Systematics, ecology, and application of Helotiales: Recent progress and future perspectives for research with special emphasis on activities within JapanHelotiales: Recent progress and future perspectives for research with special emphasis on activities within Japan

Abstract

Helotiales is one of the most diverse groups of apothecial ascomycetes, including 3000—4000 taxa. Recent progress in the systematics, ecology, and their applications through research is herein reviewed based on the experiences of the author with a special emphasis on activities in Japan. In the past 30 y, more than 50 helotialean taxa have been added to the mycobiota of Japan, including new taxa. With the advent of molecular phylogeny, some families have been revisited, such as members with stroma (Sclerotiniaceae and Rutstroemiaceae) or hairs (Hyaloscyphaceae and Lachnaceae). Although the monophyly of Helotiales has not yet been demonstrated, our understanding of its phylogeny has greatly advanced. The unexpected ecological nature represented by endophytism has been revealed through barcoding and other molecular techniques. The research history of ash dieback is also reviewed, and the endophytism/saprophytism of the pathogen on its original host is discussed. Drug discoveries within Helotiales are reviewed, and successful examples are presented. As future perspectives, both the cumulation of occurrence and sequence data of Helotiales is greatly encouraged to elucidate this important group of fungi.

1. Introduction

Among the apothecial ascomycetes with inoperculate asci, Helotiales is one of the most diverse groups. It is known to include some 3000-4000 taxa (Kirk, Cannon, Minter, & Stalpers, 2008; Baral, 2016). Most members of the Helotiales have minute apothecia, usually less than 2 mm diam. They may be sessile or stipitate, dark to bright colored, and superficial or erumpent through the plant host. The overall shape of the apothecia is cupulate-discoid, turbinate funnel-shaped, or clavate (Korf, 1973). Most are known to be saprophytic, living on fallen leaves and decaying wood, but some are parasitic to pathogenic or symbiotic with other organisms. Many members are relatively easy to isolate, but the inducing the formation of apothecia in vitro is not easy (Müller & Loeffler, 1976). Though not frequent, some members produce an asexual state, but many remain unassociated with this state. Despite these biological diversities, taxonomy and ecological studies are insufficient.

From the viewpoint of applied science, in particular discovery research, Helotiales are an attractive biological resource for the following reasons. 1) They are in different ecological groups than soil fungi. 2) Most are easily accessible and culturable. 3) They have a high level of species diversity, which may be reflected in their metabolic diversity, but relatively underexplored not only taxonomically but also in applied science. 4) They are also thought to be ecologically diverse, suggesting diverse metabolites. In short, Helotiales are underutilized but easily accessible fungi (Hosoya, 1998).

I have been working on this attractive group of fungi for more than 30 y, first as a researcher in the private sector in search of novel biological resources for metabolites, and then in a more pure science-directed position. Through my career, I have made many new discoveries and contributions to the field of systematics and discovery research. In the present article, I review recent advances and future perspectives for research on this attractive group of fungi.

2. Systematics

2.1. Infrastructure for systematics

Collecting specimens is a fundamental of systematics. In the “ in situ” activity in the National Museum of Nature and Science, the following process is usually applied. Once the specimens are collected, single spore isolation using Skerman’s manipulator (Skerman, 1968) or multisporous isolation from discharged ascospores is attempted, then the specimens are heat dried or air dried and stored as voucher specimens in the fungarium. DNA is extracted from the isolates and stored in a freezer as a sample for future use. When isolation is not successful, part of the apothecia is kept in a buffer [20% DMSO, 100 mM Tris-HCl (pH8.0), 250 mM EDTA, 100 mM Na ₂SO ₃, NaCl to saturation; Hosaka & Castellano, 2008] for DNA extraction when necessary. If asexual reproduction is recognized in culture, the culture of isolates may also be dried and registered as specimens. Because our facility is not suitable for the maintenance of cultures, we have a close collaboration with culture collection facilities that have the capacity to store intriguing isolates from the specimens (Fig. 1).

Figure 1.

Diagrammatic representation showing the research process and management of voucher specimens.

