Specific and sensitive detection tools for Xanthomonas arboricola pv. corylina, the causal agent of bacterial blight of hazelnut, developed with comparative genomics.

Xanthomonas arboricola pv. corylina (Xac; formerly Xanthomonas campestris pv. corylina) is the causal agent of the bacterial blight of hazelnuts, a devastating disease of trees in plant nurseries and young orchards. Currently, there are no PCR assays to distinguish Xac from all other pathovars of X. arboricola. A comparative genomics approach with publicly available genomes of Xac was used to identify unique sequences, conserved across the genomes of the pathogen. We identified a 2,440 bp genomic region that was unique to Xac and designed identification and detection systems for conventional PCR, qPCR (SYBR® Green and TaqMan™), and loop-mediated isothermal amplification (LAMP). All PCR assays performed on genomic DNA isolated from eight X. arboricola pathovars and closely related bacterial species confirmed the specificity of designed primers. These new multi-platform molecular diagnostic tools may be used by plant clinics and researchers to detect and identify Xac in pure cultures and hazelnut tissues rapidly and accurately.

Taxonomic Reclassification of the Fungal Pathogen Causing Dry Berry Disease of Caneberries into the Division Ascomycota as Monilinia rubi.

As molecular genetic techniques improve and sequence data becomes available for more fungal species, taxonomic classifications historically based upon growth morphology alone are being revisited and occasionally reclassified. Herein, we present such an instance for the fungal pathogen that causes dry berry disease of caneberries. The organism was previously described as the basidiomycete fungus Rhizoctonia rubi based upon the pathogen's production of Rhizoctonia-like angular branching hyphae. Utilizing molecular genetic techniques unavailable when the pathogen was first characterized in 1959, three housekeeping gene regions (ITS, ß-tubulin, and G3PDH) were sequenced across 13 contemporary dry berry isolates, as well as the original 1959 R. rubi type strain, CBS382.59. The resulting neighbor-joining, maximum likelihood, and Bayesian phylogenies for single and multilocus sequences provide strong evidence that the dry berry pathogen was misclassified. This data, in addition to revisiting in vivo macroscopic and microscopic growth morphology, again comparing contemporary dry berry isolates to the CBS382.59 type strain, suggests that the causal organism is a new species within the genus Monilinia that we propose be classified as Monilinia rubi. A transition from designation as a basidiomycete fungus to an ascomycete fungus could have implications on chemical management decisions, as well as the assumptions made about cell structure and the pathogen's putative life cycle.

A Comprehensive Characterization of Ecological and Epidemiological Factors Driving Perennation of Podosphaera macularis Chasmothecia on Hop (Humulus lupulus).

Hop powdery mildew, caused by the ascomycete fungus Podosphaera macularis, is a consistent threat to sustainable hop production. The pathogen utilizes two reproductive strategies for overwintering and perennation: (i) asexual vegetative hyphae on dormant buds that emerge the following season as infected shoots; and (ii) sexual ascocarps (chasmothecia), which are discharged during spring rain events. We demonstrate that P. macularis chasmothecia, in the absence of any asexual P. macularis growth forms, are a viable overwintering source capable of causing early season infection two to three orders of magnitude greater than that reported for perennation via asexual growth. Two epidemiological models were defined that describe (i) temperature-driven maturation of P. macularis chasmothecia; and (ii) ascosporic discharge in response to duration of leaf wetness and prevailing temperatures. P. macularis ascospores were confirmed to be infectious at temperatures ranging from 5 to 20°C. The organism's chasmothecia were also found to adhere tightly to the host tissue on which they formed, suggesting that these structures likely overwinter wherever hop tissue senesces within a hop yard. These observations suggest that existing early season disease management practices are especially crucial to controlling hop powdery mildew in the presence of P. macularis chasmothecia. Furthermore, these insights provide a baseline for the validation of weather-driven models describing maturation and release of P. macularis ascospores, models that can eventually be incorporated into hop disease management programs.

Transcriptome-Derived Amplicon Sequencing Markers Elucidate the U.S. Podosphaera macularis Population Structure Across Feral and Commercial Plantings of Humulus lupulus.

Obligately biotrophic plant pathogens pose challenges in population genetic studies due to their genomic complexities and elaborate culturing requirements with limited biomass. Hop powdery mildew (Podosphaera macularis) is an obligately biotrophic ascomycete that threatens sustainable hop production. P. macularis populations of the Pacific Northwest (PNW) United States differ from those of the Midwest and Northeastern United States, lacking one of two mating types needed for sexual recombination and harboring two strains that are differentially aggressive on the cultivar Cascade and able to overcome the Humulus lupulus R-gene R6 (V6), respectively. To develop a high-throughput marker platform for tracking the flow of genotypes across the United States and internationally, we used an existing transcriptome of diverse P. macularis isolates to design a multiplex of 54 amplicon sequencing markers, validated across a panel of 391 U.S. samples and 123 international samples. The results suggest that P. macularis from U.S. commercial hop yards form one population closely related to P. macularis of the United Kingdom, while P. macularis from U.S. feral hop locations grouped with P. macularis of Eastern Europe. Included in this multiplex was a marker that successfully tracked V6-virulence in 65 of 66 samples with a confirmed V6-phenotype. A new qPCR assay for high-throughput genotyping of P. macularis mating type generated the highest resolution distribution map of P. macularis mating type to date. Together, these genotyping strategies enable the high-throughput and inexpensive tracking of pathogen spread among geographical regions from single-colony samples and provide a roadmap to develop markers for other obligate biotrophs.

Transcriptomic Profiling of Acute Cold Stress-Induced Disease Resistance (SIDR) Genes and Pathways in the Grapevine Powdery Mildew Pathosystem.

Temperatures from 2 to 8°C transiently induce quantitative resistance to powdery mildew in several host species (cold stress-induced disease resistance [SIDR]). Although cold SIDR events occur in vineyards worldwide an average of 14 to 21 times after budbreak of grapevine and can significantly delay grapevine powdery mildew (Erysiphe necator) epidemics, its molecular basis was poorly understood. We characterized the biology underlying the Vitis vinifera cold SIDR phenotype-which peaks at 24 h post-cold (hpc) treatment and results in a 22 to 28% reduction in spore penetration success-through highly replicated (n = 8 to 10) RNA sequencing experiments. This phenotype was accompanied by a sweeping transcriptional downregulation of photosynthesis-associated pathways whereas starch and sugar metabolism pathways remained largely unaffected, suggesting a transient imbalance in host metabolism and a suboptimal target for pathogen establishment. Twenty-six cold-responsive genes peaked in their differential expression at the 24-hpc time point. Finally, a subset of genes associated with nutrient and amino acid transport accounted for four of the eight most downregulated transcripts, including two nodulin 1A gene precursors, a nodulin MtN21 precursor, and a Dynein light chain 1 motor protein precursor. Reduced transport could exacerbate localized nutrient sinks that would again be transiently suboptimal for pathogen growth. This study links the transient cold SIDR phenotype to underlying transcriptional changes and provides an experimental framework and library of candidate genes to further explore cold SIDR in several systems, with an ultimate goal of identifying novel breeding or management targets for reduced disease.