Resolving host and species boundaries for perithecia-producing nectriaceous fungi across the central Appalachian Mountains

Abstract

The Nectriaceae includes numerous canker pathogens. Due to the scarcity of ascomata on many hosts, comprehensive surveys are lacking. Here we characterize the diversity of perithecia-producing nectriaceous fungi across the central Appalachians in eastern North America. Nine species from eleven hosts were recovered including a novel Corinectria sp. from Picea rubens. Neonectria ditissima and Neonectria faginata were most abundant and associated with Fagus grandifolia with beech bark disease (BBD). Neonectria ditissima was also recovered from additional cankered hardwoods, including previously unreported Acer spicatum, Ilex mucronata, and Sorbus americana. Cross-pathogenicity inoculations of N. ditissima confirmed susceptibility of Acer and Betula spp. Neonectria magnoliae was recovered from cankered Liriodendron tulipifera and Magnolia fraseri and pathogenicity on L. tulipifera was confirmed. Fusarium babinda was consistently recovered from beech with BBD, although its role remains unclear. This survey provides a contemporary snapshot of Nectriaceae diversity across the Appalachian Mountains. The following nomenclatural changes are proposed: Neonectria magnoliae comb. nov.

Introduction

Members of the Nectriaceae occupy diverse ecological niches from mycoparasites to phytopathogens, with numerous genera and species implicated in causing annual and perennial cankers on diverse woody plant hosts with varying degrees of host specificity. One such canker disease, beech bark disease (BBD), is a disease complex occurring across the range of American beech (Fagus grandifolia) in North America and European beech (Fagus sylvatica) in Europe. The disease requires prior infestation by a scale insect (Cryptococcus fagisuga) native to the Caucasus mountain region in eastern Europe, which predisposes the host bark tissues to subsequent invasion by predominantly one of two canker-causing fungi: Neonectria ditissima (syn. Nectria galligena) and either Neonectria faginata (syn. Nectria coccinea var. faginata) on American beech in North America or Neonectria coccinea on beech in Asia and Europe (Thomsen et al., 1949; Houston, 1994b). In North America, BBD was first observed in Halifax, Nova Scotia, Canada around 1890 following the introduction of C. fagisuga and presently continues to spread throughout the continuous range of American beech (Hewitt, 1914; Ehrlich, 1934; Cale et al., 2017). Other members of Bionectriaceae and Nectriaceae have occasionally been associated with BBD, including Clonostachys rosea (syn. Bionectria ochroleuca) and Fusarium spp., but their roles, if any, in BBD are not well understood (Cotter and Blanchard, 1982; Houston et al., 1987; Kasson and Livingston, 2009).

In addition to infecting scale-infested beech, N. ditissima is a well-known perennial target canker pathogen on many hardwood tree species often co-occurring in beech forests impacted by BBD (Spaulding et al., 1936; Lohman and Watson, 1943; Booth, 1967). Unlike BBD, which requires predisposition of host tissues by beech scale or possibly Xylococculus betulae (Cale et al., 2015), no insect partner has yet been established as a causal factor for N. ditissima infection on non-beech hosts. Based on previous pathogenicity studies, N. ditissima strains occurring in eastern North America do not exhibit host specificity (Plante and Bernier, 1997), allowing unfettered interactions between inoculum produced from cankers on beech and non-beech hosts. Although this has not been confirmed among all dominant host species co-occurring with beech in BBD areas, namely black birch (Betula lenta) and striped maple (Acer pensylvanicum), mating barriers do not exist among these strains. This indicates there is likely no host specificity given an outcrossing population of strains occurring on varying host species (Stauder et al., 2020). Further investigations are warranted as unknown host specificity and mating barriers could theoretically influence the population dynamics of N. ditissima strains participating in the BBD complex by limiting or disrupting gene flow.

In contrast to N. ditissima, N. faginata has only been observed causing cankers following C. fagisuga infestation on American beech trees (Castlebury et al., 2006), leaving questions regarding its origin and ecological niche, if any, outside of BBD. Other closely related Neonectria species such as Neonectria punicea also occur in eastern North America within northern hardwood forests and exhibit morphological attributes that overlap with N. faginata including ascospore size (Booth, 1959; Castlebury et al., 2006), which has historically served as a diagnostic measure (Lohman and Watson, 1943; Houston, 1994a; Kasson and Livingston, 2009). As such, previous misidentifications of Neonectria spp. on beech and non-beech hosts are plausible when relying on morphological attributes, possibly underestimating diversity (for example see Fig. 2 in Kasson and Livingston, 2009). Likewise, the close relationships among Neonectria spp., coupled with the lack of sequence data in repositories such as NCBI, has also presented species identification challenges, especially for rarer species, which further complicates identification. For example, our recent phylogenetic analyses of mating type gene sequences for Nectria magnoliae from tulip-poplar (Liriodendron tulipifera) revealed this species formed a genealogically exclusive clade among other Neonectria species that was closely allied with N. faginata (Stauder et al., 2020). This supports previous findings by Gräfenhan et al. (2011), who showed but did not discuss that N. ditissima and N. magnoliae were genealogically exclusive. Earlier work by Castlebury et al. (2006) concluded N. magnoliae was a synonym of N. ditissima. Additionally, many former members of the genus Nectria have since been reclassified into different genera (Brayford et al., 2004, Mantiri et al., 2001, Rossman et al., 1999; Castlebury et al., 2006). These findings emphasize the need for enhanced surveys to recover and phylogenetically resolve cryptic and understudied members of Nectriaceae as well as to investigate the potential for cryptic reservoirs of known species including the possibility of N. faginata occurring on a non-beech host.

This study sought to provide a greater understanding of the ecology and genetic relationships among N. ditissima, N. faginata, and allied fungi. The first objective was to survey perithecia-producing members of Nectriaceae in American beech stands to identify possible native reservoirs of N. faginata. This is important given the uncertainty surrounding the origin of N. faginata and the potential for previous misidentifications of nectriaceous fungi recovered from non-beech hosts. The second objective was to resolve phylogenetic relationships among described and possibly undescribed members of Nectriaceae recovered from forests across the central Appalachian Mountains. This was critical given both the significant undersampling of nectriaceous fungi in these regions, the resulting lack of sequence data, and/or contradictory evidence regarding known members of Nectriaceae, such as Nectria magnoliae. A third objective was to test host specificity of N. ditissima, N. faginata, and N. magnoliae strains isolated from American beech trees and tulip-poplar. Together these aims sought to provide contemporary insights into the true diversity, phylogenetic relationships, and ecological niches of perithecia-producing nectriaceous fungi across the central Appalachian Mountains.