Given the above process, four major collections (specimens, isolates, extracted DNA and tissue samples) were obtained and managed in-house ( http://db.kahaku.go.jp/webmuseum_en/; database opened for specimens only) or ex-house (e.g., NBRC, https://www.nite.go.jp/nbrc/catalogue/NBRCDispSearchServlet?lang=en; GBIF, Global Biodiversity Information Facility, https://www.gbif.org/) databases. These collections may be linked with sequence data on publicly accessible databases (INSDC, http://www.insdc.org/). All of these sites were visited on Feb 25, 2020. The occurrence data based on the specimens were also provided to GBIF for global use. Our systematic studies were based on these infrastructures.

2.2. Advances in the systematics of Helotiales and their studies in Japan

Korf (1973) published a key to the genera of Helotiales with taxonomic comments. This monumental publication covered a wide range of taxonomic concepts for apothecial fungi and has been used since the establishment of Helotiales by Nannfeldt (1932). In his taxonomy, Helotiales were divided into eight families (Ascocorticiaceae, Dermateaceae, Geoglossaceae, Hemiphacidiaceae, Hyaloscyphaceae, Leotiaceae, Orbiliaceae, and Sclerotiniaceae), whereas a more recent treatment (Baral, 2016) recognized greater taxonomic diversity in 25 families (Table 1). Reviewing recent taxonomic studies in Japan based on the classification by Korf (1973), Ascocorticiaceae had not yet been documented. Geoglossaceae has been almost completely unstudied since Imai (1934) and is now treated as an independent class apart from Helotiales (Schoch et al., 2009). Orbiliaceae has been almost unstudied with a few exceptions (Nagao, 1996), and it is now known as a separate group (Orbiliomycetes) from Helotiales and is recognized as a primitive member of the apothecial fungi (Eriksson, Baral, Currah, Hansen, & Kurtzman, 2003; James et al., 2006). Studies are largely limited to Dermateaceae, Hemiphacidiaceae, and Leotiaceae. In my previous studies, I worked with Hyaloscyphaceae and Sclerotiniaceae sensu lato, which are reviewed below.

Table 1.

Comparisons of recent taxonomy at the family level (Baral, 2016) with the previous concept (Korf, 1973).

Baral (2016)	Clade a	Korf, 1973 b
Cenangiaceae Rehm	A	Hemiphacidiaceae Korf
Rutstroemiaceae Holst-Jensen, L. M. Kohn & T. Schumacher	A	Sclerotiniaceae
Sclerotiniaceae Whetzel	A	Sclerotiniaceae
Ascocorticiaceae J. Schröt.	Au	Ascocorticiaceae
Ascodichaenaceae D. Hawksw. & Sherwood	Au	N/A
Chaetomellaceae Johnst. & Rossman	Au	Hyalocyphaceae
Chlorociboriaceae Baral & P. R. Johnst.	Au	Helotiaceae Rehm
Cordieritidaceae (Sacc.) Sacc.	Au	Helotiaceae
Dermateaceae Fr.	Au	Dermateaceae
Godroniaceae Baral	Au	Helotiaceae
Mitrulaceae Rchb.	Au	Geoglossaceae Corda
[Stamnaria Fuckel lineage]	Au	Leotiaceae Corda
Gelatinodiscaceae S. E. Carp.	B	N/A (Helotiaceae)
Helotiaceae Rehm s. l.	B	Helotiaceae
Roesleriaceae Y. J. Yao & Spooner	B	N/A
[Discinella-Pezoloma lineage]	B	Leotiaceae
Loramycetaceae Debbus ex Digby & Goos	Cm	N/A
Mollisiaceae Rehm	Cm	Dermateaceae
Vibrisseaceae Locq. ex Korf	Cm	(Ostropales Nannf.)
[Strossmayeria Schulzer lineage]	Cm	Leotiaceae
Calloriaceae Marchand	Cp	Dermateaceae
Drepanopezizaceae Bat. & H. Maia	Cp	Dermateaceae
Heterosphaeriaceae Rehm	Cp	Dermateaceae
Ploettnerulaceae Kirschst.	Cp	Dermateaceae
[Hysteropezizella Höhn. lineage]	Cp	Dermateaceae
Arachnopezizaceae Hosoya, J. G. Han & Baral	D	Hyalocyphaceae
Hyaloscyphaceae Nannf.	D	Hyalocyphaceae
Lachnaceae Raitiv.	D	Hyalocyphaceae
Pezizellaceae Velen.	D	N/A (Hyalocyphaceae)
[Bryoglossum L. Ludw., P. R. Johnst. & Steel lineage]	D	N/A

Correspondence between the two are based on the major genera included in the families, sorted on clade,families in Baral (2016) with generic lineage at the end, and Korf (1973).

