Spinach belongs to the healthy leafy green vegetables, and the Netherlands is a market leader in breeding new spinach varieties and exporting seeds all over the world. Spinach cultivation is at severe risk because of constantly evolving races
of downy mildew that break newly introduced resistances at a high pace. The partners in this project need knowledgedriven approaches to breed new resistant spinach varieties for sustainable agriculture. In a previous TKI project, this consortium has prepared a first reference genome sequence (genome) of the spinach downy mildew Peronospora effusa (also known as P. farinosa f. sp. spinaciae, here named Pfs), which has been used to compare genomes of resistancebreaking Pfs isolates. For several defeated resistance traits, we have identified corresponding Pfs effector genes, mutation of which can explain the resistance breaking character of the corresponding isolates. Although this Pfs reference genome is still fragmented and incomplete, initial analysis has demonstrated the power and prospect of pathogen genome analysis
for disease monitoring and resistance breeding.
Aim: Develop high-quality and complete genomes of spinach downy mildew isolates to identify effectors, understand their evolution, function, and significance for spinach infection, and use this knowledge to bring resistance breeding in spinach to the next level to contribute to sustainable agriculture.
The expected outcome of the project is in depth scientific knowledge on the complete genomes and effector repertoires of all denominated Pfs races and how new emerging isolates break resistance. This will be instrumental in designing innovative
methods that the sector can use to select new resistance sources and to make educated choices for combining resistance traits in new crop varieties. These resilient varieties are key for future agriculture and can have the impact that is needed
to achieve societal goals with respects to sustainable production. Varieties with optimal disease resistance will circumvent important crop losses (in particular in organic farming) and are a prerequisite for reducing the use of agrochemicals.

Develop high-quality genomes of spinach downy mildew isolates to identify effectors, understand their evolution, function, and significance for infection of spinach, and use this knowledge to bring resistance breeding in spinach to the next level to contribute to sustainable agriculture.

The Dutch vegetable breeding companies are, together with the US, the
major exporters of high-value seeds with outstanding quality and disease resistance traits. These traits and new innovations are important to have crop varieties with an optimal genetic make-up that allows production with reduced inputs and
chemical control. For future sustainable food production, it is therefore required to have access to powerful technologies that enable the selection of optimal genetic traits, e.g. by knowledge-driven breeding using pathogen-informed strategies
based on effectors.

Scientific insights into pathogen effectors, their functions, and their evolution
are instrumental in knowledge-driven breeding for resistance. Here, we will develop genomics-based methods to understand pathogen evolution and effector function, which are needed for new approaches to breed downy mildew resistant spinach. The principles and methodologies will be developed for the spinach-Pfs system but can be explored for many other crops and diseases. It is an important technological development that will first be explored for resistance breeding in spinach because of the urgency of the disease. Concrete deliverables:
- Pfs genome sequences (including raw data) produced through Nanopore technology
- Pfs sequences of all effectors, and knowledge on their variation, identified in this project
- All generated Pfs effector constructs as a resource (effector library) to be used by the companies
- Bacterial strains, constructs optimized for delivery of effectors into spinach, and methods to use them in vivo.