure cultures.

Fungarium materials, including a dried culture of each new species, were deposited at the Fungus Reference Collection (IRAN…F) of Herbarium Ministerii Iranici Agriculturae “IRAN”, Iranian Research Institute of Plant Protection (Tehran). Living cultures (Table 1) are preserved at the Iranian Fungal Culture Collection (IRAN…C) of “IRAN” Herbarium, the Collection of Fungal Cultures, Department of Plant Protection, Shahid Chamran University of Ahvaz, Iran (SCUA), and the Westerdijk Fungal Biodiversity Institute, the Netherlands (CBS). Nomenclatural and taxonomic data of new taxa were deposited in MycoBank.

Morphological study

Colony diameter and morphology were determined on PDA up to 20 days of incubation at 25°C and 30°C in the dark. In addition, to induce ascomal formation on culture medium, all strains were also inoculated onto water agar (WA) supplemented with starch, peptone, malt, filter paper, and leaf and stem parts of the host plants and other trees such as Conocarpus erectus L., Hibiscus rosa sinensis Linn., Prosopis juliflora (Sw.) D.C., and Ziziphus spina-christi (L.) Willd. Colony colours (obverse and reverse) were assessed according to the Methuen handbook of colour (Kornerup and Wanscher 1967). Morphological characters were studied by observing cultures sporulating on PDA after 8–20 days of incubation at 25°C under a 12 h cycle of near-ultraviolet/dark conditions. Micro-morphological characteristics were obtained by mounting fungal structures (including pycnidia) in a drop of lactophenol or lactophenol cotton blue on microscopic slides. Microscopic sections of pycnidia (approx. 3 µm thickness) were prepared using a Leica RM 2235 microtome (Germany) and mounted in a drop of Hematoxylin and Eosin mix on microscopic slides (Ahmadpour et al. 2022), to study the anatomy of pycnidial walls and morphology of conidiogenous cells. Observation and measurement of fungal structures were done using a Leitz Wetzlar (SM-LUX) Basic Biological Light Microscope (Germany) at 100×, 400× and 1000× magnification. At least 50 observations for each fungal structure were measured and reported as minimum–maximum values, 95% confidence intervals and average values with standard deviations. Fungal structures were photographed using an OLYMPUS BX-50 compound microscope (Japan) fixed with a TUCSEN GT 12 digital camera (China).

Biomass production, DNA isolation and amplification

Mycelial biomass was scraped from the surface of 10-day-old colonies grown on PDA at 25°C in darkness. Genomic DNA was extracted from mycelial biomass powdered in liquid nitrogen according to the procedure described by Mehrabi-Koushki et al. (2018). 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 the primer pair ITS1 and NL4 (White et al. 1990; O’Donnell 1993). The primers Btub2Fd and T2 (O’Donnell & Cigelnik 1997; Woudenberg et al. 2009) were used for amplification and sequencing of the partial region of the β-tubulin gene (tub2). Part of the translation elongation factor 1 alpha (tef1α) gene was amplified and sequenced using the primer pair EF1-688F (Alves et al. 2008) and EF2 (O'Donnell et al. 1998) and partial RNA polymerase II second largest subunit (rpb2) with primer pair RPB2-5F2 and fRPB2-7cR (Liu et al. 1999; Sung et al. 2007). PCR amplification was carried out in a 30 µL reaction mixture containing 3 µL of 10× PCR Buffer (GenetBio, South Korea), 100–200 ng of genomic DNA, 1.2 µL of each primer (10 µM), 1.5 µL of dNTP mix (2.5 mM of each dNTP), 1.8 µL of MgCl₂ (25 mM), 3 units of Prime Taq DNA polymerase (GenetBio, South Korea) and nuclease free milii-Q water up to final volume (30 µL). PCR thermal cycles was run in a SimpliAmp ™ Thermal Cycler and consisted of pre-denaturation for 3 min at 95°C, followed by 35 cycles of 30 s denaturation at 95°C, 30 s annealing at 56°C (ITS-LSU), 57°C (tub2), 57°C (tef1a) or 58°C (rpb2) and 30 s elongation at 72°C, and a final elongation at 72 ºC for 5 min. PCR products were analysed and sequenced as described by Mehrabi-Koushki et al. (2021) and Artand et al. (2022). PCR products were sequenced at the CardiGenetics Research Center or Codon Genetics Group (Tehran Province, Iran).

Phylogenetic analyses

Phylogenetic analyses were performed as described in Safi et al. (2021). Forward and reverse sequences of each sample were assembled using DNA Baser Sequence Assembler v.4 (2013, Heracle BioSoft, http://www. dnaba ser.com) and subjected to BLASTn search algorithm against the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to find the closest matching ex-type strains, which were added to the sequence alignment. Accordingly, the sequences of representative taxa were downloaded and used for phylogenetic analyses (Table 1). The total sequences of each individual gene were aligned with ClustalW in BioEdit Alignment Editor v.7.2.5 software (Hall 1999), and trimmed based on the longest start and end gap of the alignment. Alignments were checked and edited manually where necessary. Phylogenetic analyses were performed first for each genomic region (not shown), and then for a combined alignment (ITS + LSU + tub2 + tef1a + rpb2). This composite alignment was made by concatenation of all single-region alignments using raxmlGUI 2.0 beta software (Edler et al. 2019). The unavailable sequences of each genomic region for each strain in composite alignment were treated as missing data. Seven outgroup taxa from the families Didymellaceae, Leptosphaeriaceae, Neopyrenochaetaceae, Pseudopyrenochaetaceae, Pyrenochaetopsidaceae or incertae sedis in Pleosporales were used to root phylogenetic trees, according the results of previous studies (Jaklitsch et al. 2018; Valenzuela-Lopez et al. 2018; Jaklitsch and Voglmayr 2020; Su et al. 2022). The maximum likelihood (ML) analysis was performed using raxmlGUI 2.0 beta (Edler et al. 2019) with parameters adjusted for 1000 bootstrap replications (MLBS) and the general time-reversible (GTR) substitution model with a gamma-distributed rate variation and invariant site (G + I). Maximum parsimony (MP) analyses were performed in MEGA v.7 (Tamura et al. 2013) with selecting the heuristic search for tree inference with 1,000 bootstrap replicates (MPBS). Bayesian inference analyses (BI) were performed in MrBayes v.3.2.6 software (Ronquist et al. 2012) using Markov chain Monte Carlo sampling to determine Bayesian posterior probabilities values (BPP). The best model of evolution for BI analysis was estimated for each genomic region using jModelTest 2 (Darriba et al. 2012). Accordingly, the following substitution models were used: GTR + I + G for ITS, LSU, tef1a, and rpb2 regions and HKY + I + G for tub2. Four simultaneous chains were run for 3,000,000 generations, with trees sampled every 300 generations and discarding the first 25% of sampled trees to calculate BPP values. The newly generated sequences were recorded in the GenBank nucleotide database (Table 1).

