Molecular reassessment of diaporthalean fungi associated with strawberry, including the leaf blight fungus, Paraphomopsis obscurans gen. et comb. nov. (Melanconiellaceae)

Phytopathogenic fungi in the order Diaporthales (Sordariomycetes) cause diseases on numerous economically important crops worldwide. In this study, we reassessed the diaporthalean species associated with prominent diseases of strawberry, namely leaf blight, leaf blotch, root rot and petiole blight, based on molecular data and morphological characters using fresh and herbarium collections. Combined analyses of four nuclear loci, 28S ribosomal DNA/large subunit rDNA (LSU), ribosomal internal transcribed spacers 1 and 2 with 5.8S ribosomal DNA (ITS), partial sequences of second largest subunit of RNA polymerase II (RPB2) and translation elongation factor 1-α (TEF1), were used to reconstruct a phylogeny for these pathogens. Results confirmed that the leaf blight pathogen formerly known as Phomopsis obscurans belongs in the family Melanconiellaceae and not with Diaporthe (syn. Phomopsis) or any other known genus in the order. A new genus Paraphomopsis is introduced herein with a new combination, Paraphomopsis obscurans, to accommodate the leaf blight fungus. Gnomoniopsis fragariae comb. nov. (Gnomoniaceae), is introduced to accommodate Gnomoniopsis fructicola, the cause of leaf blotch of strawberry. Both of the fungi causing leaf blight and leaf blotch were epitypified. Fresh collections and new molecular data were incorporated for Paragnomonia fragariae (Sydowiellaceae), which causes petiole blight and root rot of strawberry and is distinct from the above taxa. An updated multilocus phylogeny for the Diaporthales is provided with representatives of currently known families.


INTRODUCTION
The order Diaporthales is one of the largest and bestdefined orders in the Sordariomycetes (Castlebury et al. 2002;Zhang et al. 2006;Rossman et al. 2007). The order comprises many destructive plant pathogens causing diseases on various crops, ornamental plants and forest trees, as well as numerous endophytic and saprobic fungal species (Udayanga et al. 2011Shuttleworth and Guest 2017;Senanayake et al. 2017a;Jiang et al. 2019Jiang et al. , 2020. It currently contains approximately 31 families supported by molecular data, with many recent additions and segregations of genera and families within the order (Castlebury et al. 2002;Lumbsch and Huhndorf 2007;Rossman et al. 2007Rossman et al. , 2015Rossman et al. , 2016Yang et al. 2018;Voglmayr et al. 2017Voglmayr et al. , 2019aJiang et al. 2020). Although the phylogenetic relationships and species composition of the majority of commonly encountered pathogenic genera are known, much work remains to be done concerning more obscure taxa from various geographic locations around the world (Zhang and Blackwell 2001;Rossman et al. 2007;Yun and Rossman 2011;Crous et al. 2012aCrous et al. , 2012bWalker et al. 2012;Rossman et al. 2015).
The genus Fragaria, better known as strawberry, belongs in the plant family Rosaceae and is well known for its edible fruits (Hancock 1999). Worldwide, there are more than 25 described species, including wild species and many hybrids and cultivars (Potter et al. 2000;Staudt 2009;Zhong et al. 2018). The modern cultivated/ garden strawberry, Fragaria × ananassa (Weston) Duchesne ex Rozier is one of the most important economic fruit crops worldwide (Simpson 2018). Pre-and postharvest fungal diseases caused by various pathogens have a great impact on strawberry production, decreasing subsequent fruit yield and quality (Maas 1998;Koike et al. 2009;Xu et al. 2015; Baroncelli et al. 2015;Abdelfattah et al. 2016). Among those pathogenic fungi, three of the destructive species namely Gnomoniopsis fructicola, Paragnomonia fragariae, and Phomopsis obscurans are members of the order Diaporthales (Sordariomycetes, Ascomycota).
