Polyphasic taxonomy of four passalora-like taxa occurring on fruit and forest trees

Species of Passalora s. lat. are phytopathogenic fungi that generally cause leaf spot diseases on a broad variety of plants throughout the world. During our investigations exploring cercosporoid fungi associated with leaf spot symptoms of fruit and forest trees in northern and north-western Iran, several passalora-like fungi were isolated from symptomatic leaves of trees belonging to the Fabaceae, Malvaceae, Rosaceae, and Ulmaceae. A polyphasic taxonomic approach applying molecular data, morphological features, and host data was employed to identify the isolates. In a multi-gene phylogenetic analysis (LSU, ITS, and RPB2), these isolates are clustered in four clades in the Mycosphaerellaceae. The taxa encompassed Paracercosporidium microsorum on Tilia platyphyllos, Prathigadoides gleditsiae-caspicae gen. et. sp. nov. on Gleditsia caspica, Pruniphilomyces circumscissus on Prunus avium and Prunus cerasus, and Sirosporium celtidis on Celtis australis. The new genus Prathigadoides and its type species Prathigadoides gleditsiae-caspicae are molecularly distinct from all phylogenetically related genera, and some characteristics of the conidiophores and conidia differ from those of the morphologically similar species Prathigada condensata on the North America Gleditsia triacanthos.


Introduction
The Mycosphaerellaceae is a large and diverse family in the order Mycosphaerellales and class Dothideomycetes (Crous et al. 2009;Abdollahzadeh et al. 2020). Since the first molecular study on Mycosphaerellaceae (Stewart et al. 1999), the concept of the family and its genera has been significantly revised, and recently, based on phylogenetic data, 122 asexual and sexual genera have been accepted within this family (Videira et al. 2017;Crous et al. 2020). Most of the asexual morphs connected with this family are cercosporoid fungi ). Based on a mainly morphological approach, Crous and Braun (2003) reduced the true cercosporoids to four genera, viz. Cercospora Fresen. ex Fuckel, Pseudocercospora Speg., Passalora Fr. and Stenella Syd. Recent phylogenetic examinations of cercosporoid fungi have revealed that these genera are polyphyletic. Pantospora Cif. (Minnis et al. 2011;Braun et al. 2013), Pallidocercospora Crous, Paracercospora Deighton , and Phaeocercospora Crous (Crous et al. 2012) are genera belonging to the Pseudocercospora complex. They are morphologically similar to or only slightly different from Pseudocercospora, but they were clearly segregated as distinct genera on the basis of phylogenetic data Hyde et al. 2013). Despite previous assumption of the monophyly of the genus Cercospora Bakhshi et al. 2015a), Bakhshi et al. (2015b) introduced the genus Neocercospora M. Bakhshi, Arzanlou, Babai-ahari & Crous, characterized by being morphologically cercospora-like, but clustering in a separate clade in Mycosphaerellaceae apart from Cercospora s. str. Regarding the genus Stenella, sequencing of the type species (S. araguata Syd.) revealed that it belongs to Teratosphaeriaceae and the remaining species in Mycosphaerellaceae. Most former mycosphaerellaceous Stenella species were assigned to Zasmidium, an old genus name; other species were placed in new genera (e.g., Pseudozasmidium Videira & Crous;Videira et al. 2017). The phylogenetic structure of Passalora is, however, more complicated than the other cercosporoids.
The genus Passalora was originally described by Fries (1849) based on Passalora bacilligera Mont. & Fr., a fungus occurring on the forest tree Alnus glutinosa (L.) Gaertn. The delimitation of species within the genus as well as the circumscription of the genera has changed since its establishment. A full discussion on the history of taxonomy of passalora-like taxa was presented by Braun et al. (2013). Recent phylogenetic analyses based on a much broader sampling, including the type species of Passalora and its synonyms, revealed that Passalora s. str. forms a well-supported clade in the Mycosphaerellaceae and includes only the type species (Videira et al. 2017). As a consequence, Videira et al. (2017) resurrected several old generic names, which were previously merged with Passalora based on morphology (Crous and Braun 2003), and described many additional new genera which comprise passalora-like species. On the other hand, some Passalora species are clustered within the Cercospora or Pseudocercospora clade Videira et al. 2017). Therefore, previous generic definitions of cercosporoid fungi (Crous and Braun 2003) can no longer be applied to these genera in their current circumscriptions (Videira et al. 2017), and the identification of passalora-like taxa strongly depends on the availability of DNA sequence data.