In this study, a total of 12 isolates belonging to the family Cucurbitariaceae were obtained. Following a comprehensive morphological evaluation, these isolates were grouped in two morphologically identical types, respectively, isolated from symptomatic tissues of Crataegus sp. (seven isolates) and Quercus brantii (five isolates). Among them, two isolates of each morphologically identical types are represented in the phylogenetic reassessment of Cucurbitariaceae. Accordingly, one new genus, two new species and four combinations are reported.

DNA analyses and phylogeny

Phylogenetic analysis of combined ITS, LSU, tub2, tef1a and rpb2 dataset included 424 sequences from 97 strains, with the outgroup taxa. The combined DNA dataset consisted of a total length of 2772 nucleotide sites, including gaps (ITS: 452 bp, LSU: 820 bp, tub2: 341 bp, tef1a: 459 bp, rpb2: 700 bp). Of those sites, 1721 were constant (ITS: 274 bp, LSU: 711 bp, tub2: 171 bp, tef1a: 197 bp, rpb2: 368 bp), 203 were parsimony uninformative (ITS: 40 bp, LSU: 40 bp, tub2: 18 bp, tef1a: 51 bp, rpb2: 54 bp), and 808 were parsimony informative (ITS: 133 bp, LSU: 67 bp, tub2: 147 bp, tef1a: 183 bp, rpb2: 278 bp). The ITS, LSU, tub2, tef1a and rpb2 sequence data showed congruency in the tree topologies for the 70% reciprocal bootstrap trees, which allowed to combine the sequences to a concatenated DNA dataset. Phylogenetic analyses using ML algorithm generated tree that had a similar topology with those of obtained from MP and BI methods in major clades. Accordingly, The ML tree is here depicted to show the phylogenetic relationships of the representative taxa in Cucurbitariaceae (Fig. 1). In the phylogenetic tree (Fig. 1), 11 major lineages are supported by bootstrap and posterior probability values, of which 10 represent existing genera, and the remaining lineage is described as new genus.

Lineage 2, a low-supported clade (MLBS 62%, MPBS 92%, BPP 1.0), accommodated all the known species of the genus Parafenestella, with the exception of P. pittospori Crous (CPC 34462) which grouped with lineage 1 and is placed in Neocucurbitaria. In addition, two strains representing the new species Parafenestella quercicola (IRAN 4857C and SCUA-IS-B1-2) clustered in a well-supported clade (MLBS 100%, MPBS 98%, BPP 1.0) distinct from other previously known species of Parafenestella in the lineage 2. Lineage 5, a well-supported clade (MLBS 100%, MPBS 99%, BPP 1.0), included two species of the genus Allocucurbitaria, A. botulispora Valenz.-Lopez et al. and A. mori W.X. Su et al., and two species of Seltsamia which were recombined into this genus, A. galinsogisoli (syn. Seltsamia galinsogisoli T.Y. Zhang & Y.X. Zhang) and A. ulmi (syn. Seltsamia ulmi Jaklitsch & Voglmayr). Lineage 6, a well-supported clade (MLBS 100%, MPBS 100%, BPP 1.0), comprised two isolates obtained in this study (IRAN 4856C and SCUA-Is-A10) and the ex-type strain of Allocucurbitaria prunicola (Crous & Akulov) Magaña-Dueñas et al., which represents a novel genus Nothocucurbitaria to accommodate N. izehica and N. prunicola. The remaining lineages represent other known genera in Cucurbitariaceae, including Astragalicola, Cucitella, Cucurbitaria (MLBS 100%, MPBS 98%, BPP 1.0), Fenestella (MLBS 100%, MPBS 100%, BPP 1.0), Neocucurbitaria (MLBS 85%, MPBS 68%, BPP 0.83), Paracucurbitaria (MLBS 100%, MPBS 100%, BPP 1.0), Protofenestella, and Synfenestella (MLBS 94%, MPBS 96%, BPP 1.0).

Taxonomy

Allocucurbitaria Valenz.-Lopez et al., Stud. Mycol. 90: 51. 2017. emend. M. Mehrabi-Koushki & Eisvand

Sexual morph on Hapalocystis bicaudata Fuckel on Ulmus glabra Huds.: Ascomata pyriform, black, immersed singly or in valsoid groups beneath periderm above ascomata of its host, upright or oblique with convergent ostiolar necks, surrounded by subiculum, forming bumps, becoming visible through bark fissures. Ostiolar necks forming stout papillae. Peridium leathery, black, pseudoparenchymatous, 3-layered. Hamathecium consisting of branched paraphyses. Asci cylindrical, with a distinct ocular chamber, a slightly elongated stipe and a simple base, containing 8 uni- to partly biseriately arranged ascospores. Ascospores fusoid to subclavate, with the upper part slightly widened, first yellow, with 3 main septa, later brown, finally with numerous transverse and longitudinal septa, surrounded by a swollen sheath around each hemisphere (Jaklitsch et al. 2018).

