Introduction

The Phaeosphaeriaceae, which is characterized by coelomycetous asexual morphs, constitutes a large family in the order Pleosporales, which is the largest and most diverse order in the class Dothideomycetes (Phookamsak et al. 2014). The family was introduced by Barr (1979) and typified by Phaeosphaeria with Phaeosphaeria oryzae (Miyake 1909) as the type species.

The family Phaeosphaeriaceae has a cosmopolitan distribution, and many species in this family are important plant pathogens, infecting several major crops (Carson 2005; Stukenbrock et al. 2006; Quaedvlieg et al. 2013), while others may be saprobes (Shoemaker 1984; Schoch et al. 2006; Zhang et al. 2012; Hyde et al. 2013; Quaedvlieg et al. 2013), endophytes (Wang et al. 2005; Sánchez Márquez et al. 2007) or even lichenicolous (Lawrey et al. 2012). Furthermore, some species have been reported to cause human infections (Ahmed et al. 2017).

The taxonomy of the family Phaeosphaeriaceae has been subject of several changes in recent years. Barr (1979) introduced the family with 15 genera, but during the past 10 years, various phylogenetic studies have revealed the Phaeosphaeriaceae to be heterogeneous, and recent studies have introduced several new genera or transferred some known genera to other families (Zhang et al. 2012; Hyde et al. 2013; Quaedvlieg et al. 2013; Phookamsak et al. 2014; Trakunyingcharoen et al. 2014; Crous et al. 2015; Ertz et al. 2015). In this regard, Phookamsak et al. (2014) revised the family and published a monograph of Phaeosphaeriaceae based on morphology and phylogeny and accepted 30 genera. More recently, further novel genera were placed in the Phaeosphaeriaceae (Phukhamsakda et al. 2015; Senanayake et al. 2015; Tennakoon et al. 2016; Tibpromma et al. 2016, b; Ahmed et al. 2017; Wanasinghe et al. 2018) based on morphological characteristics and phylogenetic analyses. Presently, more than 50 sexual and asexual genera are accepted in the family. These genera include the following: Acericola, Allophaeosphaeria, Amarenomyces, Ampelomyces, Bhatiellae, Camarosporioides, Chaetosphaeronema, Dactylidina, Dematiopleospora, Didymocyrtis, Embarria, Equiseticola, Galiicola, Hawksworthiana, Italica, Juncaceicola, Leptosphaeria, Leptospora, Loratospora, Melnikia, Muriphaeosphaeria, Neosetophoma, Neostagonospora, Neosulcatispora, Nodulosphaeria, Ophiobolopsis, Ophiobolus, Ophiosimulans, Ophiosphaerella, Paraophiobolus, Paraphoma, Parastagonospora, Phaeopoacea, Phaeosphaeria, Phaeosphaeriopsis, Poaceicola, Populocrescentia, Pseudoophiobolus, Pseudophaeosphaeria, Sclerostagonospora, Scolicosporium, Septoriella, Setomelanomma, Setophoma, Sulcispora, Tintelnotia, Vagicola, Vrystaatia, Wojnowicia, Wojnowiciella, Xenoseptoria and Yunnanensis (Quaedvlieg et al. 2013; Wijayawardene et al. 2014, 2016; Phookamsak et al. 2014, 2017; Ariyawansa et al. 2015, b; Ertz et al. 2015; Li et al. 2015; Phukhamsakda et al. 2015; Senanayake et al. 2015; Tennakoon et al. 2016; Tibpromma et al. 2016, b; Ahmed et al. 2017; Wanasinghe et al. 2018).

During a recent survey exploring the fungal species associated with leaf spot diseases of herbaceous plants in Iran, a coelomycetous fungus was recovered from Black Bindweed, Fallopia convolvulus. A subsequent phylogenetic study based on different gene regions revealed this fungus to represent an undescribed genus in Phaeosphaeriaceae. The aim of this study was thus to resolve the taxonomy of this genus and elucidate the phylogenetic relationship to allied genera in Phaeosphaeriaceae.