Among various plant pathogens, Phomopsis obscurans is known to cause leaf blight and fruit rot in most of the strawberry growing regions of the world (Plakidas 1964;Sutton 1965;Eshenaur and Milholland 1989;Maas 1998;Ellis et al. 2000;Udayanga et al. 2011). Due to the implementation of one name for pleomorphic fungi, all species formerly known as Phomopsis and phylogenetically congeneric should now be placed in the genus Diaporthe (Udayanga et al. 2012(Udayanga et al. , 2014a(Udayanga et al. , 2014bRossman et al. 2014;Gomes et al. 2013;Fan et al. 2018). However, the generic placement of the strawberry leaf blight fungus has always been subject to uncertainty. Ellis and Everhart (1894) formally described the species causing leaf blight of Fragaria as Phoma obscurans based on a collection from West Virginia (USA). The fungus was reported from various regions of North America in subsequent studies. A severe outbreak of leaf blight was reported from Indiana in 1919 (Anderson 1920;Plakidas 1964). The causal agent of this outbreak was identified by Anderson (1920) as Dendrophoma obscurans. Sutton (1965) revisited the concept of Dendrophoma and suggested D. obscurans was not congeneric with the type species, D. cytisporoides. The type species of Dendrophoma, D. cytosporoides, belongs to the family Chaetosphaeriaceae (Chaetosphaeriales) based on available molecular data (Crous et al. 2012a). Phoma obscurans has been also known as Sphaeropsis obscurans and Phyllosticta obscurans in taxonomic literature (Kuntze 1898;Tassi 1902). However, the taxonomic affinity of P. obscurans to either Sphaeropsis or Phyllostica was unknown. Based on comparisons with representative Phomopsis species, the name Phomopsis obscurans was proposed for the leaf blight fungus, by Sutton (1965).
In 1916, Sphaeronaemella fragariae was reported to be the causal agent of "Sphaeronaemella" rot in strawberry (Stevens and Peterson 1916;Maas 1998). This species was not accepted in the mycoparasitic genus Sphaeronaemella by Malloch (1974), as a sexual morph was not known (Stevens and Peterson 1916). Hausner and Reid (2004) utilized nuc 18S rDNA sequence data of the ex-syntype isolate (CBS 118.16) of S. fragariae and confirmed it did not group with the type species of Sphaeronaemella, S. helvellae. Therefore, they considered it to be a synonym of Phomopsis obscurans in the Diaporthales. In the study by Senanayake et al. (2017a), the name Microascospora fragariae was proposed, based on S. fragariae and unauthenticated ITS sequences from an unpublished study. However, the name Phoma obscurans has since been found to be the oldest name for this fungus.
Similarly, confusion has existed among Gnomonia-like species associated with strawberry (Sogonov et al. 2008;Walker et al. 2010). The name Gnomonia comari is commonly used in older literature to refer to the fungus causing leaf blotch and fruit rot of strawberry. However, Sogonov et al. (2008) expanded the concept of Gnomoniopsis (Gnomoniaceae) to include G. comari as Gnomoniopsis comari. That same study revealed G. comari to be distinct from the causal agent of leaf blotch and petiole blight of strawberry in Europe and North America, known as Gnomoniopsis fructicola. Therefore, G. comari is now considered to be associated exclusively with Comarum palustre and not as a pathogen of strawberry.
The third diaporthalean fungus associated with strawberry, Paragnomonia fragariae, is known to cause petiole blight and root rot of perennial strawberry in Northern Europe and has been shown to be not congeneric with Gnomonia (Gnomoniaceae) based on molecular data (Moročko and Fatehi 2007). Morphologically, this species is similar to gnomoniaceous taxa with an apparently limited distribution in Europe and no known asexual morph (Moročko 2006;Moročko and Fatehi 2007). Recently, Moročko-Bičevska et al. (2019) lectotypified it based on illustrations from the original description, providing taxonomic and nomenclatural clarifications, and designating an epitype specimen from Latvia, The aims of this study were to infer the evolutionary relationships and revise the taxonomy of diaporthalean fungi associated with strawberry utilizing fresh collections, ex-type isolates and preserved fungal specimens from herbaria. An updated multilocus phylogeny of the order diaporthales, including fungal isolates from strawberry and modern taxonomic descriptions and illustrations are provided for the fungi reassessed in this study.