Asexual morph on OA: Conidiomata pycnidial, brown, solitary or confluent, superficial, pycnidial wall of textura angularis, glabrous, subglobose to ovoid, ostiolate. Conidiogenous cells phialidic, hyaline, smooth-walled, ampulliform. Conidia aseptate, hyaline, smooth- and thin-walled, cylindrical to allantoid, guttulate (Valenzuela-Lopez et al. 2018).

Type species

Allocucurbitaria botulispora Valenzuela-Lopez et al.

Notes: The genus Allocucurbitaria (21 Nov. 2017, Stud. Mycol. 90: 51) was emended to accommodate the genus Seltsamia (21 Nov. 2017, Stud. Mycol. 90: 111) that clustered with type species of Allocucurbitaria, A. botulispora, in the phylogenetic analyses of the present study (MLBS 100%, MPBS 99%, BPP 1.0). Features of the sexual morph of Seltsamia (Jaklitsch et al. 2018) are incorporated into the emended generic circumscription of Allocucurbitaria.

Allocucurbitaria ulmi (Jaklitsch & Voglmayr.) M. Mehrabi-Koushki & Eisvand, comb. nov.

MycoBank: MB 849859.

Basionym: Seltsamia ulmi Jaklitsch & Voglmayr, Stud. Mycol. 90: 113. 2017.

Description

Jaklitsch et al. (2018).

Material examined

Norway, Aust-Agder, Froland kommune, Ytre Lauvrak, associated with Hapalocystis bicaudata on corticated twigs of Ulmus glabra (Oleaceae), 3 Oct. 2014, H. Voglmayr & W. Jaklitsch (WU 36957, ex-holotype culture CBS 143002 = L150).

Notes: Seltsamia was published on 21 Nov. 2017, with the type species Seltsamia ulmi (CBS 143002) isolated from corticated Ulmus glabra in Norway (Jaklitsch et al. 2018). The sexual morph is exceptional among the members of the Cucurbitariaceae because of swollen ascospore sheath.

Allocucurbitaria galinsogisoli (T.Y. Zhang & Y.X. Zhang) M. Mehrabi-Koushki & Eisvand, comb. nov.

MycoBank: MB 849860

Basionym: Seltsamia galinsogisoli T.Y. Zhang & Y.X. Zhang, Scientific Reports 9 (no. 8319): 2. 2019.

Description

Zhang et al. (2019).

Material examined

China, Liaoning province, Huludao city, from the rhizosphere of Galinsoga parviflora Cav. (Asteraceae), Sep 2014 (ex-type culture CBS 140956 = CGMCC 3.17981 = SYPF 7336).

Notes: Seltsamia galinsogisoli was introduced with the description of isolate CBS 140956 obtained from the rhizosphere soil of G. parviflora in China (Zhang et al. 2019). In the multilocus phylogenetic tree (Fig. 1), this species grouped with Allocucurbitaria, and is thus transferred to this genus.

Neocucurbitaria pittospori (Crous) M. Mehrabi-Koushki & Eisvand, comb. nov.

MycoBank: MB 849861

Basionym: Parafenestella pittospori Crous, Persoonia 43: 245. 2019.

Description

Crous et al. (2019b).

Material examined

New Zealand, Auckland, Rotorua, leaf spots on Pittosporum tenuifolium Gaertn. (Pittosporaceae), 25 Aug. 2017, R. Thangavel (ex-type culture CPC 34462 = CBS 146026).

Notes: Parafenestella pittospori was introduced from Pittosporum tenuifolium in New Zealand (Crous et al. 2019b). The only isolate of this species (CPC 34462) is however phylogenetically distinct from other species of Parafenestella, but closely related to species of Neocucurbitaria (Fig. 1), where it is introduced as a new combination.

Nothocucurbitaria Eisvand & M. Mehrabi-Koushki, gen. nov.

MycoBank: MB 849862

Etymology: Notho = fake, close to Cucurbitaria but different.

Conidiomata pycnidial, brown to olivaceous black, solitary or aggregated, globose to ellipsoidal, occasionally forming a short ostiolated neck, with some hyphal outgrowths or setose; pycnidial wall of textura angularis. Conidiogenous cells phialidic, subcylindrical to doliiform. Conidia aseptate, hyaline, smooth- and thin-walled, (sub-) cylindrical to allantoid, guttulate, straight or curved.

Type species: Nothocucurbitaria izehica Eisvand & M. Mehrabi-Koushki

Nothocucurbitaria izehica Eisvand & M. Mehrabi-Koushki, sp. nov. (Fig. 2)

MycoBank: MB 849863

Typus

Iran, Khuzestan Province, Izeh, isolated from stem canker of Crataegus sp. (Rosaceae), May. 2022, P. Eisvand (holotype, IRAN 18298 F; ex-type cultures, IRAN 4856C = SCUA-Is-A10).

Etymology

Pertaining to Izeh, the Township in Iran where the fungus was collected.

Morphology on PDA: Conidiomata pycnidial, formed after a week of incubation in the light and dark, immersed or semi-immersed, solitary or aggregated, brown olivaceous to olivaceous black, globose, sub-globose, ellipsoidal or irregular in shape, with long and short hyphal outgrowths, ostiolate, 59.5–253 × (34–)44–112.5(–127) µm, 95% confidence limits = 114.5–126 × 69.5–75.5 µm, (x ± SD = 120 ± 34.8 × 72.5 ± 17.3 µm, n = 140), occasionally forming a short ostiolated neck, 9–40 (–77) × 17.5–49 µm, 95% confidence limits = 16.5–23.5 × 27.5–32 µm, (x ± SD = 20 ± 12.3 × 29.5 ± 7.4 µm, n = 50). Ostioles 1–2. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 4–7(–9) layers, outer layer brown pigmented. Conidiogenous cells phialidic, hyaline, smooth, ampulliform to dolliform, 2.9–6.3 × 1.5–3 µm. Conidia cylindrical to allantoid, straight or slightly curved, rounded at the ends, smooth- and thin-walled, aseptate, with several distinct guttules, 2.5–5 × 0.5–1.75 µm, 95% confidence limits = 3.5–3.6 × 1–1.1 µm, (x ± SD = 3.5 ± 0.43 × 1.03 ± 0.23 µm, n = 170). Conidial matrix cream to buff. Sexual morph and chlamydospores not observed.