^a Clades given by Baral (2016) who disposed each family in A-D clades. Au indicates an uncertain position in clade A. Cm represents Mollisiaceae sublineage in clade C, and Cp represents Ploettnerulaceae sublineage in clade C.

^b N/A is to indicate that the given type genus in the family was not dealt in Korf (1973), but suggested to be included in the families in the parenthesis based on the synonymy. Vibrissea was treated as a genus in the order Ostropales, and later it was disposed to newly proposed family (Vibrisseaceae in Korf, 1990).

2.3. Taxonomy of Hyaloscyphaceae

Hyaloscyphaceae is a large family (74 genera + 61 synonyms, 933 species., Kirk et al., 2008), composed of fungi with minute to small apothecia with various hairs. The generic taxonomy of these fungi was based on a combination of characters, including that of hairs, ectal excipular structure, and paraphyses. Korf (1973) subdivided the family into two subfamilies: Trichoscyphelloideae, including a single genus, Lachnellula , and subfamily Hyaloscyphoideae. The latter was subdivided into five tribes (Arachnopezizeae, Hyaloscypheae, Lachneae, Trichodisceae, and Trichopezizelleae). Most members of Hyaloscyphaceae belong to Arachnopezizeae, Hyaloscypheae, Lachneae, and Trichopezizelleae. A number of genera with similar morphology have been identified in Lachnum, one of the major genera in Lachneae, and even more genera have been proposed (e.g., Baral & Kiriegelsteiner, 1985). However, because morphological characters often show convergence, revision of generic taxonomy based on phylogeny is warranted.

Otani (1989) summarized the known occurrence in Japan and listed 41 taxa. However, we expected more undocumented members. In a series of studies, we contributed to the enumeration of undocumented taxa, including new species (Hosoya & Otani, 1997a, 1997b; Hosoya & Harada, 1999; Ono & Hosoya 2001; Tanaka & Hosoya, 2001; Tochihara & Hosoya, 2019).

Cantrell and Hanlin (1997) first conducted a phylogenetic analysis to assess the taxonomy of Hyaloscyphaceae using a single gene, ITS-5.8S. In their analysis, they clarified most of the genera in Lachneae, which formed a monophyletic group, but the phylogeny of higher clades was less supported. Hosoya et al. (2010c) conducted a multi-gene analysis using the combined data of ITS-5.8S, the D1-D2 region of LSU, and RPB2, to assess the taxonomic delimitation of the genera in Lachneae. Phylogenetic support of most genera was obtained, and genera separated from Lachnum were justified. Raitviir (2004) proposed raising the tribe Lachneae to family Lachnaceae based on suggested strong monophyly. However, whether the family should include members of Trichopezizelleae is still under debate (Hosoya et al., 2010b), and Ekanayaka et al. (2019) included it in their new family Solenopezizaceae.

In the continuation of these studies, Tochihara and Hosoya (2019) added three new species to the genus Incrucipulum, previously known to consist of a small number of species. Studies to clarify the generic limitations of these groups are still being conducted, but more data are required to clarify the generic limits to assess the type genus Lachnum.

On the other hand, the phylogenetic analysis of Hyaloscypheae revealed great phylogenetic diversity (Han, Hosoya, Sung, & Shin, 2014a). Although they used combined data of ITS-5.8S, the D1-D2 region of LSU, RPB2, and mtSSU, and obtained deeper clades to support the taxonomy of genera, clades at higher levels were not strongly supported, suggesting poly/paraphyly of the tribe. They hesitated to propose any taxonomic revision for the Hyaloscypheae because it was suggested that the tribe was divided into too many small groups of genera.

Han et al. (2014a) also showed that Arachnopezizeae was no longer acceptable as a monophyletic group, and proposed Arachnopezizaceae stat. nov., excluding the heterogeneous elements and clarified their phylogenetic differences from the rest of Hyaloscyphaceae.

