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Neonectria ditissimaandN. faginataare canker pathogens involved in an insect-fungus disease complex of American beech (Fagus grandifolia) commonly known as beech bark disease (BBD). In Europe, bothN. ditissimaandN. coccineaare involved in BBD on European beech (Fagus sylvatica). Field observations across the range of BBD indicate that new infections occur primarily via ascospores. Both heterothallic (self-sterile) and homothallic (self-fertile) mating strategies have been reported forNeonectriafungi. As such, investigations into mating strategy are important for understanding both the disease cycle and population genetics ofNeonectria. This is particularly important in the U.S. given that over timeN. faginatadominates the BBD pathosystem despite high densities of non-beech hosts forN. ditissima. This study utilized whole-genome sequences of BBD-associatedNeonectriaspp. along with other publicly availableNeonectriaandCorinectriagenomes andin vitromating assays to characterize mating type (MAT) loci and confirm thallism for select members ofNeonectriaandCorinectria. MAT gene-specific primer pairs were developed to efficiently characterize the mating types of additional single ascospore strains ofN. ditissima,N. faginata, andN. coccineaand several other related species lacking genomic data.In vitromating assays were used in combination with molecular results to confirm thallism. These assays also comfirmed the sexual compatibility amongN. ditissimastrains from different plant hosts. Maximum likelihood phylogenetic analysis of both MAT1-1-1 and MAT1-2-1 sequences recovered trees with similar topology to previously published phylogenies ofNeonectriaandCorinectria. The results of this study indicate that allNeonectriaandCorinectriatested are heterothallic based on our limited sampling and, as such, thallism cannot help explain the inevitable dominance ofN. faginatain the BBD pathosystem.

Horizontal transmission of virulence attenuating hypoviruses of Cryphonectria parasitica is restricted by an allorecognition system termed vegetative incompatibility (vic). A super donor formulation of two engineered C. parasitica strains (SD328/SD82) with gene disruptions at four of six vic loci transmitted hypovirus to strains in the laboratory independent of vic genotype. We now report the transmission of hypovirus by the SD328/82 formulation to a diverse, natural C. parasitica population infecting American chestnut in a forest setting. Hypovirulent (HV) isolates were recovered from 94% of cankers treated with the hypovirus-infected SD328/82 formulation compared to 51% of cankers treated with a hypovirus-infected EU5/6 formulation (strains having the same vic genotypes as SD strains but lacking vic gene disruptions). Overall, the SD328/82 formulation transmitted hypovirus into more divergent vic genotypes compared to the EU5/6 formulation. These results demonstrate the SD328/82 formulation can serve as an enhanced hypovirus vector for highly divergent C. parasitica populations.

In Mediterranean Europe and the United States, oak species (Quercus spp.) have been in various states of decline for the past several decades. Several insect pests and pathogens contribute to this decline to varying degrees including Phytophthora cinnamomi, Armillaria spp., various insect defoliators, and additionally in the U.S., the oak wilt pathogen, Bretziella fagacearum. More recently, two emerging canker pathogens, Diplodia corticola (Dc) and Diplodia quercivora (Dq) have been implicated in causing dieback and mortality of oak species in Europe and in several regions in the United States. In 2019, a fungal survey was conducted in the Mid-Atlantic region of the Eastern U.S., including Maryland, Pennsylvania, Virginia, and West Virginia to determine the range and impact of Dc and Dq on forest health within the U.S. A total of 563 oak trees between red and white oak family members were evaluated across 33 forests spanning 18 counties. A total of 32 Diplodia isolates encompassing three Diplodia spp. were recovered from 5,335 total plugs collected from the 13 of 18 sampled counties. Recovered Diplodia species included Dc, Dq, and D. sapinea (Ds), as well as Botryosphaeria dothidea (Bd), a closely related canker pathogen in the Botryosphaeriaceae. Both Dc and Ds were recovered from red and white oak family members, whereas Dq was exclusive to white oak family members and Bd to red oak family members. Of these species, Dc was most frequently isolated followed by Dq, Ds, and Bd. Overall, mortality was relatively low across all sampled counties, indicating that these fungi, at the levels that were detected, are not widely inciting oak decline across the region, but more likely are acting opportunistically when the environment is conducive for disease. In an attempt to better understand the relationships among Dc and potentially their geographic origin(s), a multi-gene phylogenetic study and corresponding morphological study were conducted. A total of 49 Diplodia isolates from Spain, France, Italy, and the U.S. were assessed. Across all isolates and geographic regions, Dc formed a strongly supported monophyletic clade sister to Dq and included two strongly supported subclades, one that included isolates from Spain and California and a second that included isolates from Italy, Maryland, and West Virginia. Both subclades also exhibited overlapping spore measurements. These results support Dc as a cosmopolitan pathogen, native to both Europe and the U.S. with the possibility of secondary introductions.