Culture characteristics

Colonies on PDA reaching 37 mm diam. after 14 days of incubation at 25 ± 0.5°C and 30 ± 0.5°C, circular to ovoid with filiform margin, olivaceous black to dull black with greenish grey in the margin, floccose, often zonate with sectors; reverse olivaceous greenish grey, paler towards margin, with colourless concentric and sectoring lines.

Additional material examined

Iran, Khuzestan Province, Izeh, isolated from stem canker of Crataegus sp. (Rosaceae), Jun. 2022, P. Eisvand (SCUA-Is-A10-2).

Notes: Nothocucurbitaria izehica is phylogenetically closely related to N. prunicola (MLBS 100%, MPBS 100%, BPP 1.0). Nucleotide comparison of these species revealed a difference of 0.1.6% (7/434 bp) in the ITS region, 6% (20/329 bp) in tub2 and 4.4% (31/700 bp) in rpb2. Morphologically, N. izehica can be easily distinguished from N. prunicola by having a short ostiolated neck in some pycnidia and more elongated conidia (2.5–5 × 0.5–1.75 µm vs (2–)3–3.5(–4) × 1.5(–2) µm). No ascomatal structures were observed on the collected specimens of this species and other new species Parafenestella quercicola. All attempts mentioned above to induce ascomatal production by these species on culture medium (supplemented with starch, peptone, malt, filter paper, and leaf and stem parts of several trees) failed, even after two months of incubation.

Nothocucurbitaria prunicola (Crous & Akulov) M. Mehrabi-Koushki & Eisvand, comb. nov.

MycoBank: MB 849864

Basionym: Neocucurbitaria prunicola Crous & Akulov, Fungal Systematics and Evolution 3: 91. 2019.

Description

Crous et al. (2019a).

Material examined

Ukraine, Ternopil region, Dniester Canyon N.P., forest, fallen twigs of Prunus padus L. (= Padus avium) (Rosaceae), 6 Oct. 2016, A. Akulov (ex-type culture CPC 33709 = CBS 145033).

Notes

This species, originally described by Crous et al. (2019a), was first placed in the genus Neocucurbitaria based on its phoma-like asexual morph and its phylogenetic relationship with other members of Cucurbitariaceae. Subsequently, it was transferred to Allocucurbitaria based on phylogenetic analyses of four loci including ITS, LSU, tub2 and rpb2 (Magaña-Dueñas et al. 2021). However, more representative strains of all known genera in Cucurbitariaceae were used in the present phylogenetic analyses, in which this species clusters with new genus Nothocucurbitaria. Accordingly, this species was treated as N. prunicola.

Parafenestella quercicola Eisvand & M. Mehrabi-Koushki, sp. nov. (Fig. 3)

MycoBank: MB 849865

Typus

Iran, Khuzestan Province, Izeh, isolated from leaf spot of Quercus brantii Lindl. (Fagaceae), May. 2022, P. Eisvand (holotype, IRAN 18299 F; ex-type cultures, IRAN 4857C = SCUA-Is-B1).

Etymology

Name refers to the host genus Quercus from which it was isolated.

Morphology on PDA: Conidiomata pycnidial, formed after a week of incubation, immersed, semi-immersed or superficial, rarely on aerial mycelium, solitary or aggregated in clusters of 2–several pycnidia, pale brown to brown, globose, sub-globose or ellipsoidal, sometimes irregular in shape, with some hyphal outgrowths, rarely glabrous, ostiolate, 43–184(–284.5) × 39–141.5(–161) µm, 95% confidence limits = 105.5–122.5 × 76.5–87 µm, (x ± SD = 114 ± 39.5 × 82 ± 24 µm, n = 60), mostly forming a short ostiolated neck, 13.5–42 × 23–60.5 µm, 95% confidence limits = 25–29. × 34–38. µm, (x ± SD = 27 ± 7 × 36 ± 8 µm, n = 60). Ostioles 1–2(–4), non-papillate. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 2–3(–5) layers, thin-walled, outer layer more pigmented. Conidiogenous cells phialidic, hyaline, smooth, ampulliform or lageniform, 3.5–5.5 × 1.8–3.3 µm. Conidia cylindrical to allantoid, straight or slightly curved, rounded at both ends, hyaline to grey, smooth- and thin-walled, aseptate, (1.6–)2.3–4.6 × 0.5–1.4(–1.7) µm, 95% confidence limits = 3.37–3.53 × 0.88–0.95 µm, (x ± SD = 3.4 ± 0.47 × 0.9 ± 0.2 µm, n = 130). Conidial matrix whitish to grey. Sexual morph and chlamydospores not observed.

Culture characteristics

Colonies on PDA reaching 31 mm diam. after 14 days of incubation at 25 ± 0.5°C, and 30 mm diam at 30 ± 0.5°C, circular with filiform margin, dull green to greenish glaucous, with age becoming darker at the centre due to the forming of concentric rings of sporulation, floccose; reverse greenish glaucous with paler margin, with light and dark concentric zones.

Additional material examined

Iran, Khuzestan Province, Izeh, isolated from leaf spot of Quercus brantii Lindl. (Fagaceae), May 2022, P. Eisvand (SCUA-Is-B1-2).

Notes: Parafenestella quercicola is closely related to P. tetratrupha (Berk. & Broome) Jaklitsch & Voglmayr (MLBS 73%, MPBS 68%, BPP 0.91). A nucleotide comparison of these two species revealed a difference of 0.5% (2/431 bp) in the ITS region, 5.5% (18/330 bp) in tub2 and 2.4% (10/409 bp) in tef1a. Morphologically, Parafenestella quercicola can be distinguished from P. tetratrupha by its less globose and smaller pycnidia (43–184 × 39–141.5 µm vs 100–200 µm) having a short ostiolated neck, smaller conidiogenous cells (3.5–5.5 × 1.8–3.3 µm vs (4.5–6.7 × 2.2–4 µm), and slightly curved conidia with rounded ends.