2.4. Taxonomy of Lambertella and its allies in Sclerotiniaceae sensu lato

The family Sclerotiniaceae was proposed by Whetzel (1945) for members of Helotiales that produce stroma, which included two types, sclerotial stroma (sclerotia) forming “a more or less characteristic form and a strictly hyphal structure under the natural conditions of its development” and substratal stroma of “diffuse or indefinite form,” often recognized as a thin black layer on the surface of the substrate or lines in a vertical section. Both were supposed to be an overwintering structure to survive low temperatures. Members of these fungi have been primarily studied in Japan because of agricultural interests (e.g., Harada, 1977; Willetts & Harada, 1984).

Later, Holst-Jensen et al. (1997) illustrated the phylogenetic differences of members with substratal stroma from members with sclerotial stroma and proposed the family Rutstroemiaceae for members with substratal stroma.

The genus Lambertella, first known in Sclerotiniaceae and later transferred to Rutstroemiaceae, is chiefly characterized by substratal stroma and ascospores that turn brown when mature (Whetzel, 1943). Following Whetzel (1943), Dumont (1971) published a monograph of Lambertella in which 29 species were included, but more potential members were expected because browning of the ascospores sometimes occurred only after discharge and were overlooked in the dried specimens. In fact, we made transfers from other genera to Lambertella (Hosoya & Otani, 1997c) or justified previous transfers (Hosoya, Otani, & Furuya, 1993) based on the observation of fresh specimens. However, browning of the spores may be a convergent feature and molecular phylogenetic assessment is required.

Zhao et al. (2016) conducted a molecular phylogenetic analysis of Lambertella, incorporating other members of Rutstroemiaceae, Sclerotiniaceae, and Helotiaceae, based on LSU and RPB2, and demonstrated the following facts. 1) Sclerotiniaceae is monophyletic but is placed within the clade of Rutstroemiaceae. 2) Stromata have multiple origins, occurring both in Rutstroemiaceae and Helotiaceae. 3) Lambertella is a polyphyletic genus that requires taxonomic revision. Based on the common features observed in the strongly supported clade including the type species Lambertella corni-marisHöhn., they redefined the genus Lambertella with ectal excipulum composed of brown, rectangular cells, and ascospores becoming brown within the asci before discharge (Zhao, Hosaka, & Hosoya, 2016), in addition to the substratal stroma. Their exclusion resulted in several necessary transfers of current members of Lambertella to appropriate genera, including new genera that needed to be proposed, but these have been left untreated to avoid further confusion under the present unstable taxonomy. Their findings also provided a new concept regarding sclerotia as a special type of substratal stroma and the merging of Rutstroemiaceae into Sclerotiniaceae s. l.(Johnston et al., 2019).

2.5. Advances in helotialean mycobiota exploitation in Japan

As already described, exploration and enumeration of mycobiota are among the bases of our systematic studies. Otani (1989) enumerated the Japanese mycobiota of Discomycetes, a taxon no longer used but still practical in the modern taxonomy, and enumerated 77 genera with 214 species in the order. Through our exploration and enumeration, we added 57 newly documented taxa in Japan, and 30 new taxa, including 2 genera (Otani, Hosoya, & Furuya, 1991; Hosoya et al, 1993; Kubono & Hosoya, 1994; Hosoya & Otani 1995, 1997a, 1997b, 1997c; Hosoya & Harada 1999; Hosoya, 2000, 2002, 2004, 2005, 2009; Ono & Hosoya, 2001; Tanaka & Hosoya, 2001; Hosoya & Huhtinen 2002; Narumi-Saito, Hosoya, Sano, & Harada, 2006; Hosoya et al., 2010a, 2010c, 2011; Hosoya, Sung, Han, & Shin, 2010d; Han, Hosoya, & Shin, 2011; Hosoya et al., 2012; Hosoya, Hosaka, Saito, Degawa, & Suzuki, 2013a; Hosoya et al., 2013b; Hosoya, Saito, & Sasagawa, 2013c; Zhao, Hosoya, Shirouzu, Kakishima, & Yamaoka, 2013; Han et al., 2014a; Hosoya, Zhao, & Degawa, 2014; Zhao & Hosoya, 2014, 2015; Degawa et al., 2015; Gross, Hosoya, Zhao, & Baral, 2015; Hosoya & Zhao, 2016; Itagaki, Nakamura, & Hosoya, 2019; Tochihara & Hosoya, 2019). These included new species, new genera of both sexual and asexual states, now being merged under single names (some examples are presented in Fig. 2).

