As part of Work Package 4 “Genetic effects and genotyping tools in legumes”, on June 21, 2024, Bernadette Julier led a meeting with the companies Barenbrug, Cérience, DLF, and RAGT. The purpose was to evaluate the experimental capacity for phenotyping resistance to Verticillium and Ditylenchus dipsaci, in 400 alfalfa accessions from the INRAE collection.
The plant material had already been genotyped during the previous EUCLEG project. The partners CER, DLF, and R2N scheduled phenotyping tests on the accessions between 2025 and 2027. INRAE sent seed lots and all required control varieties to each partner during the summer of 2024.
During the Teams meeting, DLF committed to conducting phenotyping for resistance to alfalfa stem nematodes (Ditylenchus dipsaci) on 100 accessions provided by INRAE.
Ditylenchus dipsaci, commonly referred to as alfalfa stem nematode or “bulb and stem nematode,” is one of the few nematode species that feed on above-ground plant parts. This nematode is present in nearly every alfalfa-growing region worldwide.
The screening experiments began in January 2025. Two tests were conducted until April, with three more scheduled for the end of 2025.
Before screening the BELIS accessions, DLF adopted the GEVES method, which differs from our internal protocol. GEVES shared its procedure with DLF for screening stem nematode resistance. Ditylenchus dipsaci larvae are obtained from GEVES prior to each inoculation.
Throughout January, several preliminary experiments were carried out to gain confidence in the new protocol. Adjustments were needed for climate conditions, pre-germination days, the number of days before inoculation, inoculum concentration, and phenotyping date.
For the screening, five control varieties are required: Europe (as the susceptible control), Daisy and Ludelis (as intermediate controls), and Mezzo and Salsa (as resistant controls).
The most critical part of the protocol involves laying the seedlings horizontally on paper and carefully rolling them in preparation for inoculation. This time- and patience-consuming step is performed by the team while listening to a variety of background music. This phase is essential for successful inoculation. Two days later, 3,000 plants are inoculated.
Phenotyping takes place 21 days after inoculation. The scoring scale is qualitative, based on the presence of “swollen” plants. Stunted plants typically show swollen nodes and shortened internodes.
For results and analysis, the percentage of swollen plants (G) is calculated for each variety.
These tests will be repeated during 2026 and 2027 as now we are starting the observation season on the field and at nurseries. Ready to see what happens next!