In this study, we identified 12 isolates belonging to the family Cucurbitariaceae which were isolated from disease symptoms in two genera of forest trees in Khuzestan province, Iran. The family Cucurbitariaceae was reassessed using phylogenetic analyses based on DNA sequences from five nuclear regions (ITS, LSU, tub2, tef1a and rpb2) to clarify generic and species concepts in this family. These genomic regions have been shown to be suitable to reflect phylogenetic relationships and delimit genera and species within Cucurbitariaceae (Wanasinghe et al. 2017; Jaklitsch et al. 2018; Valenzuela-Lopez et al. 2018; Jaklitsch and Voglmayr 2020; Su et al. 2022). The major clades of phylogenetic trees (Fig. 1) constructed in this study showed the same overall topology with those obtained in previous studies. Isolates in the present study were shown to represents one new genus, Nothocucurbitaria, and two new species, Nothocucurbitaria izehica, and Parafenestella quercicola. Furthermore, the genus Allocucurbitaria was emended to accommodate the genus Seltsamia which clustered with the type species of Allocucurbitaria. Similarly, in phylogenetic analyses performed by Jaklitsch et al. (2018), the type species of both genera Allocucurbitaria and Seltsamia grouped in a strongly-supported clade, but Seltsamia was introduced as a new genus based on morphological differences. The use of more cultures of these two genera in the present study showed that they should be synonymised. Accordingly, S. galinsogisoli and S. ulmi were recombined into Allocucurbitaria. Furthermore, Allocucurbitaria prunicola grouped in the new genus Nothocucurbitaria, and Parafenestella pittospori in the genus Neocucurbitaria, for which new combinations were introduced.

Comparative analysis of single-locus trees (not shown) revealed that the ITS and LSU phylogeny showed a low resolution at both generic and species level. The ITS phylogeny was able to separate seven of the 11 generic lineages with sufficient support, including Allocucurbitaria, Astragalicola, Cucurbitaria, Fenestella Nothocucurbitaria, Paracucurbitaria, and Protofenestella. The LSU phylogeny was only able to distinguish three generic lineages, including Astragalicola, Cucurbitaria, and Paracucurbitaria. The tub2 phylogeny was only able to distinguish nine of the 11 generic lineages and lacked resolution for Allocucurbitaria and Neocucurbitaria. The tef1a tree was not able to distinguish the genera Neocucurbitaria and Synfenestella from other generic lineages in Cucurbitariaceae. The rpb2 tree displayed the best resolution at the generic and species level among these genomic regions. This phylogenetic tree was able to distinguish all generic and most species lineages in Cucurbitariaceae (Fig. 1). Previous studies have been shown that the rpb2 phylogeny results in much higher bootstrap and posterior probabilities support for most taxa of Ascomycota from class to species level (Liu et al. 1999; Chen et al. 2015; Crous et al. 2021). Because rpb2 is a single-copy gene with a large size and modest rate of evolutionary changes, it generates a high phylogenetic resolution to distinguish different taxa of Ascomycota (Liu et al. 1999). However, there were some gaps in sequence data for some published species represented in our phylogenetic analyses, and thus we could not include all of them in our analyses.

In present study Nothocucurbitaria izehica was isolated from necrotic tissues of stem cankers in Crataegus sp. Species of Crataegus were reported to be host several fenestelloid fungi from the family Cucurbitaceae, including Fenestella crataegi (Niessl) Jaklitsch & Voglmayr, Parafenestella austriaca Jaklitsch & Voglmayr and P. rosacearum Jaklitsch & Voglmayr on C. monogyna Jacq. (Jaklitsch and Voglmayr 2020), and F. fenestrata (Berk. & Broome) J. Schröt. on C. laevigata (Poir.) DC. (Mulenko et al. 2008). However, the fenestelloid fungi are phylogenetically and morphologically distinct from other genera in Cucurbitariaceae, including Nothocucurbitaria (Jaklitsch and Voglmayr 2020). Interestingly, another species of this genus, N. prunicola, has also been isolated from the fallen twigs of Prunus padus (Crous et al. 2019a). Because of insufficient data about the pathogenicity of these species, we cannot conclude that this new genus is pathogenic. Furthermore, Cucurbitaria and other cucurbitaria-like fungi, including Allocucurbitaria, Paracucurbitaria, and Neocucurbitaria, are mainly saprobic on bark, branches, leaves, stems, twigs, and/or wood of different trees and shrubs (Bubak and Kabat 1912; Doilom et al. 2013; Jaklitsch et al. 2018; Valenzuela-Lopez et al. 2018; Crous et al. 2019a; Su et al. 2022). A small number of them are opportunistic pathogens of animals and humans, including A. botulispora infecting human leg (Valenzuela-Lopez et al. 2016), N. chlamydospora H.Y. Jung & S.Y. Lee infecting Hygia lativentris Motschulsky (Hong et al. 2023), N. irregularis Valenz-Lopez et al. infecting human arm (Valenzuela-Lopez et al. 2018)d keratinophila (Verkley et al.) Valenz.-Lopez et al., and N. unguis-hominis (Punith. & M.P. English) Wanas. et al. infecting the skin and nails of humans (Punithalingam and English 1975; Verkley et al. 2010). The remaining species have been isolated from plant debris in freshwater, rhizosphere, soil, and sea water (Jaklitsch et al. 2018; Zhang et al. 2019; Magaña-Dueñas et al. 2021). These reports shows that saprophytism is the most common lifestyle reported among members of cucurbitaria-like taxa to date.

