Breanne Kisselstein worked in David Gadoury‘s Cornell plant pathology epidemiology lab and Lance Cadle-Davidson‘s USDA Grape Genetics Research Unit (GGRU) lab while completing her Plant Pathology PhD at Cornell University. She has had the pleasure of working closely with other plant pathologists, plant breeders, computational biologists, and mechanical and software engineers on both the VitisGen and VitisGen2 projects, as this lab has hosted the phenotyping center for these multi-institutional SCRI-funded grape breeding projects.
Due to Breanne’s proximity to this research, she was able to adapt the project’s Amplicon Sequencing (AmpSeq) genotyping platform and the semi-automated computational pipelines developed by VitisGen2 for exploring the pathogen side of the grape-powdery mildew relationship. With this, she was able to rapidly sequence >100 amplicons of ~4,500 Erysiphe necator samples collected across 10 commercial sites, 1 Cornell fungicide trial site, and wild vines in the Northeastern United States each year between 2015 and 2020. Since powdery mildew fungi are obligate biotrophs, most researchers are required to subculture these samples onto sterilized host tissue to obtain enough biomass and DNA for subsequent analyses. However, Breanne was able to show that the sensitivity of AmpSeq allows the DNA from these 1-cm field-collected samples to be genotyped without tedious subculturing, despite being undetectable by a nano drop spectrophotometer. Researchers were previously limited to only genotyping isolates they could maintain in the laboratory which requires countless hours in daily greenhouse management, biweekly spore transfers under a microscope, and frequent observation for potential culture contaminants, allowing researchers to sequence far more samples than previously possible for obligate biographic pathogens!
Project 1: Clonality, pairing of sexually compatible isolates, and winter survival of chasmothecia in E. necator
The E. necator fungus is heterothallic and has a mixed mode of reproduction here in the cold climate of the Northeastern U.S., surviving winter solely as chasmothecia (in its sexual state). Using AmpSeq data and disease assessments from multiple snapshots in time during the grape growing season, we found that 1-cm foliar samples from the field are more likely to be genetically clonal in the early stages of the epidemic, while disease incidence and severity are still low. We were also able to develop a conservative model that showed that given an estimated leaf abscission date of November 1st and a maximal level of disease severity, chasmothecia were still unlikely to fully maturate prior to leaf fall if initiation (i.e. pairing of sexually compatible individuals) began on or after September 23rd.
Project 2: Genetics and diversity of E. necator populations in the Northeastern United States
The Northeastern United States is a center of diversity for the E. necator fungus, yet we commonly grow susceptible vines like the Eurasian Vitis vinifera for its wine qualities. Having a diverse genetic pool and mixed mode of reproduction contribute to the pathogen’s high evolutionary potential, which is seen in this pathosystem at a practical level by its demonstrated ability to overcome certain forms of host resistance and several fungicide groups. Using AmpSeq data, we found that on average 95% of individuals collected from commercial vineyards contained at least one of two mutations known to confer resistance to DMI fungicides (CYP51 A495T and A1119C), while 45% had both. However, these mutations were rarely found on unmanaged native vines in the same region.
Project 3: Illuminating the durability of deploying RUN1 resistant grapevines in a center of diversity of the grape powdery mildew pathogen, Erysiphe necator
Unfortunately, this pathogen’s propensity for rapid genetic evolution also works to overcome qualitative resistance genes in disease-resistant grape cultivars, which can lead to a boom-and-bust cycle. Three NLR (nucleotide-binding site−leucine-rich repeats) resistance genes of different effect sizes used in the Cornell grape breeding program were evaluated, the qualitative resistance gene RUN1, found in V. rotundifolia, and two quantitative resistance genes, REN2 and REN3, found in other sources. Interestingly, multiple E. necator isolates found here are able to grow on RUN1 vines despite V. rotundifolia not being grown within 800 km of Geneva. However, RUN1 continues to be relevant in breeding programs despite evidence that these naïve pathogen populations are able to overcome RUN1 resistance, because its durability can be enhanced by pyramiding multiple resistance genes and by planting in grape growing regions with less diverse pathogen populations.