Sample sources and morphology
Strains of pathogenic fungi causing leaf blight of strawberry (Phomopsis obscurans) were obtained from a conventionally managed, matted-row production system at a private farm in Germantown, MD, USA (Black et al. 2002). In addition, samples were collected from two locations at the Beltsville Agriculture Research Center (USDA-ARS) in Beltsville, MD, where neither fumigants nor fungicides had been used: the Student Discovery Garden and yield-trial plots for the strawberry breeding program (Lewers et al. 2019). Pure cultures of the pathogens were isolated by single spore isolation (Udayanga et al. 2012) from leaf specimens with typical mature disease symptoms. Other fresh specimens and pure cultures were obtained from culture collections and various contributors (Table 1). Holotype and other specimens were obtained from the United States National Fungus Collections (BPI) and other herbaria.
Morphological descriptions were based on pycnidia or perithecia formed on inoculated alfalfa stems placed on 2% water agar (WA), as well as from type specimens. Digital images of fruiting bodies were captured using a Discovery V20 stereomicroscope and AxioCam HrC digital camera (Carl Zeiss Microscopy, Thornwood, New York, USA) imaging system. Whenever possible, 20-30 measurements were made of the structures mounted in 5% KOH using a Carl Zeiss Axioplan2 compound light microscope. The sample sizes are given in parentheses with mean and standard deviation. Triplicates of the cultures for each isolate were used for determining colony characters on Potato Dextrose Agar (PDA), Malt Extract Agar (MEA, Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and V8 juice agar (V8A) (Dhingra and Sinclair 1985) at 25°C in indoor light. After 1 wk., and color of the colonies were recorded. The colony color codes are given within the parenthesis according to the charts by Rayner (1970). For determination of growth rates, triplicate PDA plates were inoculated with 5 mm in diam plugs of actively growing fungal cultures. Mycelial growth was measured daily along two perpendicular lines drawn at the center of the colonies and continued for two weeks. Radial growth rates were calculated and expressed in mm day − 1 . Digital images were captured and cultural characteristics were observed as described in Udayanga et al. (2014aUdayanga et al. ( , 2014b.

DNA extraction, PCR and sequencing
Mycelial scrapings (50-60 mg) from the leading edge of cultures on PDA, incubated for 4-5 d at 25°C were harvested and lysed in tubes containing 500 μm garnet media and a 6mm zirconium bead (OPS Diagnostics, Lebanon, New Jersey) with the Fast Prep FP120 benchtop bead-beating instrument (Thermo Fischer Scientific Inc., Waltham, Massachusetts) for 60 s (20 s × 3 with 10 s intervals). Genomic DNA was extracted with the DNeasy Plant Mini Kit (Qiagen, Inc., Valencia, California) according to the manufacturer's instructions. DNA was eluted from DNAeasy mini spin column using 50 μl of elution buffer and visualized with agarose gel electrophoresis in 1% agarose gels stained with SYBR Safe DNA Gel Stain (Invitrogen, Eugene, Oregon).
Isolates were selected to represent each of the 31 known families in the Diaporthales based on the latest available literature. Each taxon selected was represented by at least an LSU sequence. In addition to fungal isolates from Fragaria, several new sequences were generated for representative taxa of the order: Phylogenetic reconstructions of concatenated and individual gene regions were performed using Maximum Likelihood (ML) and Bayesian Inference (BI) (Felsenstein 1981;). Individual datasets were tested for congruency using the 70% reciprocal bootstrap (BS) threshold method as described by Gueidan et al. (2007). ML gene trees were estimated using the software RAxML 8.2.8 Black Box (Stamatakis 2006;Stamatakis et al. 2008) in the CIPRES Science Gateway platform (Miller et al. 2010). For the concatenated dataset, all free-modal parameters were estimated by RAxML with an ML estimate of 25 per site rate categories. The concatenated dataset was partitioned by locus and the gaps were treated as missing data. The RAxML analysis utilized the GTRCAT model of nucleotide substitution with the additional options of modeling rate heterogeneity (G) and proportion invariable sites (I).
