The Relationship Between Fungal Diversity and Invasibility of a Foliar Niche—The Case of Ash Dieback

Journal of Fungi Article The Relationship between Fungal Diversity and Invasibility of a Foliar Niche—The Case of Ash Dieback Ahto Agan 1,* , Rein Drenkhan 2, Kalev Adamson 2, Leho Tedersoo 1, Halvor Solheim 3, Isabella Børja 3, Iryna Matsiakh 4, Volkmar Timmermann 3, Nina Elisabeth Nagy 3 and Ari Mikko Hietala 5 1 Natural History Museum and Institute of Ecology and Earth Sciences, University of Tartu, 50411 Tartu, Estonia; [email protected] 2 Institute of Forestry and Rural Engineering, Estonian University of Life Sciences, 51006 Tartu, Estonia; [email protected] (R.D.); [email protected] (K.A.) 3 Norwegian Institute of Bioeconomy Research, p.b. 115, 1431 Ås, Norway; [email protected] (H.S.); [email protected] (I.B.); [email protected] (V.T.); [email protected] (N.E.N.) 4 Institute of Forestry and Park Gardening, Ukrainian National Forestry University, 79057 Lviv, Ukraine; [email protected] 5 Norwegian Institute of Bioeconomy Research, p.b. 2609, 7734 Steinkjer, Norway; [email protected] * Correspondence: [email protected] Received: 24 July 2020; Accepted: 24 August 2020; Published: 26 August 2020 Abstract: European ash (Fraxinus excelsior) is threatened by the invasive ascomycete Hymenoscyphus fraxineus originating from Asia. Ash leaf tissues serve as a route for shoot infection but also as a sporulation substrate for this pathogen. Knowledge of the leaf niche partitioning by indigenous fungi and H. fraxineus is needed to understand the fungal community receptiveness to the invasion. We subjected DNA extracted from unwashed and washed leaflets of healthy and diseased European ash to PacBio sequencing of the fungal ITS1-5.8S-ITS2 rDNA region. Leaflets from co-inhabiting rowan trees (Sorbus aucuparia) served as a reference. The overlap in leaflet mycobiomes between ash and rowan was remarkably high, but unlike in rowan, in ash leaflets the sequence read proportion, and the qPCR-based DNA amount estimates of H. fraxineus increased vigorously towards autumn, concomitant with a significant decline in overall fungal richness. The niche of ash and rowan leaves was dominated by epiphytic propagules (Vishniacozyma yeasts, the dimorphic fungus Aureobasidion pullulans and the dematiaceous hyphomycete Cladosporium ramotenellum and H. fraxineus), and endophytic thalli of biotrophs (Phyllactinia and Taphrina species), the indigenous necrotroph Venturia fraxini and H. fraxineus. Mycobiome comparison between healthy and symptomatic European ash leaflets revealed no significant differences in relative abundance of H. fraxineus, but A. pullulans was more prevalent in symptomatic trees. The impacts of host specificity, spatiotemporal niche partitioning, species carbon utilization profiles and life cycle traits are discussed to understand the ecological success of H. fraxineus in Europe. Further, the inherent limitations of different experimental approaches in the profiling of foliicolous fungi are addressed. Keywords: Hymenoscyphus fraxineus; Venturia fraxini; mycobiome; epiphytic and endophytic fungi; qPCR; PacBio 1. Introduction Globalization has created an unprecedented movement of species across continents. These concern also fungi that can hitchhike along with imported seed, plants, soil (potted plants) or plant material J. Fungi 2020, 6, 150; doi:10.3390/jof6030150 www.mdpi.com/journal/jof J. Fungi 2020, 6, 150 2 of 27 like timber [1]. Some of the imported fungi that have pathogenic potential can cause huge ecological and economic damage on evolutionary naïve plants that, due to lack of co-evolution with the incomer, have no effective defense mechanisms against it. Classic examples of pandemics caused by invasive alien tree pathogens include Dutch elm disease caused by Ophiostoma ulmi (Buisman) Melin and Nannf. and O. novo-ulmi Brasier, and chestnut blight caused Cryphonectria parasitica (Murrill) Barr, diseases that initiated during early 20th century and decimated billions of elm and chestnut trees in North America and Europe [2,3]. Conventionally, in the frame of the disease triangle [4], the focus of the scientific community engaged with tree pandemics, and plant diseases in general, has been to decipher how host defense responses and developmental phases, pathogen biology (life cycle, pathogenicity factors) and abiotic factors, like weather influence the outcome of the tree–pathogen interaction. However, an emerging view is that microorganisms critically affect host physiology and performance, suggesting that the evolution and ecology of plants and animals can only be understood in a holobiont (host and its associated microorganisms) context [5]. The host microbiome is hypothesized to extend the phenotype of the host organism [6]. Besides this role, the fungi associated with plants often occupy