Species of Passalora s. lat. are mostly well-known phytopathogenic fungi that cause typical leaf spots, necrosis, or chlorosis, on a wide range of woody and herbaceous host plants growing in semi-arid or wet environments under a wide range of climates (Chupp 1954;Crous and Braun 2003;Videira et al. 2017), while some display endophytic (Douanla-meli et al. 2013) or mycophilic life styles Videira et al. 2017). Passalora-like fungi include important plant pathogenic species, such as Nothopassalora personata (Berk. & M.A. Curtis) U. Braun, C. Nakash., Videira & Crous (syn. Pass. personata), the causal agent of late leaf spot disease on peanut (Clevenger et al. 2018;Bakhshi and Zare 2020), Pass. vaginae (W. Krüger) U. Braun & Crous causing a foliar disease on Saccharum officinarum L. (Crous and Braun 2003) (Thomma et al. 2005) and Passalora spp. associated with a leaf spot disease on Ficus spp. (Singh et al. 2013  . Owing to the strong relevance of such diseases in agriculture, horticulture, and forestry, understanding and stabilizing the taxonomy of species of the genus Passalora s. lat. are, therefore, of particular importance. The mainland of Iran is well-known as an area with enormous biodiversity, including a very wide range of vascular plants which are the basis for a huge diversity of foliicolous fungi, including cercosporoid hyphomycetes. Some genera of cercosporoid fungi of Iran, e.g., Cercospora (Bakhshi et al. 2015a(Bakhshi et al. , b, 2018Bakhshi 2019;Bakhshi and Zare 2020) and Pseudocercospora (Bakhshi et al. 2014;Braun et al. 2020), have been relatively well-documented in recent years based on recent molecular revisions of these genera Crous et al. 2013). Nevertheless, except for Nothopassalora personata (Bakhshi and Zare 2020), no molecular studies have so far been conducted on other passalora-like species known from Iran. The aim of this study was to characterize passaloralike taxa associated with some fruit and forest trees in the northern zone of Iran, in terms of morphology, cultural characteristics, ecology, and DNA phylogeny.

Sample collection and isolates
Plant samples with cercosporoid leaf spot symptoms were collected in Iran from different biotopes, including natural forests and agricultural orchards. Fresh samples were immediately taken to the laboratory in paper bags. To establish axenic cultures originating from single conidia, samples were examined using a Zeiss Stemi 305 (Carl Zeiss, Jena, Germany) stereo microscope, and cercosporoid conidia from lesions were suspended in sterilized distilled water and spread on malt-extract agar (MEA; Fluka, Hamburg, Germany) medium using a flame-sterilized micro-spatula as explained in Bakhshi et al. (2021). Representative axenic cultures were deposited in culture collection of the Iranian Research Institute of Plant Protection (IRAN C), Tehran, Iran, and the Westerdijk Fungal Biodiversity Institute (formerly CBS), Utrecht, The Netherlands. Diseased plant specimens were dried in a plant press and deposited in fungal herbarium of the Iranian Research Institute of Plant Protection (IRAN F).

Phylogenetic analyses
The raw trace files were inspected and edited with MEGA v. 6 software (Tamura et al. 2013), and consensus sequences were manually generated from the forward and reverse sequences. Following BLAST searches of the NCBI's Gen-Bank nucleotide database (www. ncbi. nlm. nih. gov) for preliminary identifications, a multi-gene tree was constructed using LSU, ITS, and RPB2 sequences from Mycosphaerellaceae taxa (Videira et al. 2017;Crous et al. 2020). At first, the alignments for each locus were performed by MAFFT v. 7 (http:// maff. tcbrc. jp/ align ment/ server/.) (Katoh and Standley 2013) followed by manual adjustments using MEGA v. 6. Multiple alignments were combined with Mesquite v. 3.61 (Maddison and Maddison 2018). Phylogenetic reconstructions of the combined gene trees were performed using maximum parsimony (MP) and Bayesian inference (BI) criteria.