Materials and methods

Fungal isolates

During a field excursion in the Jowkandan region, Talesh country, Guilan province, Iran, symptomatic Black Bindweed (Fallopia convolvulus) leaves were collected and returned to the laboratory. Leaves were examined directly under a Nikon SMZ 1500 dissecting microscope to observe sporulation. Single conidial isolates were obtained in pure culture by direct transfer of spores onto plates containing 2% malt extract agar (MEA; Fluka, Hamburg, Germany) using a procedure previously described by Bakhshi et al. (2011).

Dried specimens were preserved in the Fungarium of the Iranian Research Institute of Plant Protection, Tehran, Iran (IRAN). Representative cultures were deposited in the culture collection of the Westerdijk Fungal Biodiversity Institute (CBS), Utrecht, The Netherlands, and the culture collection of Tabriz University (CCTU), Tabriz, Iran.

DNA extraction, amplification and sequencing

Fungal genomic DNA was extracted from fresh mycelium harvested from colonies grown on MEA for 10 days at 25 °C in the dark, according to the protocol described by Möller et al. (1992). Seven genomic loci were targeted for PCR amplification and sequencing, namely the 28S nrRNA gene (LSU), internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS) of the nrDNA operon, actin (actA), histone H3 (his3), translation elongation factor 1-α (tef1), calmodulin (cmdA) and DNA-directed RNA polymerase II second largest subunit (rpb2). PCR amplifications were performed in a total volume of 12.5 μL solutions on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, California, USA). The primers, protocols and conditions for standard amplification and subsequent sequencing of the loci were according to Bakhshi et al. (2015) for LSU, ITS and his3 loci, and Quaedvlieg et al. (2013) for the remaining loci (actA, tef1, cmdA and rpb2).

Sequence alignment and phylogenetic analyses

The reference nucleotide sequences (Table 1) of representative genera in the Phaeosphaeriaceae were retrieved from GenBank and recently published alignments (Quaedvlieg et al. 2013; Ertz et al. 2015; Jayasiri et al. 2015; Phukhamsakda et al. 2015; Tennakoon et al. 2016; Tibpromma et al. 2016; Karunarathna et al. 2017; Phookamsak et al. 2017; Wanasinghe et al. 2018) (Table 1). The obtained sequences from GenBank, together with the generated sequences in this study, were aligned with the MAFFT v. 7 online interface using default settings (http://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013) for each gene and improved manually where necessary using MEGA v. 6.06 (Molecular Evolutionary Genetics Analysis) (Tamura et al. 2013). The alignments were concatenated with Mesquite v. 3.10 (Maddison and Maddison 2015). The best nucleotide substitution model for each data partition was determined by MrModeltest v. 2.3 (Nylander 2004), and a Bayesian phylogenetic reconstruction was performed with MrBayes v. 3.2.2 (Ronquist et al. 2012). The heating parameter was set at 0.15, and the Markov Chain Monte Carlo (MCMC) analysis of four chains was started in parallel from a random tree topology and lasted until the average standard deviation of split frequencies reached a value of 0.01. Burn-in was set to 25%, and trees were saved each 1000 generations. The resulting phylogenetic tree was printed with Geneious v. 8.1.8 (Kearse et al. 2012). All new sequences generated in this study were deposited in GenBank (www.ncbi.nlm.nih.gov).

Table 1 Taxa used in the phylogenetic analysis and GenBank accession numbers. Type species are indicated with boldface type
Full size table

Taxonomy

Morphological descriptions were based on isolates sporulating in vitro and in planta. In this regard, colonies were sub-cultured onto synthetic nutrient-poor agar plates (SNA; Crous et al. 2009) containing sterile Urtica dioica (stinging nettle) stems (Quaedvlieg et al. 2013). Cultures were incubated at 25 °C under continuous near-ultraviolet light for 14–30 days to promote sporulation. Freehand sections of fungal conidiomata were prepared, and fungal structures were mounted in clear lactic acid. For the morphological study in planta, hand sections were made from infected leaves and mounted in lactic acid. Observations were made with a Nikon Eclipse 80i compound microscope with differential inference contrast (DIC) illumination at 1000× magnification and a mounted Nikon digital sight DS-f1 high-definition colour camera. Thirty measurements were made of all relevant morphological features, and the 95% percentiles are presented, with extremes given between brackets. Photographic plates were edited and combined using Adobe Photoshop CS5. Growth rates and culture characters were noted on MEA and Oatmeal Agar (OA; Crous et al. 2009) after 20 days in the dark at 25 °C. Colony colour was rated according to the mycological colour charts of Rayner (1970).