Bayesian analysis was performed using MrBayes v. 3.1.2  and substitution models were determined in MrModeltest v. 2.3 (Nylander 2004). Bayesian reconstructions were performed using MrBayes 3.1.2. Six simultaneous Markov chains were run for 1,000,000 generations and trees were sampled every 100 generations, resulting in 10,000 total trees. The first 25% of the trees, representing the burn-in phase of the analyses, were discarded, and the remaining trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree. Trees were visualized in

Phylogenetic analyses, limits and boundaries of genera and families
In total, 60 new DNA sequences were generated in this study. The approximate sizes of the target fragments of ITS, LSU, RPB2 and TEF1 were observed to be 600 bp, 1200 bp, 1000 bp and 650 bp, respectively. The remaining sequences were downloaded from GenBank (Table 1). Each gene region was aligned individually before concatenation in a sequence alignment consisting of 103 taxa representing 48 genera in 31 families of Diaporthales, including the isolates of fungi associated with Fragaria obtained in this study. The final combined four gene alignment consisted of 3899 total characters including gaps. Each taxon is represented by at least the LSU sequence. The ML tree resulting from the RAxML analysis had a final ML Optimization Likelihood of − 61, 871.952114 and the following model parameters: alpha = 0.344711, pi(A) = 0.239113, pi(C) = 0.263167, pi(G) = 0.271106, and pi(T) = 0.226613. This tree was used to represent the phylogeny of the order Diaporthales (Fig. 1).
The phylogeny inferred from the combined analysis of four loci resolved deeper nodes where confusion has remained at familial and generic boundaries when using only LSU data or other single gene regions (trees available at https://doi.org/10.15482/USDA.ADC/1518737). Major monophyletic groups representing families and genera were resolved with well-supported branches. Both BI and ML trees resolved the 31 families and 48 genera including the new genus described herein.
Multilocus phylogeny generated in this study placed the Fragaria isolates in the Melanconiellaceae, Sydowiellaceae and Gnomoniaceae. Based on the combined analysis, we determined that the isolates from strawberry causing leaf blight known to date as Phomopsis obscurans, are distinct from their closest relatives classified in Melanconiella, Microascospora or Greeneria. Therefore, a new genus Paraphomopsis is described below to accommodate the species formerly known as Phomopsis obscurans. The combined analysis further revealed that Paraphomopsis obscurans appears to be a sister taxon to Microascospora rubi, the type species of Microascospora. However, in the LSU and TEF1 single gene analyses, Microascospora rubi and Paraphomopsis obscurans were found to be non-monophyletic, and they were diverged based on ITS and RPB2 single gene trees. The ML bootstrap and BPP values for the node that groups Microascospora rubi and Paraphomopsis obscurans in the combined analysis were 65% and 0.68, respectively (≤90%/0.90, not shown in Fig. 1). Therefore, the taxa were not considered to be congeneric based on combined phylogeny. The three representative ITS sequences (HM854850, HM854852, HM854849), used by Senanayake et al. (2017a) to propose the name Microascopora fragariae (synonymized under Paraphomopsis obscurans below) were not included in the analyses due to lack of LSU sequences for those isolates. However, the ITS sequences for these isolates were 100% identical with the isolates of Paraphomopsis obscurans generated for this study.
The leaf blotch pathogen of strawberry, Gnomoniopsis fructicola, is currently placed in Gnomoniaceae with new molecular data from multiple isolates. The genus Gnomoniopsis (including syn. Sirococcus) represents a basal lineage to the rest of the genera in Gnomoniaceae, which contains the genera Gnomonia, Plagiostoma, Cryptodiaporthe, Apiognomonia, Discula, Cryptosporella, Ophiognomonia and Anisogramma. However, to preserve the historical concept of the widely accepted family Gnomoniaceae, which includes the major non-stromatic lineages in the Diaporthales, it is considered as a diverse single taxonomic entity with the assumption that intermediate genera in this family remain to be discovered. The closest family Melanconidaceae is clearly distinct from Gnomoniaceae. The new sequences generated for the fresh collection of the petiole blight and root rot pathogen, placed it within the Sydowiellaceae and conspecific with the recently designated epitype of Paragnomonia fragariae (F129/P3/1) with high ML bootstrap and BPP.

Taxonomy
Based on the molecular phylogenetic assessment of the order we introduce a new genus and combination to accommodate the strawberry leaf blight fungus, with lectoand epitypification of the taxon. A new combination is introduced for Gnomoniopsis fructicola, with lecto-and epitypification providing a revision of synonyms. The remaining strawberry isolates collected in this study belonged to Paragnomonia fragrariae, for which we provide a description based on fresh collections from France.
