Genetic and Phenotypic Characterization of the Fungal Pathogen Cytospora plurivora from Western Colorado Peach Orchards and the Development of a ddPCR Assay for Detection and Quantification.

Cytospora canker is one of the most important diseases affecting peach production in Colorado, yet previous efforts to characterize Cytospora species diversity in Colorado have relied exclusively on morphological traits. Recently, several new Cytospora species were described from peach orchards within the United States using molecular and morphological data, prompting the need to reexamine Cytospora spp. present on peach trees in Colorado. A total of 137 isolates of Cytospora spp. were collected from eight orchards in western Colorado. Isolates were sequenced at the internal transcribed spacer region and elongation factor 1-α and assessed with reference sequences in phylogenetic analyses. All isolates from western Colorado peach trees resolved with the newly described Cytospora plurivora. In addition to molecular characterization, temperature growth and virulence assays were conducted to assess phenotypic variation among the isolates from western Colorado. Variation across isolates was found both in growth at different temperatures and in virulence. Ancestral state reconstruction analyses resolved the most virulent (and most often collected) haplotypes together in a well-supported clade from which a single monophyletic origin of high virulence can be inferred. Finally, a droplet digital PCR assay was developed for use in ongoing and future studies to detect and quantify C. plurivora from field and laboratory samples.

Virulence of Genetically Distinct Geosmithia morbida Isolates to Black Walnut and Their Response to Coinoculation with Fusarium solani.

Geosmithia morbida is well documented as the causal agent of thousand cankers disease of black walnut trees. However, it is not well understood how G. morbida strains differ in virulence and how their interactions with co-occurring pathogens contribute to disease severity. In this study, we systematically investigated virulence of genetically distinct G. morbida strains. Overall, we found varying degrees of virulence, although differences were not related to genetic groupings. Furthermore, the pathogen Fusarium solani is also commonly isolated from thousand canker-diseased trees. The degree of disease contribution from F. solani is unknown, along with interactions it may have with G. morbida. This research shows that coinoculation with these pathogens does not yield a synergistic response.

Metalloproteases secreted by Actinobacillus suis.

Actinobacillus suis secretes metalloproteases into its medium. These secreted proteins, when concentrated by precipitation with 70% (NH4)2SO4 or methanol, displayed proteolytic activity at >200 kDa molecular mass bands in 10% polyacrylamide gels copolymerized with bovine casein (1%). They showed activity in a broad pH range (from pH 5 to pH 10) and were inhibited by 20 mM EDTA or EGTA, but could be reactivated by calcium. They were found heat stable at 40 degrees C, 50 degrees C, 60 degrees C, and 70 degrees C, but their activity diminished at 80 degrees C or higher. They degraded pig and bovine IgG and cross-reacted with a polyclonal serum against a high molecular mass secreted protease from A. pleuropneumoniae. Extracellular proteases could play a role in diseases caused by A. suis.