Morphological and Phylogenetic Resolution of Diplodia corticola and D. quercivora, Emerging Canker Pathogens of Oak (Quercus spp.), in the United States.

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, in the United States, the oak wilt pathogen Bretziella fagacearum. More recently, two emerging canker pathogens, Diplodia corticola and D. quercivora, 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 United States, including Maryland, Pennsylvania, Virginia, and West Virginia, to determine the range and impact of D. corticola and D. quercivora on forest health in the United States. 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 D. corticola, D. quercivora, and D. sapinea, as well as Botryosphaeria dothidea, a closely related canker pathogen in the Botryosphaeriaceae. Both D. corticola and D. sapinea were recovered from red and white oak family members, whereas D. quercivora was exclusive to white oak family members and B. dothidea to red oak family members. Of these species, D. corticola was most frequently isolated, followed by D. quercivora, D. sapinea, and B. dothidea. Overall, mortality was 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 probably are acting opportunistically when the environment is conducive to disease. To better understand the relationships between D. corticola and potentially their geographic origins, a multigene phylogenetic study and corresponding morphological study were conducted. A total of 49 Diplodia isolates from Spain, France, Italy, and the United States were assessed. Across all isolates and geographic regions, D. corticola formed a strongly supported monophyletic clade sister to D. quercivora 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 D. corticola as a cosmopolitan pathogen, native to both Europe and the United States, with the possibility of secondary introductions.

Multilocus PCR Assays Elucidate Vegetative Incompatibility Gene Profiles of Cryphonectria parasitica in the United States.

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.

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

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

Enhanced hypovirus transmission by engineered super donor strains of the chestnut blight fungus, Cryphonectria parasitica, into a natural population of strains exhibiting diverse vegetative compatibility genotypes.

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.

Diversity and function of fungi associated with the fungivorous millipede, Brachycybe lecontii.

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, including 176 genera, 39 orders, four phyla, and 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.

Psychoactive plant- and mushroom-associated alkaloids from two behavior modifying cicada pathogens.

Entomopathogenic fungi routinely kill their hosts before releasing infectious spores, but a few species keep insects alive while sporulating, which enhances dispersal. Transcriptomics- and metabolomics-based studies of entomopathogens with post-mortem dissemination from their parasitized hosts have unraveled infection processes and host responses. However, the 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 likely 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.