Results

Phylogenetic analyses

The final concatenated alignment contained 92 ingroup taxa within the family Phaeosphaeriaceae with 1427 characters including gaps (gene boundaries of LSU, 1–790; ITS, 792–1427). These characters contained 586 unique site patterns (195 and 391 for LSU and ITS, respectively). One taxon of Coniothyriaceae (Coniothyrium carteri, CBS 105.91) was used as outgroup.

The results of MrModeltest recommended a SYM + I + G for ITS and GTR + I + G for LSU. All partitions had Dirichlet base frequencies. The Bayesian analysis lasted 4,405,000 generations and saved a total of 8812 trees. After discarding the first 25% of sampled trees, the consensus trees and posterior probabilities were calculated from the remaining 6610 trees (Fig. 1).

Fig. 1
figure 1figure 1

Consensus phylogram (50% majority rule) of 8812 trees resulting from a Bayesian inference analysis of the combined two-loci (LSU and ITS) sequence alignment showing the phylogenetic relationship of the new genus Parastagonosporella among the other Phaeosphaeriaceae genera. Type species are indicated with boldface type. The scale bar indicates 0.02 expected changes per site. The tree was rooted to Coniothyrium carteri (CBS 105.91)

Full size image

Taxonomy

In the multi-locus phylogeny inferred from the combined dataset shown in Fig. 1, the two isolates occurring on Fallopia convolvulus clustered in a separate clade, distinct from other genera in the family Phaeosphaeriaceae, suggesting that they represent a novel genus in this family. Therefore, a monotypic genus Parastagonosporella, typified by Parastagonosporella fallopiae, is introduced in the family Phaeosphaeriaceae.

Parastagonosporella M. Bakhshi, Arzanlou & Crous, gen. nov.

MycoBank: MB 826900.

Diagnosis: Morphologically distinct from the genus Parastagonospora by having conidiomata with more or less papillate neck, and walls of 4–8 layers of brown textura angularis.

Type species: Parastagonosporella fallopiae M. Bakhshi, Arzanlou & Crous, sp. nov.

Etymology: Morphologically resembling to the genus Parastagonospora, but distinct.

Parastagonosporella fallopiae M. Bakhshi, Arzanlou & Crous, sp. nov. Fig. 2

Fig. 2
figure 2

Parastagonosporella fallopiae (CBS 135981). a Leaf spot; b culture on MEA; c culture on OA; d Conidiomata on stinging nettle stem on SNA; e–k Conidiomata, conidiophores and conidia; l–p Conidia. Scale bars = 10 μm

Full size image

MycoBank: MB 826901.

Type: IRAN, Guilan Province, Talesh, Jowkandan, on Fallopia convolvulus, Jul. 2012, M. Bakhshi (holotype IRAN 17010 F, culture ex-type CBS 135981). GenBank accessions for sequences obtained from ex-type culture: LSU = MH460545; ITS = MH460543; actA = MH460537; his3 = MH460541; tef1 = MH460549; cmdA = MH460539; rpb2 = MH460547.

Etymology: Named after the host genus from which it was isolated, Fallopia.

