Resistance of Botrytis cinerea to anilinopyrimidine fungicides: A novel ARMS-PCR method for the detection of Bcpos5 mutations and characterization of resistance using CRISPR/Cas9 editing.

BACKGROUND: Anilinopyrimidine (AP) fungicides have been widely used against Botrytis cinerea, yet their resistance mechanisms have only recently been clarified. Resistance is primarily linked to mutations G408V, L412V, and L412F in the Bcpos5 gene, whose encoded protein is localized to the mitochondria. In this study, we developed a detection method and tested the fitness of L412F/V mutants obtained by using the CRISPR/Cas9 editing technique. RESULTS: For rapid and cost-effective mutation identification, a TETRA-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) was developed to rapidly detect the nucleotide alterations that lead to L412F and L412V mutations, producing a 702 bp band in all isolates, with additional 470 bp (F) or 252 bp (V) fragments. Isolates harboring only the 702 bp band were further digested with MlyI to confirm the mutation leading to the amino acid substitution G408V mutation (467 bp + 235 bp). Results were validated by Sanger sequencing. Application of the assay to 170 isolates from strawberry and tomato revealed mutation frequencies of 70.2% (L412F), 8.3% (L412V), and 4.7% (G408V) within the resistant fraction of the population (n = 82 resistant isolates). Furthermore, sequencing analysis revealed also a low frequency of the E407K mutation in Bcmdl1, along with evidence of additional, yet undefined, resistance mechanisms. To further characterize the mutations, the B. cinerea reference strain B05.10 was transformed with L412F and L412V alleles via CRISPR/Cas9 and homologous recombination. The resulting mutants displayed resistance to cyprodinil, and potential fitness costs were assessed in both field-derived and CRISPR/Cas9-generated isolates through measurements of mycelial growth and sporulation in vitro, and pathogenicity in planta. The L412F and L412V transformants did not differ significantly from the parental B05.10 strain in any of the evaluated fitness parameters. CONCLUSION: Overall, the developed ARMS PCR offers a fast, cost-effective tool for resistance monitoring aiming to identify the most common mutations conferring resistance to APs, while CRISPR/Cas9 provides an efficient approach for functional validation of resistance mutations in B. cinerea. Using this approach, we confirmed that L412F and L412V mutations in Bcpos5 confer resistance to APs, while are not associated with fitness cost.