The Nectriaceae contains numerous canker pathogens. Due to scarcity of ascomata on many hosts, comprehensive surveys are lacking. Here we characterize the diversity of perithecia-producing nectriaceous fungi across the central Appalachians. Ten species from twelve hosts were recovered including a novel Corinectria sp. from Picea rubens. Neonectria ditissima and N. faginata were most abundant and associated with Fagus grandifolia with beech bark disease (BBD). N. 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.

Fungivorous millipedes (subterclass Colobognatha) likely represent some of the earliest known mycophagous terrestrial arthropods, yet their fungal partners remain elusive. Here we describe relationships between fungi and the fungivorous millipede, Brachycybe lecontii. Their fungal community is surprisingly diverse with 176 genera, 39 orders, and four phyla and includes several undescribed species. Of particular interest are twelve genera conserved across wood substrates and millipede clades that comprise the core fungal community of B. lecontii. Wood decay fungi, long speculated to serve as the primary food source for Brachycybe species, were absent from this core assemblage and proved lethal to millipedes in pathogenicity assays while entomopathogenic Hypocreales were more common in the core but had little effect on millipede health. This study represents the first survey of fungal communities associated with any colobognath millipede, and these results offer a glimpse into the complexity of millipede fungal communities.

Most wood-boring insects compete with wood decaying basidiomycetes for woody biomass. One clade of ambrosia beetles gained access to rotten wood – an abundant resource unsuitable to most wood-boring insects – by evolving a farming-like mutualism with a white rot polypore. Here we show the mutualist of Ambrosiodmus/Ambrosiophilus, the polypore Flavodon ambrosius, is superior in lignocellulolytic capacity compared to Ascomycota ambrosia fungi and other white rot Basidiomycota. This mutualism facilitated the evolution of large, long-lived, communal colonies with overlapping generations and egg-laying by pre-dispersal progeny females. F. ambrosius resembles other white rot Polyporales in that it causes significant weight loss in wood decay assays and strong polyphenol oxidase reactions, indicative of lignin-modifying enzymes. The symbiosis is asymmetrical: there are many species of Ambrosiodmus and Ambrosiophilus but all use a single known species of Flavodon, which determines the ecological strategy of the entire insect clade.

Entomopathogenic fungi routinely kill their hosts before releasing infectious spores, but select species keep insects alive while sporulating, which enhances dispersal. Transcriptomics and metabolomics studies of entomopathogens with post-mortem dissemination from their parasitized hosts have unraveled infection processes and host responses, yet mechanisms underlying active spore transmission by Entomophthoralean fungi in living insects remain elusive. Here we report the discovery, through metabolomics, of the plant-associated amphetamine, cathinone, in four Massospora cicadina-infected periodical cicada populations, and the mushroom-associated tryptamine, psilocybin, in annual cicadas infected with Massospora platypediae or Massospora levispora, which appear to represent a single fungal species. The absence of some fungal enzymes necessary for cathinone and psilocybin biosynthesis along with the inability to detect intermediate metabolites or gene orthologs are consistent with possibly novel biosynthesis pathways in Massospora. The neurogenic activities of these compounds suggest the extended phenotype of Massospora that modifies cicada behavior to maximize dissemination is chemically-induced.

Chestnut blight is a devastating disease of Castanea spp. Mycoviruses that reduce virulence (hypovirulence) of the causative agent, Cryphonectria parasitica , can be used to manage chestnut blight. However, vegetative incompatibility ( vic ) barriers that restrict anastomosis-mediated virus transmission hamper hypovirulence efficacy. In order to effectively determine the vegetative incompatibility genetic structure of C. parasitica field populations, we have designed PCR primer sets that selectively amplify and distinguish alleles for each of the six known diallelic C. parasitica vic genetic loci. PCR assay results were validated using a panel of 64 European tester strains with genetically determined vic genotypes. Analysis of 116 C. parasitica isolates collected from five locations in the eastern United States revealed 39 unique vic genotypes and generally good agreement between PCR and tester strain coculturing assays in terms of vic diversity and genotyping. However, incongruences were observed for isolates from multiple locations and suggested that the coculturing assay can overestimate diversity at the six known vic loci. The availability of molecular tools for rapid and precise vic genotyping significantly improves the ability to predict and evaluate the efficacy of hypovirulence and related management strategies.