Maximum parsimony analysis was performed in PAUP v. 4.0 (Swofford 2003) and involved 10,000,000 replicates of fast stepwise search option using the tree bisection reconnection (TBR) as the branch-swapping algorithm with 100 random sequence additions. All characters were unordered and given equal weight, and alignment gaps were treated as fifth character state. Branches of zero length were collapsed, and all multiple, equally most parsimonious trees were saved. Tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for the resulting tree. For Bayesian phylogenetic reconstruction, the best-fit evolutionary models were selected independently for each locus using MrModeltest v. 2.3 (Nylander 2004) and applied to each gene partition. Bayesian inference analysis (BI) was conducted with MrBayes v. 3.2.6 (Ronquist et al. 2012) by Markov Chain Monte Carlo sampling (MCMC) in two parallel runs for a maximum of 10,000,000 generations, and trees were sampled every 100th generation. The analyses were stopped when the two runs converged, and the average standard deviation of split frequencies came below 0.01. The first 25% of generated trees representing the burn-in phase were discarded, and the remaining trees were used to calculate posterior probabilities of the majority rule consensus tree. Phylogenetic trees were visualized with Geneious v. 8.1.8 (Kearse et al. 2012) and edited in Adobe ® Illustrator v. CC 2017. The alignments and phylogenetic trees were deposited in TreeBASE (Study ID S28243) (www. TreeB ASE. org). The accession numbers of the sequences used for the phylogenetic analyses are listed in Table 1.

Morphology
Detailed morphological descriptions are provided for all taxa based on structures observed in herbarium material. Specimens were processed and examined with a Zeiss Stemi 305 stereo microscope, and fungal structures (stromata, hyphae, conidiophores, and conidia) were mounted on glass slides in clear lactic acid for microscopic studies and microphotography. Specimens were examined with an Olympus BX51 (Olympus, Tokyo, Japan) light microscope with differential inference contrast (DIC) illumination at × 1000 magnification and photographed with an Olympus DP25 camera fitted to the microscope. Size ranges of morphological characters were derived from at least 30 measurements and images used for figures processed with Adobe Photoshop CS5 (Adobe Systems, USA). Colony morphology on MEA was determined after 20 days at 25 °C in the dark in duplicate, and colony color was described using the mycological color charts of Rayner (1970). Nomenclatural novelties and descriptions were deposited in MycoBank (www. mycob ank. org; Crous et al. 2004a).

Molecular phylogenetic analyses
The final concatenated alignment comprised 130 ingroup taxa within the family Mycosphaerellaceae, with 2250 characters including alignment gaps (gene boundaries of LSU: 1-745, RPB2: 746-1555, and ITS: 1556-2250). The five  (Fig. 1). Phylogenetic trees obtained from BI and MP analyses yielded trees with similar overall topologies at the generic level and agree with previous studies, such as Videira et al. (2017). The results of MrModeltest recommended that Bayesian analysis should use dirichlet (1, 1, 1, 1) state frequencies and the GTR + I + G with an invgamma-shaped rate variation for all loci (LSU, RPB2, and ITS). The Bayesian analysis resulted in 4942 trees after 2,470,000 generations. The first 1234 trees, representing the burn-in phase of the analyses, were discarded, while the remaining 3708 trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree (Fig. 1). The alignment contained a total of 1230 unique site patterns (217, 557, and 456 for LSU, RPB2, and ITS, respectively).
The maximum parsimony (MP) analyses generated a maximum of 1000 equally most parsimonious trees, and the bootstrap support values (MP-BS) were mapped on the Bayesian tree as the second value in the tree nodes (Fig. 1). From the analyzed characters, 1074 were constant, 206 were variable and parsimony-uninformative, and 970 were parsimony-informative. A parsimony consensus tree was calculated from the equally most parsimonious trees, and the branches were mapped with a thicker line on the Bayesian tree (length = 14,442, CI = 0.170, RI = 0.500, RC = 0.085, HI = 0.830).

Taxonomy
Based on phylogenetic analyses, the isolates collected in this study could be assigned to four different taxa in the Mycosphaerellaceae. The taxa are treated as follows.
In any case, the North American cercosporoid species on G. triacanthos neither pertain to Cercospora nor to Passalora in the current sense and circumscription (Videira et al. 2017).
Cultural characteristics: Colonies on MEA, surface flat, folded, with moderate aerial mycelium and entire margin, dark olivaceous green, reverse olivaceous black, reaching 20 mm after 20 days at 25 °C. Notes: To our knowledge, this study is the first molecular confirmation of Pruniphilomyces circumscissus for the Middle East. Furthermore, part of the ACT , TEF1-α, CAL, and HIS loci is sequenced for the first time in this species.
