A detached leaflet assay to determine the pathogenicity of diverse Botrytis spp. causing chocolate spot disease in faba bean (Vicia faba L.)

Chocolate spot (CS) disease, caused by certain Botrytis species, is one of the most destructive diseases of faba bean worldwide. This research set out to determine whether different Botrytis species cause different responses in faba bean genotypes that display varied levels of CS susceptibility. Three faba bean inbred lines – Kontu, Mélodie/2, and ILB 938/2 – identified as highly susceptible, moderately susceptible, and moderately resistant, respectively, were chosen as hosts, and 21 Botrytis isolates, representing six species ( B. fabae , B. fabiopsis , B. cinerea , B. pseudocinerea , B. medusae , and B. euroamericana ), were used as disease agents. A PCR assay using primers targeting the 10 NEP protein genes was used to genotype Botrytis isolates. The Botrytis isolates were grown on half-strength PDA medium and treated with near-UV light to induce sporulation. Spore suspensions were inoculated onto detached, fully expanded leaflets. Results revealed a significant difference in virulence reactions among the inbred lines, isolates and their interaction. All the 21 Botrytis species/isolates tested induced CS symptoms. B. fabae 19B053-4 was the most virulent, causing the largest lesions, followed by B. fabiopsis 19B175, B. fabae 17B4, B. fabiopsis 17B24, and B. cinerea 19B048. At the other extreme, lesions developed most slowly with B. cinerea 17B10-1, B. fabiopsis 19B024, B. fabae 17B28, and B. cinerea 19B014. The DNA analysis revealed two main clusters and four sub-clusters among the Botrytis species, showing species-level groupings. The detached leaflet assay enabled a rapid and effective comparison of the pathogenicity of the studied Botrytis isolates.