Notes: Paraphomopsis can be distinguished from its closely related genera (Greeneria, Melanconiella, Microascopsora) in Melanconiellaceae based on both molecular phylogeny and morphology. The genus Paraphomopsis is morphologically described herein, exclusively based on the characters of the asexual morph. The asexual morph of Melanconiella usually consists of both dark brown melanconium-like conidia as well as hyaline discosporinalike conidia (Voglmayr et al. 2012). Similarly, the genus Greeneria, which is typified by G. uvicola, forms pale brown conidia, smooth, variously shaped ranging from fusiform, oval, to ellipsoidal, each with a truncate base and obtuse to bluntly pointed apex (Farr et al. 2001). In Paraphomopsis, although the appearance of conidia is superficially similar to Diaporthe (syn. Phomopsis), micropscopic examination revealed that the shape and overall appearance are distinct from those in Diaporthe species. In general, conidia of Paraphomopsis are fusiform with minute guttules toward the end of the conidia, whereas most Diaporthe species form ovate to clavate conidia with no or prominent biguttulate or multiguttulate conidia. The morphology of sexual morph of the new genus described here remains unknown and is not available for comparison with other closely related genera. Although, the genus Paraphomopsis represents a sister clade to Microascopora in the phylogeny presented (Fig. 1), the asexual morph of the latter remains undetermined. The sexual morph of Microascospora distinct from other genera in the same family having immersed, solitary ascomata with narrow papilla with smaller hyaline, aseptate ascospores bearing long appendages (Senanayake et al. 2017a(Senanayake et al. , 2017b. However, the sexual morph of the saprobic genus Melanconiella is identified by its inconspicuous ectostroma projecting above the substrate and the hyaline, yellow or brown ascopsores, with or without short, blunt appendages and occasionally with a thin gelatinous sheath (Voglmayr et al. 2012;Senanayake et al. 2017aSenanayake et al. , 2017b. Paraphomopsis obscurans (Ellis & Everh.) Udayanga & Castl. comb. nov. Fig. 2. (See figure on previous page.) Fig. 1 ML tree generated based on combined LSU, ITS, RPB2, and TEF1 alignment of representative taxa in the order Diaporthales. Isolates from Fragaria are indicated in red. Ex-type/epitype isolates are in bold and marked with asterisk (*). The ML bootstrap values/Bayesian PP greater than 90% /0.90 are indicated above or below the respective branches. The tree is rooted with Pyricularia grisea (M83) and Ceratosphaeria aquatica (MFLU 18-2323) (Magnaporthaceae, Magnaporthales)  Description: Pycnidia on alfalfa stems on WA: globose, ostiolate, scattered over the substrate, 40-55 μm diam, embedded in tissue, erumpent at maturity, with a slightly elongated, black neck 60-100 μm high, wider towards the apex at maturity, often with a yellowish, conidial cirrus extruding from ostiole; walls parenchymatous, consisting of 3-4 layers of medium brown textura angularis. Conidiophores hyaline, smooth, branched, ampulliform, long, slender, wider at the base, 9-12 μm long and wide. Conidiogenous cells phialidic, cylindrical, terminal, slightly tapering towards apex, 1.5-2.5 μm diam at the widest point. Collarette present and conspicuous. Paraphyses absent. Alpha conidia 5-7 × 1.5-2.2 μm (avg. ± SD = 6 ± 0.5 × 2 ± 0.2, n = 30), abundant in culture and on alfalfa stems, aseptate, hyaline, smooth, ellipsoidal to fusiform, often biguttulate and rarely multiple guttules and confined to minute particles clumped towards the vertices of the spore, base subtruncate. Beta conidia unknown.
Notes: Although the appearance of conidia is superficially similar to Phomopsis (Syn. Diaporthe), microscopic examination revealed that the shape and overall appearance of guttules are distinct from those in Diaporthe species. In general, conidia of Paraphomopsis obscurans are fusiform with minute guttules toward the end of the conidia, whereas most Diaporthe species bear ovate to clavate conidia with no or prominent biguttulate or multiguttulate conidia. Paraphomopsis obscurans can be distinguished from the closely related species Microascospora rubi and other genera in the family Melanconiellaceae based on its morphology and robust support of the multilocus phylogeny. Due to confusion of nomenclature and taxonomy, previous records of the pathogen from various geographic locations were linked to multiple names: Phoma obscurans, Sphaeronaemella fragariae and Phomopsis obscurans, or misidentified as Gnomonia fragariae, Gnomonia comari and Gnomoniopsis fragariae. Therefore, the actual distribution of the fungus may be largely underestimated.