Cultural characteristics: Colonies on MEA, surface brown-gray, with sparse aerial mycelium and entire margin, colony surrounded by a red-brown halo, reverse dark brown, reaching 4 mm after 20 days at 25 °C.
Material Notes: To our knowledge, this study is the first molecular confirmation of Sirosporium celtidis for Asia. Furthermore, first sequences of the ACT , TEF1-α, CAL, and HIS loci have also been generated for this species.

Discussion
The present study aimed at identifying passalora-like strains associated with fruit and forest trees from the northern and northwestern zones of Iran based on a combination of DNA phylogeny, host range, ecology, and morphological characteristics. Members of Passalora s. lat. are devastating plant pathogens on a wide range of economically important woody and herbaceous plants worldwide, including Iran (Crous and Braun 2003;Thomma et al. 2005;Singh et al. 2013;Fernandes et al. 2013;Bakhshi and Zare 2020). Recent developments in the phylogeny of the Mycosphaerellaceae have revealed the polyphyly of Passalora s. lat. and the distribution of passalora-like clades throughout the Mycosphaerellaceae (Videira et al. 2017;Crous et al. 2020). As a consequence, several generic names with passaloroid morphology, previously reduced to synonymy with Passalora (Crous and Braun 2003), e.g., Cercosporidium Earle, Fulvia Cif., Mycovellosiella Rangel, Phaeoramularia Munt-Cvetk., and Ragnhildiana Solheim, have been reinstated, and many additional new genera, e.g., Pleuropassalora U. Braun, C. Nakash., Videira & Crous, Graminopassalora U. Braun, C. Nakash., Videira & Crous, Coremiopassalora U. Braun, C. Nakash., Videira & Crous, and Neocercosporidium Videira & Crous, have been described. Some of these genera, e.g., Exopassalora Videira & Crous, clustered within the Phaeothecoidiellaceae clade (Videira et al. 2017). There are very few distinctive morphological features at generic level within the Passalora complex, and most of these genera are morphologically similar to or even indistinguishable from Passalora s. str. (Videira et al. 2017).
Among taxa of Passalora s. lat. known from Iran (Bakhshi et al. 2012;Pirnia 2019), so far only the identity of one species, Nothopassalora personata, has been confirmed based on DNA sequence data (Bakhshi and Zare 2020). During the present study, passalora-like fungi associated with leaf spot symptoms of different trees of the Fabaceae, Malvaceae, Rosaceae, and Ulmaceae were subjected to a multi-gene phylogeny (LSU, ITS, and RPB2). According to our findings, these isolates clustered in four different genus clades in the Mycosphaerellaceae, of which one taxon was associated with cherry fruit trees (Prunus spp.) and the three remaining taxa were associated with forest trees.
Recently, Crous et al. (2020) studied an isolate of Passalora circumscissa (Sacc.) U. Braun, a significant foliar pathogen of Prunus spp., and revealed that the sequence clustered apart from sequences retrieved from the type species of Passalora (P. bacilligera), so that the new genus Pruniphilomyces was introduced to accommodate Passalora circumscissa. In Iran, the causal agent of the leaf spot disease of Prunus spp. has been reported as Passalora circumscissa based on morphological data (Pirnia 2019). During this study, we prepared living cultures of several isolates of a passalora-like taxon associated with leaf spot diseases of cherry and sour cherry from different provinces of Iran, and the isolates were morphologically and molecularly analyzed. Both Bayesian and maximum parsimony analyses of concatenated alignments of three loci (LSU, ITS, and RPB2) showed that the Iranian isolates obtained from Prunus spp. formed a highly supported clade assignable to Pruniphilomyces circumscissus (Fig. 1). Crous et al. (2020) described Pruniphilomyces on the basis of a single isolate. Our analyses were based on sequence data of nine isolates from different localities of Iran, which further confirmed the phylogenetic status of this taxon.
