Characterization of mating type genes in heterothallic <i>Neonectria</i> species, with emphasis on <i>N. coccinea, N. ditissima</i>, and <i>N. faginata</i>

<i>Neonectria ditissima</i> and <i>N. faginata</i> are canker pathogens involved in an insect-fungus disease complex of American beech (<i>Fagus grandifolia</i>) in North America commonly known as beech bark disease (BBD). In Europe, both <i>N. ditissima</i> and <i>N. coccinea</i> are involved in BBD on European beech (<i>Fagus sylvatica</i>). Field observations across the range of BBD indicate ascospores to be the dominant spore type in the environment. Several studies report a heterothallic (self-sterile) mating strategy for <i>Neonectria</i> fungi, but one study reported homothallism (self-fertility) for <i>N. ditissima</i>. As such, investigations into mating strategy are important for understanding both the disease cycle and population genetics of <i>Neonectria</i>. This is particularly important in the United States given that over time <i>N. faginata</i> dominates the BBD pathosystem despite high densities of nonbeech hosts for <i>N. ditissima</i>. This study utilized whole-genome sequences of BBD-associated <i>Neonectria</i> spp. along with other publicly available <i>Neonectria</i> and <i>Corinectria</i> genomes and in vitro mating assays to characterize mating type (MAT) locus and confirm thallism for select members of <i>Neonectria</i> and <i>Corinectria</i>. MAT gene–specific primer pairs were developed to efficiently characterize the mating types of additional single-ascospore strains of <i>N. ditissima, N. faginata</i>, and <i>N. coccinea</i> and several other related species lacking genomic data. These assays also confirmed the sexual compatibility among <i>N. ditissima</i> strains from different plant hosts. Maximum likelihood phylogenetic analyses of both <i>MAT1-1-1</i> and <i>MAT1-2-1</i> sequences recovered trees with similar topology to previously published phylogenies of <i>Neonectria</i> and <i>Corinectria</i>. The results of this study indicate that all <i>Neonectria</i> and <i>Corinectria</i> tested are heterothallic based on our limited sampling and, as such, thallism cannot help explain the inevitable dominance of <i>N. faginata</i> in the BBD pathosystem.