critical roles in carbon and nutrient cycling of terrestrial ecosystems as well [7]. Concerning invasive alien plant pathogens and their interaction with the native fungal community, the ‘Diversity Resistance’ hypothesis argues that species rich communities should be highly competitive and readily resist invasion [8]. The underlying assumption is that niche space in diverse natural communities is a limiting factor, and that such communities are structured by interspecific competition [6,8]. Several observational and/or experimental studies have suggested that local biodiversity [9], the presence and absence of niche specialists and generalists, and the type and number of incoming colonists into the recipient ecosystem [10], influence the outcome of species introductions. Additional factors that can influence the local establishment and subsequent invasive phase of an introduced species include propagule pressure [11], the similarity of recipient community structure at different locations [12], the viability of small introduced populations [13], and the presence of specific ecological drivers [14]. Dieback of European ash represents the most recent pandemic in trees that is currently threatening the tree species at a continental scale in Europe. The disease agent Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz and Hosoya [15] (syn. H. pseudoalbidus V. Queloz, C.R. Grünig, R. Berndt, T. Kowalski, T.N. Sieber and O. Holdenrieder [16], Helotiales, Ascomycetes) is a relatively recently discovered pathogen in Europe [15,17]. The disease, first observed in Poland in the early 1990s [18], has spread rapidly across the continent [19,20]. Its main host, European ash, is growing naturally in mixed forests with other broadleaved trees over a wide range of altitudes and soil moisture conditions across Europe [21]. European ash is one of the most important pioneer tree species in western, central and northern regions of the continent, and is considered a principal tree species in the prospect of biological diversity and industrial use for furniture and construction [22]. Ash dieback has emerged as one of the most serious problems in European forests, as the disease is threatening this keystone tree species at a continental scale [22]. Thus, H. fraxineus is listed as one of the most serious invasive forest disease agents [23], accompanied with huge economical losses as well; the total cost of the dieback of European ash is estimated at £15 billion for Britain alone [24]. The pathogen probably originates from East Asia, where it is associated with leaves of native Asian ash species [25–31], and causes only minor shoot dieback symptoms in the Russian Far East [29]. In Europe, besides European ash, this invasive fungus causes shoot dieback in the native narrow-leafed ash F. angustifolia Vahl. [31,32], which is still unaffected in its southern range. The pathogen is also able to colonize leaves, and to a varying extent, shoot tissues of the North American ash species F. nigra Marsh., F. pennsylvanica Marsh. and F. americana L., and the Asian ash species F. mandshurica Rupr., F. chinensis Roxb. and F. Sogdiana Bunge growing in European parks and arboreta [30,32–35]. The infected ash trees die eventually, although the progress of the disease is much slower in older trees [36–38]. Importantly, there are obvious genetic differences between different individuals of J. Fungi 2020, 6, 150 3 of 27 European ash in the susceptibility to shoot infection by H. fraxineus—field observations suggest that ~1%–5% of the European ash trees possess tolerance against this fungus [19]. Invasive pathogens may deteriorate the health of evolutionary naïve host populations that have not co-evolved with the pathogen. The success of invaders depends also on their traits relative to those of the native species competing for the same niche [39]. Concerning H. fraxineus, the traits of indigenous species associated with ash leaves are the most relevant, as leaf infection serves apparently as a route to shoot colonization by this pathogen [40]. Moreover, the pathogen forms its fruiting bodies on petioles, rachis and leaflet vein tissues of the compound ash leaf on the forest floor in the season following leaf shed. The mycobiome of healthy ash leaves is formed by basidiomycetous yeasts with low host specificity, a few highly-specific biotrophic fungi, and numerous endophytes or facultative parasites, which may have competence for endophytic overwintering in buds and/or saprobic overwintering in ash leaf petioles [41]. Hymenoscyphus fraxineus has been considered to be specific for Fraxinus spp., while most of the endophytic and epiphytic species associated with foliage of angiosperm trees are shared among many species [42–44]. The level of richness and the degree of functional

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