Other new species introduced in this study include P. quercicola from leaf spots of Quercus brantii in Zagrosian forests of Izeh, Iran. The genus Quercus also hosts several other species of Cucurbitaceae, namely F. parafenestrata Jaklitsch & Voglmayr on Q. robur L. and Q. petraea (Matt.) Liebl., Neocucurbitaria juglandicola Jaklitsch & Voglmayr on Q. rubra, N. quercina (Kabát & Bubák) Wanas. et al. on Q. robur, N. cava (Schulzer) Valenz.-Lopez et al. on Q. cerris L. and Q. robur (Valenzuela-Lopez et al. 2018; Jaklitsch and Voglmayr 2020; Jankowiak et al. 2022). Parafenestella is one of the more species-rich genera among the members of Cucurbitariaceae. Most species of Parafenestella are chiefly fungicolous, parasitising (or colonising) the sexual and asexual morphs of other fungi. For example, some species are reported from Cytospora sp. on Salix spp., including P. parasalicum Jaklitsch & Voglmayr, P. salicis (Rehm) Jaklitsch & Voglmayr, P. salicum Jaklitsch & Voglmayr, and P. vindobonensis Jaklitsch & Voglmayr (Jaklitsch and Voglmayr 2020). Some of them are associated with perithecial ascomata on woody plants, including P. alpina Jaklitsch & Voglmayr, P. austriaca Jaklitsch & Voglmayr, P. germanica Jaklitsch & Voglmayr, P. pseudoplatani Jaklitsch & Voglmayr, P. pseudosalicis Jaklitsch & Voglmayr, and P. rosacearum (Jaklitsch et al. 2018; Jaklitsch and Voglmayr 2020). In addition, some species of Parafenestella were reported to be saprobic on dead stems and twigs, including P. faberi (J. Kunze ex Sacc.) Jaklitsch & Voglmayr (syn. Fenestella mackenziei Wanas. et al.) on Rosa canina L., P. ostryae (Wanas. et al.) Jaklitsch & Voglmayr on Ostrya carpinifolia Scop., P. ulmi W.X. Su et al. and P. ulmicola W.X. Su et al. on Ulmus pumila L., and P. ontariensis Ilyukhin & Markovsk. on Acer negundo L. (Wanasinghe et al. 2017; Jaklitsch and Voglmayr 2020; Ilyukhin et al. 2022; Su et al. 2022). Other fenestella-like fungi, including Cucitella, Fenestella and Protofenestella, have a similar lifestyle (Jaklitsch et al. 2018; Jaklitsch and Voglmayr 2020). All species colonise Cytospora spp. on different trees except the species Cucitella opali Jaklitsch & Voglmayr, Fenestella fenestrata (Berk. & Broome) J. Schröt., and Protofenestella ulmi Jaklitsch & Voglmayr which have been directly isolated from branches and twigs of Acer opalus Mill., Alnus glutinosa (L.) Gaertn., and Ulmus spp., respectively (Jaklitsch et al. 2018). These reports indicate that mycoparasitism and saprophytism are possibly a common lifestyle in fenestella-like fungi.

Some genera and species related to the family Cucurbitariaceae do not have DNA sequence data in GenBank, and therefore their phylogeny and taxonomy remains unconfirmed. More molecular data and representative cultures of Cucurbitariaceae and allied taxa are needed to truly elucidate the taxonomy of the genera and species in this family and among all related taxa within Pleosporales. Given that the new species were isolated from plant disease symptoms, their pathogenicity and ecology should be further investigated to clarify whether they are plant pathogens.

Ethics approval and Consent to participate

Not applicable

Consent for publication

Informed consent was obtained from all individual participants included in the study.

Availability of data and materials

New sequences generated in the current study are deposited in NCBI GenBank and new species in MycoBank.

Competing interests

The authors confirm that they have no conflict of interest to declare.

Funding

This work was financially supported by grant (SCU.AP1400.294) from Research Council of Shahid Chamran University of Ahvaz.

Authors' contributions

Payam Eisvand carried out sample preparation, fungal isolation and purification, morphometric and morphological determination, DNA isolation, PCR amplification and contribution to describe the novel species. Mehdi Mehrabi-Koushki carried out the design and implementation of the research, DNA and phylogenetic analyses and the writing of the manuscript. Pedro W. Crous contributed to the writing of the manuscript.

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Table I Strains used in this study and their GenBank accession numbers. The new taxa and combinations are designated in bold.