Figure 2. Representatives of Helotiales with taxonomic significance studied over the past 30 y in Japan. A–C: Gelatinipulvinella astraeicola. A monotypic genus with the yeast-like asexual state (B) with annellidic conidiation (C). It is a mycoparasite of Astraeus hygrometricus (Hosoya et al., 1995). D: Dasyscyphella longistipitata, a fungus confused with Lachnum virgineum for a long time in Japan. It is currently regarded as an endemic species occurring in the cupules of Fagus crenata, an endemic tree. Although the fungus is strongly connected with the distribution of F. crenata (Hosoya et al., 2010b), it does not show any population differentiation in contrast to its host. E: Venturiocistella japonica, occurring on Cercidiphyllum japonicum leaf. Its close phylogenetic relationship with Glutinomyces, a root endophytic genus, is indicated (Nakamura et al., 2018). F–I: Hyphodicus hyaloscyphoides, a new species with a chalara-like asexual state. Hyphodiscus are frequently detected in living plant roots. White apothecia on the substrate (F). Cross section of the apothecium, showing a typical structure of apothecium consisting of the stipe, excipulum, and hymenium composed of paraphyses and asci (G). Chalara-like conidiophores and conidia (H). Conidia are borne in chains or drops (I). J: Hymenoscyphus fraxineus, the pathogen of ash dieback. The photo was taken in a place where the first specimen of this fungus was collected (Sugadaira, Nagano Pref., Japan). Note that the blackened region of the host surface is the external view of the stroma. K: Poculum pseudosydowianum, originally recognized as an isolate of an unidentified endophyte, later connected with the specimens collected in the field, showing that barcoding data works (Hosoya et al., 2014). L: Lambertella corni-maris, the type of Lambertella, chiefly defined by having substratal stroma and spores that turn brown before germination. M: Arachnopeziza aurelia, the type species of its genus in the family Arachnopezizaceae. N: Mollisia sp. This genus may contain a number of potentially undescribed species and are scarcely distinguishable in morphology because of their resemblances. Bars: B, C, G, H 10 μm; I 50 μm.

2.6. Monophyly of order and familial taxonomy

With the increasing molecular phylogenetic data for a wide range of ascomycetes, it became possible to study large-scale phylogeny at the order or higher level. After the exclusion of heterogeneous elements, Helotiales became relatively homogeneous, but its monophyly was still not supported (Wang et al., 2006a; Wang, Johnston, Takamatsu, Spatafora, & Hibbett, 2006b) based on the combination of SSU, LSU, and 5.8S rDNA data. On the other hand, Johnston et al. (2019), using up to 15 concatenated genes, confirmed the monophyly of Leotiomycetes, the higher taxa that includes Helotiales, but also demonstrated the paraphyly of the order. However, they also demonstrated a strong phylogenomic structure within the order when genome information was used. Apparently, more informative data are required to determine the entire taxonomic structure of Helotiales, whereas ambitious informal proposals to separate some members in other orders (such as “Sclerotiniales”) have been made (Ekanayaka et al., 2019).

3. Ecology

3.1 Expanding realm for roles of Helotiales in ecosystems

Although some members have been known as plant pathogens (e.g., Sclerotinia spp.) or symbiotic partners of plants (e.g., Rhizoscyphus ericae(D.J. Read) W.Y. Zhuang & Korf), most of the members of Helotiales were considered weak saprophytes in the past (Alexopoulos, Mims, & Blackwell, 1996). The advent of molecular techniques in ecological studies made meta-genomic analysis possible (e.g., Toju et al., 2013b). As a result, more environmental DNA data have accumulated, and a wider range of occurrence of Helotiales has been suggested in the environment. In recent years, two topics have been remarkable: their role as endophytes and as a virulent invasive pathogen, Hymenoscyphus fraxineus, causing ash dieback.

