Chrysanthemum is one of the most important ornamental crops worldwide with most of the breeding companies
concentrated in NW-Europe (mainly in the Netherlands, but also in Belgium and Germany). This region also hosts
a significant number of commercial growers, making it an important area for chrysanthemum breeding and
production. While considerable progress is being made in breeding for resistance to important fungal diseases
like fusarium and white rust, viral diseases are becoming an increasing problem that remains difficult for breeding
companies to tackle. Tomato spotted wilt virus (TSWV) is a prominent example, with a very wide host range.
Although simultaneous and efficient virus detection methods are available, there is an urgent need for a reliable
TSWV disease test that can be employed in a consistent bio-assay to screen for variation in susceptibility to TSWV,
thereby paving the possibility to breed for TSWV-resistant pot, garden and cut-flower chrysanthemums. Next to
the bio-assay, we aim to develop a screening method to identify potential specific resistances based on HR
response triggered by variation in viral (coat) proteins, and to obtain a comprehensive overview of the diversity
of chrysanthemum-infecting TSWV isolates across different production regions (both for cuttings and flowers).
Depending on the outcomes of the bio-assay and effectoromics studies, a forward or reverse genetics approach
will be employed to genetically characterize the TSWV disease resistance presence in chrysanthemum breeding
material.

The project aims to tackle the existing hurdles for research and breeding in chrysanthemum with regard to TSWV
disease resistance through the following objectives:
- Objective 1A: Develop and apply TSWV disease assay
Although TSWV is a well characterized virus, the disease it causes in chrysanthemum remains understudied.
Currently, there is no controlled disease assay protocol for TSWV disease resistance screening (in
chrysanthemum) that is reliable and allows high throughput screening, which is indispensable for targeted
breeding. Here, we will develop a reliable and accessible disease assay for the TSWV-chrysanthemum
pathosystem.
- Objective 1B: Identification of TSWV resistance sources and implementation plus harmonization of the test
Once a disease assay has been developed, wild species (mainly diploid) and hexaploid varieties will be tested
to identify putative resistant resources. A ring test of the developed TSWV disease assay will be performed
by all our involved partners to validate the resistant sources.
- Objective 2: Identification of R-genes by effector screening
A virus effector pipeline will be established, and viral proteins or peptides will be used to screen the same
set of wild species and varieties that were used in our TSWV disease bio-assay for HR responses.
- Objective 3: Genetic diversity of chrysanthemum-infecting TSWV isolates
Sequencing-based genotyping of TSWV isolates found in chrysanthemum raised at different geographic
locations will be done to gain insight into the genetic variation of viral genes relevant to the disease assays.

This project contributes to ST2, Biotechnology and breeding, as it aims to generate the knowledge and tools
necessary to develop chrysanthemum varieties with resistance against TSWV

➢ Deliverable 1.1: Method for maintenance and stable production of TSWV
➢ Deliverable 1.2: Disease assay protocol for TSWV inoculation in chrysanthemum
➢ Deliverable 1.3: Screening method for TSWV resistance in chrysanthemum wild species and hexaploid
breeding material/Identification of resistance resources
➢ Deliverable 1.4: Training for breeding companies on bio-assay and ring test harmonization
➢ Deliverable 2.1: Effector-based screening assay/ Effectoromics pipeline in chrysanthemum
➢ Deliverable 2.2: Selection of chrysanthemum wild species and hexaploid breeding material that respond
to TSWV proteins
➢ Deliverable 2.3: Comparison report providing the correlation between disease resistance and viral
protein recognition in chrysanthemum wild species and hexaploid breeding material
➢ Deliverable 2.4: Training for breeding companies on effector-based screening (Industrial partners);
Peptides (or proteins) for easy screening of TSWV resistance by partners.
➢ Deliverable 3.1: Overview of genetic variability of chrysanthemum infecting TSWV isolates