Taxon	Strain^a	Source	Origin	GenBank accession numbers
Taxon	Strain^a	Source	Origin	ITS	LSU	*tub2*	*tef1α*	*rpb2*
Allocucurbitaria botulispora	CBS 142452 T	Human	USA	LT592932	LN907416	LT593001	-	LT593070
*A. galinsogisoli*	CBS 140956	Galinsoga parviflora	China	KU759584	KU759581	-	-	-
A. mori	HMJAU 60183	Morus alba	China	OL996120	OL897171	OL898725	-	OL944505
*A. ulmi*	CBS 143002 T	Ulmus glabra	Norway	MF795794	MF795794	MF795918	MF795882	MF795836
Astragalicola amorpha	CBS 142999 T	Astragalus angustifolius	Greece	MF795753	-	MF795883	MF795842	MF795795
Cucitella opali	CBS 142405 T	Acer opalus	France	MF795754	MF795754	MF795884	MF795843	MF795796
Cucurbitaria berberidis	C241	Berberis sp.	Austria	MF795756	MF795756	MF795886	MF795845	MF795798
C. berberidis	CB	Berberis vulgaris	Austria	MF795757	MF795757	MF795887	MF795846	MF795799
C. ephedricola	HA 42	dead wood	Italy	-	KT313007	-	-	-
C. oromediterranea	C229 T	Berberis cretica	Greece	MF795761	MF795761	MF795890	MF795849	MF795803
C. oromediterranea	CB2	Berberis cretica	Greece	MF795763	MF795763	MF795892	MF795851	MF795805
Fenestella crataegi	C287	Crataegus monogyna	Austria	MK356281	MK356281	MK357598	MK357554	-
F. crataegi	CBS 144857 T	Crataegus monogyna	Czech Republic	MK356282	MK356282	MK357599	MK357555	MK357512
F. fenestrata	FP9 T	Alnus glutinosa	Austria	MF795765	MF795765	MF795893	-	MF795807
F. gardiennetii	FM T	Acer saccharum	France	MK356283	MK356283	MK357600	MK357556	MK357513
F. granatensis	C279 T	Acer granatense	Spain	MK356284	MK356284	MK357601	MK357557	MK357514
F. media	FP T	Corylus avellana	Austria	MK356285	MK356285	MK357602	MK357558	MK357515
F. media	FP1	Corylus avellana	Austria	MK356287	MK356287	MK357603	MK357560	MK357517
F. parafenestrata	C306 T	Quercus robur	Austria	MK356291	MK356291	MK357607	MK357564	MK357521
F. parafenestrata	C317	Salix sp.	Austria	MK356292	MK356292	MK357608	MK357565	MK357522
F. subsymmetrica	FP4	Corylus avellana	Austria	MK356296	MK356296	MK357609	MK357568	MK357524
F. subsymmetrica	FP6 T	Acer campestre	Austria	MK356297	MK356297	MK357610	MK357569	MK357525
F. viburni	FP2	Viburnum lantana	France	MK356299	MK356299	MK357612	MK357571	MK357527
F. viburni	FVL T	Viburnum lantana	Austria	MK356300	MK356300	MK357613	MK357572	MK357528
Neocucurbitaria acanthocladae	C225 T	Genista acanthoclada	Greece	MF795766	MF795766	MF795894	MF795854	MF795808
N. acerina	C255	Acer pseudoplatanus	Austria	MF795768	MF795768	MF795896	MF795856	MF795810
N. acerina	C26a	Acer pseudoplatanus	Austria	MF795767	MF795767	MF795895	MF795855	MF795809
N. aetnensis	C261 T	Genista aetnensis	Italy	MF795769	MF795769	MF795897	MF795857	MF795811
N. aetnensis	C270	Genista aetnensis	Italy	MF795770	MF795770	MF795898	MF795858	MF795812

Table I Continued

Taxon	Strain^a	Source	Origin	GenBank accession numbers
Taxon	Strain^a	Source	Origin	ITS	LSU	*tub2*	*tef1α*	*rpb2*
N. aquadulcis	FMR 17840 T	plant debris in freshwater	Spain	LR897770	-	LR897794	-	LR897793
N. aquatica	CBS 297.74 T	Sea water	Serbia and Montenegro	LT623221	EU754177	LT623238	-	LT623278
N. cava	CBS 115979	Unknown	The Netherlands	AY853248	EU754198	LT623234	-	LT623273
N. cava	CBS 257.68 T	Wheat-field soil	Germany	JF740260	EU754199	KT389844	-	LT717681
N. chlamydospora	KNUF-22-1-8-B T	Hygia lativentris	Sout Korea	OQ060587	OQ060588	OQ148365	-	OQ148364
N. cinereae	KU9 T	Genista cinerea	Spain	MF795771	MF795771	MF795899	MF795859	MF795813
N. cisticola	C244 T	Cistus monspeliensis	Spain	MF795772	MF795772	MF795900	MF795860	MF795814
N. hakeae	CPC 28920 T	Hakea sp.	Australia	KY173436	KY173526	KY173613	-	KY173593
N. irregularis	CBS 142791 T	Human arm	USA	LT592916	LN907372	LT592985	-	LT593054
N. juglandicola	BW6 T	Juglans regia	Austria	NR156358	MF795773	MF795901	MF795861	MF795815
N. juglandicola	C316	Quercus rubra	Austria	MK356301	MK356301	MK357614	MK357573	MK357529
N. keratinophila	CBS 121759 T	Human	Spain	NR137017	NG070610	LT623236	-	LT623275
*N. pittospori*	CPC 34462 T	Pittosporum tenuifolium	New	MN562098	MN567606	-	-	-
N. populi	C28 T	Populus sp.	Sweden	MF795774	MF795774	MF795902	MF795862	MF795816
N. quercina	CBS 115095 T	Quercus robur	Italy	LT623220	GQ387619	LT623237	-	LT623277
N. rhamni	C1 T	Rhamnus frangula	Austria	MF795775	MF795775	-	MF795863	MF795817
N. rhamni	C112	Rhamnus frangula	Austria	MF795776	MF795776	MF795903	MF795864	MF795818
N. rhamnicola	C185 T	Rhamnus lycioides	Spain	MF795780	MF795780	MF795906	MF795868	MF795822
N. rhamnicola	KRx	Rhamnus alaternus	Spain	MF795781	MF795781	MF795907	MF795869	MF795823
N. rhamnioides	C118 T	Rhamnus myrtifolius	Spain	MF795782	MF795782	MF795908	MF795870	MF795824
N. rhamnioides	C223	Rhamnus saxatilis subsp. prunifolius	Greece	MF795784	MF795784	MF795910	MF795872	MF795826
N. ribicola	C155	Ribes rubrum	Austria	MF795786	MF795786	MF795912	MF795874	MF795828
N. ribicola	C55 T	Ribes rubrum	Austria	MF795785	MF795785	MF795911	MF795873	MF795827
N. salicis-albae	CBS 144611	Salix alba	Germany	NR163365	MK442535	MK442738	-	MK442669
N. unguis-hominis	CBS 111112	Agapornis sp.	The Netherlands	LT623222	GQ387623	LT623239	-	LT623279
N. vachelliae	C192 T	Vachellia gummifera	Morocco	MF795787	MF795787	MF795913	MF795875	MF795829
N. variabilis	FMR 17552 T	plant debris in freshwater	Spain	LR897768	-	LR897792	-	LR897791
N. variabilis	VMD-2020	plant debris in freshwater	Spain	LR897784	-	LR897807	-	LR897806