3.2 Root endophytes

In contrast to mycorrhizae, which have a long research history (e.g., Peterson, Massicotte, & Melville, 2004), endophytes have a relatively shorter history in research (e.g., Stone, Polishook, & White, 2004). Regarding Helotiales (Wang et al., 2006b; Sieber, 2007), root endophytes are among the topics of a more recent focus (Vandenkoornhuyse, Baldauf, Leyval, Straczek, & Young, 2002). Toju et al. (2013b) recognized the frequent occurrence of Helotiales in plant roots based on a metagenomic analysis. Our study (Hosoya, Hosaka, & Nam, 2017) on Fagus crenata, an endemic tree species in Japan, also suggested the occurrence of Helotiales in F. crenata roots, while the majority of endophytes from leaves belonged to Dothideales and Diaporthales. Currently, more researchers are paying attention to Helotiales as root endophytes. The connection between Phialocephala, a frequently occurring genus of dark septate endophytes (DSE), and its mollisioid sexual state is well known (Tanney, Bouglas, & Seifert, 2016). Rhizoscyphus ericae, which shows apparent symbiosis with plants, has recently been shown to be congeneric with Hyaloscypha (Fehrer, Réblová, Bambasová, & Vohník, 2019; but see Baral & Kriegelsteiner, 2006).

Our preliminary studies showed that the majority of endophytes from F. crenata and Quercus crispula belonged to Dermateaceae s.l. and Hyaloscyphaceae s.l. (unpublished data). Thus, the above reports seem reasonable. In fact, Bizabani and Dames (2015) showed that an isolate of Lachnum from Vaccinium roots, identified by molecular evidence, had plant growth promotion effects when re-inoculated. In most cases, identification of root endophytes is based on molecular evidence because most of the Helotiales lack an asexual state and no morphologically distinguishing characteristic is available under culture.

Nakamura et al. (2018) detected a group in Hyaloscyphaceae (close to Hyphodicsus and Hyalopeziza) that was dominant and widely distributed in the roots of fagaceous trees, in addition to Lachnum (Lachnaceae or Hyaloscyphaceae s.l.) and Rhizodermea (Dermateaceae). In this study, I provided hitherto obtained sequences of Helotiales used to determine environmental data to link specimens, but no close match was found. Recognizing that the group obtained from the roots is phylogenetically distinguishable without any known reproductive morphology, Nakamura et al. (2018) proposed several new species in Glutinomyces, originally proposed by Nakamura (2017) for a helotialean fungus for which only the hyphal state was known. Later, Nakamura et al. (2019) demonstrated that Glutinomyces exchanges its genetic material through a parasexual process. These findings suggested a lack of information to link the members occurring as root endophytes, but they also suggested that there are groups of Helotiales that have adapted to underground life, losing morphologically differentiated sexual/asexual reproductive structures. Another good example is Meliniomyces, described based on sterile morphotypes with characteristic pinwheel-like colonies. These are common root endophytes and were recently combined with Hyaloscypha (Fehrer et al., 2019). Fehrer et al. (2019) also reported the first observation of an asexual state for Hyaloscypha bicolor, induced in vitro after incubation at 6 °C.

It is also worthwhile to mention that Hyalopeziza in Nakamura et al. (2018) was represented by Hyalopeziza pygmaea previously transferred to propose a new genus Hyphopeziza (Han et al. 2014a) showing an intermediate feature between Hyalopeziza and Hyphodicus. For the group including these genera, Hyphodiscaceae was proposed (Ekanayaka et al. 2019).

3.3 Ash dieback

Ash dieback is an emerging plant tree disease with severe mortality for European or common ash trees ( Fraxinus excelsior). Because F. excelsior is one of the key components of forest ecosystems and also economically important for timber production, the spread of ash dieback is very concerning. Nevertheless, the disease started to spread from Poland in 1992, and now covers almost the complete distribution range of Fraxinus in Europe (for a recent review, see Enderle, Stenlid, & Vasaitis, 2019).