Description in planta: Leaf spots numerous, small, 2–3 mm in diameter, circular to angular, and often merging to form irregular patterns, amphigenous, brown in centre, surrounded by raised dark brown margin, diffuse outward to form a halo. Conidiomata pycnidial, dark brown, subepidermal, amphigenous, several in each leaf spot, subglobose, immersed, up to 200 μm in diameter, releasing conidia in creamy to white cirrhi; wall of 4–8 layers of brown textura angularis; ostiolum central, circular, with papillate neck, 15–35 μm wide. Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, smooth, lining the inner cavity, broadly to narrowly ampulliform to subcylindrical, occasionally phialidic, with prominent periclinal thickening or annellidic, proliferating percurrently with more or less distinct annelations, 7–13 × 2.5–6 μm. Conidia hyaline, smooth, thin-walled, scolecosporous, subcylindrical, granular to multi-guttulate, with obtuse apex and truncate to subtruncate base, 3–10-septate, (18–)35–40(−50) × (3–)4–6 μm.

Description in vitro: On sterile Urtica dioica stems on SNA. Conidiomata as in planta, pycnidial, dark brown, erumpent, up to 250 μm in diameter, exuding pale orange to creamy conidial mass. Conidiophores reduced to conidiogenous cells. Conidiogenous cells as in planta, 7–15 × 3–6 μm. Conidia similar in shape as in planta, 5–12-septate, (24–)32–45(−55) × (3–)4.5–6 μm.

Culture characteristics: Colonies on MEA after 20 days in the dark at 25 °C, up to 40 mm in diameter, flat, with even margin and white aerial mycelium, surface olivaceous, reverse iron-grey; on OA surface flat, smooth, entire edge, with aerial mycelium, ochraceous white in centre, olivaceous grey in outer part, reaching 35 mm in diameter after 20 days at 25 °C.

Additional material examined: IRAN, Guilan Province, Talesh, Jowkandan, on Fallopia convolvulus, Jul. 2012, M. Bakhshi (CCTU 1151.1). GenBank accessions: LSU = MH460546; ITS = MH460544; actA = MH460538; his3 = MH460542; tef1 = MH460550; cmdA = MH460540; rpb2 = MH460548.

Discussion

A coelomycetous fungus associated with leaf spot disease of Black Bindweed was subjected to phylogenetic study and morphological analyses. By combining LSU and ITS sequence data as well as detailed morphological data, we were able to delimit a new genus Parastagonosporella, among the coelomycetous genera in the family Phaeosphaeriaceae within the order Pleosporales.

Morphological characters traditionally used to delineate genera in coelomycetes include conidiomatal structure, structure of the conidiophores, conidiogenous cells and conidial features such as septation, pigmentation, and conidial appendages (Sutton 1980; Nag Raj 1993). Recent molecular studies have shown that these features are not always appropriate to delineate genera as natural units, and they may vary even between sibling species (Crous et al. 2012; Quaedvlieg et al. 2013; Phookamsak et al. 2014).

As is the case with many other coelomycetous genera, the lack of useful morphological characters combined with the high level of variation therein and the need for high levels of expertise in morphology-based identification makes it difficult to distinguish individual genera within the Phaeosphaeriaceae based solely on their morphological characters (Quaedvlieg et al. 2013). Hence, genera and species recognition using the molecular phylogeny of several unlinked DNA loci has already resulted in the natural and reliable delimitation of genera within this family as well as in other fungal families of Dothideomycetes, such as the Botryosphaeriaceae (Phillips et al. 2013), Mycosphaerellaceae (Videira et al. 2017) and Pleosporaceae (Ariyawansa et al. 2015). By using molecular techniques, several novel taxa have been described in the family Phaeosphaeriaceae in recent years (Wijayawardene et al. 2014, 2016; Phookamsak et al. 2014, 2017; Ariyawansa et al. 2015; Ertz et al. 2015; Phukhamsakda et al. 2015; Senanayake et al. 2015; Tennakoon et al. 2016; Tibpromma et al. 2016, b; Ahmed et al. 2017; Moslemi et al. 2018; Wanasinghe et al. 2018). The loci used in these and similar recent studies typically include LSU and ITS data as these loci can distinguish most of the presently known genera within the Phaeosphaeriaceae. In the combined (LSU/ITS) phylogenetic tree, these data were sufficient to clearly separate the novel genus Parastagonosporella from other known genera within the Phaeosphaeriaceae. Furthermore, data for the additional loci generated in this study (actA, his3, tef1, rpb2 and cmdA) were deposited in GenBank, as this would aid future studies on the family.