Notes: The name of the leaf blotch fungus was documented in phytopathological literature as Gnomonia comari (syn. Gnomoniopsis comari) before Sogonov et al. (2008) identified it as Gnomoniopsis fructicola. However, the earlier name Zythia fragariae (1908) represents the oldest name for this taxon as the asexual state of G. fructicola (Fall 1951). Although Arnaud (1931) identified the asexual state as a Gloeosporium sp., Fall (1951) mentions it as identical to Z. fragariae. Attempts to find type material for Zythia fragariae in European herbaria were unsuccessful. Therefore, the illustration available from the protologue is designated as a lectotype herein with a modern epitype designated. Microscopic observation of the isotype specimens of Phyllosticta grandimaculans housed in BPI, S and CUP and comparison of symptoms revealed that this species is conspecific with Gnomoniopsis fragariae. This pathogen appears to occur both in Europe and North America and is commonly associated with cultivated and wild species and varieties of Fragaria (Bolton 1954;van Adrichem and Bosher 1958;Maas 1998 Description: Perithecia on crown and petioles of Fragaria, non stromatal, black, globose, arranged in immersed clusters on the base of the crown or solitary on petioles of the infected plants, 200-300 μm diam, bearing tapering black perithecial necks protruding from infected tissue 130-150 × 20-25 μm. Asci 50-60 × 8-10 (avg. ± SD = 56 ± 4 × 9 ± 1) μm unitunicate, 8-spored, sessile on defined hymenium or freely arranged with aging, elongate to clavate with conspicuous refractive ring at the terminals. Ascospores 14-17 × 3.5-5 (avg. ± SD = 16 ± 1.3 × 4 ± 0.4) μm, hyaline, fusiform to ellipsoid, straight to slightly curved, one septate or bicellular, with a conspicuous septum, slightly constricted at the septum, often 4-guttulate, two mucilaginous appendages present at the either ends of the ascospores. Asexual morph not seen in culture.
Culture on PDA under artificial light at 25°C for 1 wk., growth rate: 2.8 ± 0.2 mm/day (n = 3) white, with sparse aerial mycelium, with irregular margins, rhizoid form of growth and in center and at edges with grayish yellow (57) pigmentation with age, dull green (70) pigmentation developing in reverse.
Geographic distribution: Confirmed distribution in Germany: Hamburg (Klebahn 1918), Switzerland:Vaud, Les Barges, Valais, Tessin (Bolay 1971Monod 1983), United Kingdom, Latvia (all across the country), Sweden:Uppsala, Vastra (Moročko 2006;Moročko 2006), Lithuania:Kaunas, Siauliai and Finland: Parainen (Moročko-Bičevska et al. 2019), France (in this study). Notes: The holotype specimen of Gnomonia fragariae was not available in Klebahn's collection in BREM (pers. comm. Michael Stiller). The specimen (IMI 100647) linked to CBS 146.64 housed in K consists of a dry culture and slides, which may not contain conspicuous fungal structures to observe (pers. comm. Angela Bond & Paul Cannon); however, molecular data are available. Senanayake et al. (2017b) described the taxon without typifications and clarifications of affiliated names or specimens. Therefore, Moročko-Bičevska et al. (2019) designated original drawings by Klebahn (1918) specified in his original publication as a lectotype of G. fragariae and a freshly collected specimen from Latvia as an epitype, based on its morphology on the host and in culture.