The pathogen is now known as Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz & Hosoya(Baral, Queloz, & Hosoya, 2014), but it has a confusing nomenclatural history. Originally it was described as an asexual fungus, Chalara fraxineusT. Kowalski(Kowalski, 2006), once connected with Hymenoscyphus albidus (Gillet) W. Phillips (Kowalski & Holdenrieder, 2009) in its sexual state. Later, Queloz et al. (2011) discovered a cryptic entity within H. albidus corresponding to C. fraxineus and proposed a new species H. pseudoalbidusQueloz, Grünig, Berndt, T. Kowalski, T.N. Sieber & Holdenr. distinguished from H. albiduson a molecular basis. Baral (pers. comm.) kindly informed me of the possibility of the conspecificity of H. pseudoalbidus and Lambertella albida [one of the synonyms of H. albidus, documented as a new record from Japan (Hosoya et al., 1993)]. As a result of our genetic comparative studies, the conspecificity of L. albida documented in Japan and H. pseudoalbidus was demonstrated Zhao, Hosoya, Baral, Hosaka, & Kakishima, 2012). Later, because of the change in nomenclature following the One Fungus One Name (1F = 1N) policy (Hawksworth, 2011; Norvell, 2011), the new combination, H. fraxineus, was proposed (Baral et al., 2014).

Zhao et al. (2012) also demonstrated greater genetic diversity in the Japanese population compared to that of the European population. This supported the possible introduction of H. fraxineus into Europe from Asia, including Japan, which was suggested by Husson et al. (2011), Queloz et al. (2011), and Timmermann et al. (2011). Our discovery of H. fraxineus in Japan long before the emergence of the fungus in Europe partially supported this hypothesis. Later, the distribution of H. fraxineus in Asia became even wider after being found in Korea (Han et al., 2014b) and China (Zheng & Zhuang, 2014). Furthermore, Gross et al. (2014) conducted a detailed population genetic analysis to compare European and Japanese populations, and postulated that the establishment of the European population was based on the transfer of a small population through the “founder effect.” This was later confirmed by genomic analysis by McMullan et al. (2018), who postulated that the initial population was founded by only two individuals. Their studies also suggested the presence of more than 1000 effectors that dispute the resistance of the plant host. Together with the discovery research of bioactive metabolites (see Surup et al. 2018 and other papers cited herein), the process for symptom development has been elucidated.

Furthermore, the possibility of the endophytic occurrence of the fungus was examined, because no symptoms have been observed in Fraxinus trees where the first occurrence of H. fraxineus in Japan was reported. With a clear experiment in combination with field observations, Inoue et al. (2018) confirmed the occurrence of H. fraxineus as an endophyte in living leaves of F. mandshurica in Japan.

A combination of the characteristics of H. fraxineus, mainly having substratal stroma and ascospores turning brown after being discharged before germination, apparently satisfied the disposition of this fungus as Lambertella (Zhao et al., 2012). However, as shown by Zhao et al. (2016), these characters were the consequence of convergent evolution, and the combination of these characters no longer supports the disposition of this fungus to Lambertella. Since Hymenoscyphus is a large genus it may be divided into multiple genera in the future.

4. Discovery Research

Because fungi produce a great number of metabolites, they have been a significant resource in natural product research, and a number of compounds with novel structures and/or activities have been discovered (Dreyfuss & Chapela, 1994). Regarding the motivation of recent research, three major interests are recognized: natural product chemistry, chemicals from intermediate plant-fungal interactions, and discovery research for compounds for drugs (Hosoya, 1998). These research interests overlap and lead to discoveries of numerous new natural compounds (Bycroft & Payne, 2013).

Regarding discovery research, the majority of the results have come from soil fungi, in particular Aspergillus and Penicillium, and little research has focused on Helotiales. Although current environmental DNA studies suggest the occurrence of Helotiales in the soil, they cannot be isolated from soils by conventional techniques, whereas obtaining isolates from ascospores is relatively easy. Helotiales are therefore a good example of an “underutilized, but easily accessible fungi,” as proposed by Bergstom et al. (1995). Another expectation for Helotiales is that they may produce metabolites with different structures from those of Aspergillus and Penicillium because they significantly differ in their phylogenetic position.