Phylogenetic analyses of combined LSU and ITS sequence data (Fig. 1) indicated that Parastagonosporella is a distinct genus in Phaeosphaeriaceae, which is closely related to the genera Paraphoma, Pseudophaeosphaeria, Setomelanomma and Xenoseptoria. Paraphoma is distinctly different from Parastagonosporella in having ellipsoid and aseptate conidia (Quaedvlieg et al. 2013). Pseudophaeosphaeria (Hyde et al. 2016) and Setomelanomma (Wu et al. 2014) also accommodate species that reproduce sexually. Xenoseptoria differs from Parastagonosporella in having (1–)3-septate conidia tapering to subobtuse apex and obtuse base (Quaedvlieg et al. 2013). Phylogenetically, these genera are also clearly distinct (Fig. 1). Parastagonosporella is morphologically similar to the genus Parastagonospora by having pycnidial conidiomata, hyaline, smooth, ampulliform to subcylindrical conidiogenous cells, with euseptate, hyaline, granular to multi-guttulate conidia with truncate bases, but distinct in having conidiomata with more or less papillate necks and walls of 4–8 layers of brown textura angularis, versus 2–3 layers in Parastagonospora and phialidic or annellidic conidiogenous cells, versus phialidic in Parastagonospora (Quaedvlieg et al. 2013). Phylogenetically, these genera are also clearly distinguishable from each other (Fig. 1). Based on these clear morphological and phylogenetic data, Parastagonosporella is introduced as a new genus.

Quaedvlieg et al. (2013) comprehensively studied the phylogeny of the genus Septoria and other morphologically similar genera such as Stagonospora, Sphaerulina and Phaeosphaeria. Their results surprisingly revealed that “Stagonospora” nodorum (causal agent of nodorum blotch of cereals), clustered in a distinct genus, unrelated to Stagonospora s. str. within the family Massarinaceae. Consequently, they introduced the genus Parastagonospora (with P. nodorum as the type species) in the family Phaeosphaeriaceae, based on multi-locus molecular data to accommodate several cereal pathogens that could not be placed in Stagonospora or Phaeosphaeria. Current literature further indicates that the sole morphology-based classification of coelomycete families as well as their associated genera and species can be misleading. Here we introduce the novel genus Parastagonosporella to accommodate the isolates occurring on Black Bindweed which are parastagonospora-like in morphology, but cluster apart from Parastagonospora by forming a well-supported separate clade with high Bayesian posterior probability.

Based on literature, several coelomycetous fungi have been reported to be present on the host genus Fallopia within the family Polygonaceae, including Discosia sp. (Amphisphaeriaceae, Amphisphaeriales), Phyllosticta fallopiae, Phyllosticta polygonorum (Phyllostictaceae, Botryosphaeriales), Pilidium lythri (Chaetomellaceae, Chaetomellales) and Septoria polygonorum (Mycosphaerellaceae, Capnodiales) (Farr and Rossman 2018). To our knowledge, the new species Parastagonosporella fallopiae is the first association of a fungus belonging to the family Phaeosphaeriaceae on the plant genus Fallopia.

The present study adds a new genus to the Phaeosphaeriaceae, which is a family that has been intensively studied in recent years due to its economic importance (Quaedvlieg et al. 2013; Wijayawardene et al. 2014, 2016; Phookamsak et al. 2014, 2017; Ariyawansa et al. 2015; Ertz et al. 2015; Phukhamsakda et al. 2015; Senanayake et al. 2015; Tennakoon et al. 2016; Tibpromma et al. 2016, b; Ahmed et al. 2017; Wanasinghe et al. 2018). Here, we further demonstrate that the delimitation of taxa in this family based solely on morphological features is not feasible and emphasize the necessity of using DNA sequence data along with morphology and ecology to facilitate the accurate identification in the Phaeosphaeriaceae.