DISCUSSION
Post-and pre-harvest fungal diseases of strawberry cause significant annual losses to strawberry production (Maas 1998;Garrido et al. 2011). Phytopathogenic fungi are able to infect each and every part of the strawberry plant including leaves, petioles, fruits, sepals, stolon, crown and root systems at any age of the growth (Garrido et al. 2016). Although the fungal genera Botrytis, Colletotrichum, Fusarium and Verticillium causes major diseases of strawberry, several other pathogens also have significant impact on annual production (Leroch et al. 2013;Baroncelli et al. 2015). Species in the order Diaporthales also have been generally associated with strawberry diseases, although much confusion exists regarding the taxonomy, nomenclature, and evolutionary relationships of the taxa (Maas 1998;Garrido et al. 2016). In this study, evolutionary relationships of the leaf blight and leaf blotch pathogens widely known from North American strawberry fields and other strawberry growing regions in the world were revisited. Fresh collections of diseased specimens, pure cultures and multilocus phylogenetic analysis were used to resolve taxonomic problems. Type and other historic specimens from herbaria were observed and compared with fresh fungal collections to provide comprehensive nomenclatural clarification. Leaf blight of strawberry was initially identified by characteristic large V-shaped necrotic lesions along major veins bearing black protruding necks of the pycnidia when examined under the stereo microscope (Fig. 2). Although the fungus infects leaves early in the growing season, leaf blight symptoms are more common on older leaves near or during harvest. The pathogen can weaken the plants through the destruction of older foliage and can also infect runner stems, calyxes, and fruits in some varieties (Maas 1998). The leaf blotch fungus, Gnomoniopsis fragariae is characterized by purplish to brown blotches and in later stages by large necrotic spots with abundant conidiomata around the major veins of the leaf (Fig. 3). The spots often occur on the end of a leaflet and are rounded to wedge shaped. This fungus can be found on the petiole, calyx, fruit stalk, and fruit. New collections of petiole blight and root rot pathogens were found in France occurring on stalks of perennial Fragaria sp. The symptoms often are confused with the early stages of leaf scorch caused by Diplocarpon fragariae (Helotiales) and leaf blotch caused by G. fragariae. Weakened plants may overwinter, which can result in reduced yields in the following season in commercial cultivations. Under conditions highly favorable for disease development, leaf blight can cause severe defoliation leading to plant death. Leaf blight fungus is often listed as a leading threat to strawberry and commonly co-occurs with other pathogens causing leaf blotch, leaf scorch and numerous leaf spots (Maas 1998). Close inspection of symptoms of various fresh specimens and historical collections housed in the U. S. National Fungus Collections revealed that it is possible to distinguish these taxa based on symptomology as well as microscopic examination of the fungal structures when present.
One additional species associated with strawberry included in the analysis is Ophiogonomia rosae (Gnomoniaceae) as identified by Walker et al. (2012) and represented by isolate CBS 128442 isolated from Fragaria vesca (Fig. 1). The same study reported the occurrence of O. rosae on overwintered leaves of F. vesca, Comarum palustre, Rosa sp., and Rubus sp. (Rosaceae) from various geographic regions of the world. Pathogenicity on these hosts is unknown, but it is likely O. rosae either possesses a saprobic lifestyle or is perhaps a weakly opportunistic pathogen. No specific reports of it as a pathogen of strawberry are known to exist and symptomology remains unknown.
The family composition of the order Diaporthales has changed with various classification systems originally based on morphology and later based on phylogenetic analyses (Wehmeyer 1975;Barr 1978;Castlebury et al. 2002), with Diaporthaceae, Gnomoniaceae, Valsaceae, Melanconidaceae and Pseudovalsaceae as the earliest defined families based on morphological characters (Wehmeyer 1975;Barr 1978;Vasilyeva 1987;Castlebury et al. 2002;Gryzenhout et al. 2006;Cheewangkoon et al. 2010;Crous et al. 2015). We confirmed that the Melanconiellaceae, which is broadly defined in this study, is a well-resolved family distinct from other closely related families. However, it is widely known that Melanconium-like taxa are polyphyletic and scattered throughout the order, and therefore need to be redefined with reference to the placement of the type species. The genus Melanconiella was considered as Diaporthales incertae sedis until recently and placed in Sphaeriales in early classifications (Clements and Shear 1931). It is now classified within Melanconiellaceae with numerous other species (Voglmayr et al. 2012;Du et al. 2017). Melanconiella species were known to be associated with the host family Betulaceae, including Betula, Carpinus, Corylus and Ostrya, and considered to be highly host specific. Du et al. (2017) described M. cornuta associated with canker and dieback of Cornus controversa (Cornaceae) and Juglans regia (Juglandaceae) from China. Greeneria uvicola causes bitter rot and necrotic fleck of grapes (Vitis spp., Vitaceae) in North America, Australia and elsewhere in the world and often misidentified and is often confused with other common diaporthalean pathogens on grapevines including Diaporthe ampelina (Diaporthaceae) (Farr et al. 2001;Steel et al. 2007;Longland and Sutton 2008). Microascospora rubi is associated with Rubus ulmifolia from Italy but appears to be a saprobe (Senanayake et al. 2017a). However, the generic delimitation and species diversity within the family Melanconiellaceae are yet to be resolved with more collections and molecular data of closely related taxa.