Stadler and Anke (1993, 1995) were among the pioneers in utilizing Helotiales and discovered a series of chlorinated metabolites. Our attempts succeeded in the discovery of various enzyme inhibitors, such as squalene synthase (Hosoya et al., 1997; Tanimoto et al., 1997), sphingomyelinase (Tanaka, Nara, Suzuki-Kongai, Hosoya, & Ogita, 1997; Nara, Tanaka, Hosoya, Suzuki-Konagai, & Ogita, 1999), sphigosine kinase (Kono, Tanaka, Ogita, Hosoya, & Kohama, 2000; Kono et al., 2001), testosterone reductase (Hosoya et al., 1999), antibacterials (Matsumoto, Hosoya, & Shigemori, 2010; Matsumoto, Hosoya, Tomoda, Shiro, & Shigemori, 2011; Tanabe et al., 2015; Kawashima, Hosoya, Tomoda, Kita, & Shigemori, 2018), plant growth regulation factors (Tanabe, Matsumoto, Hosoya, Sato, & Shigemori, 2013), and antifungals (Ohyama et al., 2002).

Concerning ash dieback, several metabolites from H. fraxineus,including cytotoxic compounds and mycotoxins have been discovered [see Surup et al. (2018) for hyfraxins (cytotoxic) and other compounds].

With the advent of high-throughput screening coupled with natural product libraries, the research interest in search of new natural products has declined (Harvey, 2008). However, there is the expectation that natural products are not lost with the application of combinatorial chemistry, genetic engineering, and drug design technology (Pawar, 2014).

In addition, multiple species of Lachum have been shown to produce pigments and polysaccharides that exhibit antioxidant and antimicrobial activities (Ming, Wei, Cong, Shi-yan, & Yang, 2009; Ye, Peng, Fang, Li, & Yang, 2009; Qiu, Ma, Ye, Yuan, & Wu, 2013) providing remarkable support for Helotiales as a good biological resource.

5. Future perspectives

As a result of exploration, collected specimens, whether they are identified or unidentified, have cumulated in the museum fungaria. The occurrence data, based on specimens or observations, are being accumulated in public databases (e.g., GBIF, https://www.gbif.org/), and can be utilized for various purposes, including taxonomic and ecological research. Cumulation of isolates and/or parts of specimens and genetic data are also compiled in separate databases (see 2.1). However, once digitized, interoperability between the data increases, and more integrated research is possible.

Fungi occur in various environments, including the Helotiales. Metagenomic research has revealed the potential occurrence of Helotiales in various environments where we have seldom suspected them (Peay, 2014) and provide an opportunity to uncover ecological functions (Almario et al., 2017). Rapidly increasing data generated by next-generation sequencing provides insights into the interactions between fungi and plants (Toju et al., 2013a, 2013b). Therefore, when the collected specimens are accompanied by genetic data, such as barcoding sequences, the specimens could be exploited as vouchers to provide biological data. We have established links between specimen occurrence, morphological information, and DNA barcoding data (Hosoya, Jinbo, & Tanney, 2015). In the near future, it will be more important to exploit the distributed data in a more integrated manner, using DNA barcoding sequences as possible clues for linkages. In fact, we succeeded in linking unnamed endophytic fungi with newly described species (Hosoya et al., 2014).

Although some shortcomings are indicated by Hofstetter et al. (2019), barcoding for fungi using ITS-5.8S sequences has been widely accepted (Schoch et al., 2012), and there are ongoing initiatives to link the DNA sequence data with the data from specimens (e.g., UNITE, Kõljalg et al., 2005). However, whether all fungal entities should have Linnean binominals is a matter of debate (Money, 2013). Fungal biodiversity is vast, and it is apparent that millions of species could be recognized. It is therefore of substantial value to provide a common identifier, such as a digital objective identifier (DOI), as has been done in UNITE (Kõljalg et al., 2005) to the recognized groups of possible species (species hypothesis) to recognize the biological group as an entity. However, more specimens must be collected to ensure the actual existence of groups. As Korf (2005) encouraged the mycologist, we must “collect, collect, and collect” information to understand the biodiversity of Helotiales. After all, it seems for a discosystematist, Japan is still a “relatively unexplored paradise” (Korf, 1958).

Disclosure

The authors declare no conflicts of interest. All the experiments undertaken in this study complied with the current laws of the county where they were performed.