Table I Continued

Taxon	Strain^a	Source	Origin	GenBank accession numbers
Taxon	Strain^a	Source	Origin	ITS	LSU	*tub2*	*tef1α*	*rpb2*
*Nothocucurbitaria prunicola*	CBS 145033 T	Prunus padus	Ukraine	MK442594	MK442534	-	-	MK442668
*Nothocucurbitaria izehica*	IRAN 4856C; SCUA-IS-A10	*Crataegus* sp.	Iran	OR440607	OR440612	OR450816	-	OR450808
*Nothocucurbitaria izehica*	SCUA-IS-A10-2	*Crataegus* sp.	Iran	OR440608	OR440613	OR450817	-	OR450809
Paracucurbitaria corni	CBS 248.79	Fraxinus excelsior	The Netherlands	LT903672	GQ387608	LT900365	-	LT903673
P. italica	CBS 234.92 T	Olea europaea	Italy	LT623219	EU754176	LT623235	-	LT623274
Parafenestella alpina	C198 T	Cotoneaster integerrimus	Austria	MK356302	MK356302	MK357615	MK357574	MK357530
P. alpina	C249	Salix appendiculata	Austria	MK356303	MK356303	MK357616	MK357575	MK357531
P. austriaca	C152 T	Rosa canina	Austria	MK356304	MK356304	MK357617	MK357576	MK357532
P. changchunensis	HMJAU 60182	Populus	China	OL996119	OL897170	OL898719	-	-
P. faberi	MFLUCC 16-1451	Rosa canina	Germany	KY563071	KY563074	-	-	-
P. germanica	C307 T	Corylus avellana	Germany	MK356305	MK356305	MK357618	MK357577	MK357533
P. ontariensis	EI-6	Acer negundo	Canada	OM286882	OM286884	-	-
P. ostryae	MFLU 16-0184	Ostrya carpinifolia	Italy	KY563072	KY563075	-	-	-
P. parasalicum	C318 T	Salix cinerea	Austria	MK356306	MK356306	MK357619	MK357578	MK357534
P. pseudoplatani	C26 T	Acer pseudoplatanus	Austria	MF795788	MF795788	MF795914	MF795876	MF795830
P. pseudosalicis	C301 T	Salix cf. alba	Ukraine	MK356307	MK356307	MK357620	MK357579	MK357535
P. rosacearum	C203	Pyrus communis	Austria	MK356308	MK356308	MK357621	MK357580	MK357536
P. rosacearum	C309 T	Pyracantha coccinea	Austria	MK356311	MK356311	MK357624	MK357583	MK357539
P. salicis	C303	Salix alba	Austria	MK356316	MK356316	MK357628	MK357588	MK357544
P. salicis	C313 T	Salix alba	Austria	MK356317	MK356317	MK357629	MK357589	MK357545
P. salicum	C311 T	Salix alba	Austria	MK356318	MK356318	MK357630	MK357590	MK357546
*Parafenestella quercicola*	IRAN 4857C; SCUA-IS-B1	*Quercus* *brantii*	Iran	OR440609	-	OR450818	OR450812	-
*Parafenestella quercicola*	SCUA-IS-B1-2	*Quercus* *brantii*	Iran	OR440610	-	OR450819	OR450813	-
Parafenestella tetratrupha	C304 T	Alnus glutinosa	Austria	MK356319	MK356319	MK357631	MK357591	MK357547
P. ulmi	HMJAU 60179 T	Ulmus pumila	China	OL996116	OL897167	OL898717	-	OL944502
P. ulmi	HMJAU 60178	Ulmus pumila	China	OL996115	OL897166	-	-	OL944501
P. ulmicola	HMJAU 60180	Ulmus pumila	China	OL996117	OL897168	OL898724	-	OL944503
P. ulmicola	HMJAU 60181 T	Ulmus pumila	China	OL996118	OL897169	OL898718	-	OL944504

Table I Continued

Taxon	Strain^a	Source	Origin	GenBank accession numbers
Taxon	Strain^a	Source	Origin	ITS	LSU	*tub2*	*tef1α*	*rpb2*
P. vindobonensis	C302 T	Salix babylonica	Austria	MK356320	MK356320	MK357632	MK357592	MK357548
Protofenestella ulmi	FP5 T	Ulmus minor	Austria	MF795791	MF795791	MF795915	MF795879	MF795833
Synfenestella pyri	C297 T	Pyrus communis	Austria	MK356321	MK356321	MK357633	MK357593	MK357549
S. sorbi	C196	Sorbus aucuparia	Austria	MK356324	MK356324	MK357635	MK357596	MK357552
S. sorbi	FR T	Sorbus aucuparia	Austria,	MK356322	MK356322	MK357634	MK357594	MK357550
Pyrenochaetopsis leptospora	CBS 101635 T	Secale cereale	Unknow	MF795793	MF795793	MF795917	MF795881	MF795835
Pyrenochaeta nobilis	CBS 407.76 T	Laurus nobilis	Italy	MF795792	MF795792	MF795916	MF795880	MF795834
Pseudopyrenochaeta terrestris	CBS 282.72 T	Soil	The Netherlands	LT623228	LT623216	LT623246	-	LT623287
Neopyrenochaeta acicola	CBS 812.95 T	Waterpipe	The Netherlands	LT623218L	GQ387602	LT623232	-	T623271
Neopyrenochaetopsis hominis	UTHSC: DI16-238 T	Human	USA	LT592923	LN907381	LT592992	-	LT593061
Leptosphaeria doliolum	CBS 505.75	Urtica dioica	The Netherlands	JF740205	GU301827	JF740144	-	KT389640
Leptosphaerulina australis	CBS 317.83	Eugenia aromatica	Indonesia	GU237829	GU301830	GU237540	-	GU371790

^a Abbreviation of culture collections: CBS, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; IRAN, Iranian Fungal Culture Collection, Iranian Research Institute of Plant Protection, Tehran, Iran; MFLUCC, Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; Others are not registered abbreviations. ^T ex-type strains.

A revision of the family Cucurbitariaceae with additional new taxa from forest trees in Iran

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Material and Methods

Sample collection and fungal isolation

Morphological study

Phylogenetic analyses

Results

DNA analyses and phylogeny

Taxonomy

Discussion

Declarations

References

Tables

Status:

Journal Publication

Version 1