Early morphology-based classification systems placed species that occur singly within the substrate without any stromatic development in the family Gnomoniaceae (Wehmeyer 1975;Barr 1978;Monod 1983). However, molecular data and large-scale sampling of taxa have revealed that gnomoniaceous taxa sensu Wehmeyer (1975) and Monod (1983) are polyphyletic. Improvements in phylogenetic understanding have ultimately resulted in a more natural classification, leading to better insights into the evolutionary history of the Diaporthales and other Sordariomycetes (Zhang et al. 2006;Hongsanan et al. 2017;Guterres et al. 2019). These methods have also led to improvements of the understanding of the seemingly minor morphological differences of the sexual morphs of these ascomycete genera for identification purposes. Therefore, finding and utilizing phylogenetically informative genes are critical to obtain compelling, yet previously unrecognized, data to develop new evolutionarily significant insights and to encourage innovative practices in modern fungal systematics.
Due to the morphological plasticity of both asexual and sexual morphs, confusion has remained in generic and family-level classifications of many diaporthalean fungi. Phylogenetic analyses based on single gene trees have been often problematic. The conventionally used nuc 28S rDNA roughly distinguished taxa at generic and family levels, but several genera and families were poorly supported or otherwise not distinguished. Single morphological characters previously used to segregate genera or families in ascomycetes have often been found to be discordant with multilocus phylogenies and phylogenomic analyses (Choi and Kim 2017;Yang et al. 2018;Voglmayr et al. 2019b).
The best approach for developing knowledge about species in this diverse group of plant-associated fungi is through a consolidated platform utilizing morphological data, multigene phylogeny, as well as host associations and historical background information connected to voucher specimens in herbaria. For instance, correct identification of Paraphomopsis obscurans required the time-consuming process of sifting through the complicated historical literature of various genera within Diaporthales as well as unrelated genera and observation of numerous specimens. From this historical research, it was evident that previous authors observed morphological and physiological distinctions from other genera including Dendrophoma, Diaporthe, Phoma, Phyllostica, Sphaeronaemella, and Zythia. As the taxonomic opinions were based on the observation of the vouchered specimens, it was possible to reassess these opinions based on the same or other authentic specimens. To this end, a consolidated approach of multilocus phylogenetic analyses and morphological observations will provide the best resolution for taxonomists, evolutionary biologists, plant pathologists, and quarantine officials in their efforts to address issues regarding accurate identification, host plant associations and interactions, and disease management.

CONCLUSIONS
Molecular phylogeny based on newly generated DNA sequences of diaporthalean fungi associated with strawberry diseases revealed that the leaf blight pathogen represents a new evolutionary lineage within the family Melanconiellaceae, distinct from closely related taxa. The combined phylogeny based on four loci (ITS, LSU, RPB2, and TEF1) together with morphological data illustrate the generic and family-level relationships in this diverse order of fungi. Although, leaf blight, leaf blotch, petiole blight and root rot fungi of strawberry are frequently encountered, the taxonomy, accurate naming and geographic distribution were largely overlooked until recently. Therefore, this study highlights the need for revisiting poorly known genera of phytopathogenic diaporthalean fungi in order to establish their evolutionary relationships and provide reference DNA sequences for accurate identification purposes. Week names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture or any of the other coauthors' institutions.
Adherence to national and international regulations Not applicable.
Authors' contributions DU and LAC designed the research. DU performed the experiments. DU, DSM, SDM performed data analysis. LAC, KL and AG contributed with specimens and/or funds for research. All authors contributed to data interpretation and manuscript writing. All authors read and approved the final manuscript.

Funding
This project was funded by USDA-ARS Projects 8042-21220-257-00-D and 8042-22000-298-00-D and was supported in part by the appointment of Dhanushka Udayanga to the U.S. Department of Agriculture (USDA) Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and USDA (contract number DE579AC05-06OR23100).

Availability of data and materials
The datasets generated and analysed during the current study are available in the Ag Data Commons, U.S. Department of Agriculture https://doi.org/10. 15482/USDA.ADC/1518737