The other three taxa studied during this research were associated with leaf spot diseases of three forest trees in the Hyrcanian forest of northern Iran, including large leaf linden (Tilia platyphyllos), Caspian locust (Gleditsia caspica), and Mediterranean hackberry (Celtis australis). The Hyrcanian forests have always attracted the attention of biologists because they are ancient and an important biodiversity "hot spot" (Scharnweber et al. 2007;Yousefzadeh et al. 2017). In recent years, several molecular studies of fungal taxa occurring on forest trees in Hyrcanian forests of Iran revealed several novel mycosphaerellaceous species on some important trees of this area, e.g., Ramularia taleshina M. Bakhshi & Arzanlou (Bakhshi and Arzanlou 2017) (Bakhshi et al. 2014;Braun et al. 2020). The introduction of a novel taxon, Prathigadoides gleditsiae-caspicae on Caspian locust, an important native forest tree in Hyrcanian region, further confirms the diversity of mycosphaerellaceous taxa in this area.
An important and intriguing aspect of this study was the clear evidence that Prathigadoides gleditsiae-caspicae, although morphologically similar to species previously assigned to Prathigada, clustered in a separate clade within the Mycosphaerellaceae, reflecting a genus of its own. Previous molecular studies Videira et al. 2017) have shown that Prathigada cratevae (current name = Pseudocercospora cratevicola C. Nakash. & U. Braun), the type species of this genus, clusters within the Pseudocercospora s. str. clade; thus, Prathigada was reduced to synonymy with Pseudocercospora ). These findings further illustrate the importance of using sequence data to clarify the phylogeny and taxonomy of taxa previously assigned to Passalora s. lat.
The genus Paracercosporidium includes two species on Tilia, namely P. microsorum and P. tiliae (Peck) U. Braun, C. Nakash., Videira & Crous, which were previously placed in Passalora due to obclavate conidia (Crous and Braun 2003;Videira et al. 2017). Based on previous molecular studies, the two species clustered apart from the type species of the genus Passalora in a well-supported clade; therefore, Videira et al. (2017) introduced the new genus Paracercosporidium. Based on the phylogenetic analyses, the Iranian isolates obtained from large leaf linden (Tilia platyphyllos) in this study clustered within the Paracercosporidium microsorum clade.
The isolates obtained from Mediterranean hackberry in this study clustered together with sequences of Sirosporium celtidis. The genus Sirosporium is passalora-like in morphology, but treated as a separate genus confined to species with thick-walled dictyosporous conidia (Braun 1995;Crous and Braun 2003;Braun et al. 2013). So far, sequence data are available for only two species previously assigned to Sirosporium, viz., S. celtidis (treated as "Sirosporium" celtidis), and S. diffusum (treated as Ragnhildiana diffusa (Heald & F.A. Wolf) Videira & Crous), which clustered in separate clades within the Mycosphaerellaceae (Videira et al. 2017). However, sequence data are not available for the type species of the genus (S. antenniforme (Berk. & M.A. Curtis) Bubak & Serebrian), so that the status of this genus remained unresolved (Videira et al. 2017). The fact that the Iranian isolates of S. celtidis obtained from different provinces, also clustered with the isolates of this species collected from Algeria, Portugal and Italy (Videira et al. 2017; Fig. 1), further confirms the phylogenetic status of this species and its identification. Sequences retrieved from collections of S. antenniforme are urgently needed to reveal the true phylogenetic affinity of Sirosporium. If S. celtidis is not congeneric with S. antenniforme, and sequences retrieved from the cercosporoid fungus on Celtis would form a distinct clade, the name Helicoceras Linder (Linder 1931) would be available for a separate genus. Monilia celtidis Biv. (≡ Sirosporium cletidis) is the type species of Helicoceras. Furthermore, the type species of Sirosporium, S. antenniforme, described from North America, and Monilia celtidis, described from Italy, need to be epitypified with ex-epitype cultures and ex-epitype sequences in order to determine these species and the corresponding genera genetically. We confirmed the Sirosporium celtidis in Asia for the first time using molecular data.
Most passalora-like taxa obtained from Iran have been identified based only on morphological features and host range data, but living cultures and DNA data are still lacking. Accurate identifications of plant pathogenic fungi are pre-requisites for proper disease managements. As emphasized by different researchers, species identifications based solely on morphological characteristics often led to confusion in the taxonomy of cercosporoid fungi Groenewald et al. 2013;Bakhshi et al. 2014Bakhshi et al. , 2015aBakhshi et al. , 2018. Hence, comprehensive attempts to collect, cultivate, and examine cercosporoid plant pathogens from diverse host plants in various geographical regions of Iran are urgently needed.