Molecular droplet digital PCR diagnostics and bioassays for monitoring insecticide resistance status in Myzus persicae populations from Greece

Abstract BACKGROUND Myzus persicae (Sulzer), is a major global pest whose control is challenged by widespread insecticide resistance. This study assessed the resistance status of M. persicae populations collected in Greece (2021–2025), using diagnostic bioassays with insecticides (acetamiprid, flupyradifurone, flonicamid, sulfoxaflor and FliPPER) and a newly developed, highly sensitive droplet digital polymerase chain reaction (ddPCR) panel targeting seven key resistance mutations (vgsc: super‐kdr M918T/L and kdr L1014F – pyrethroid resistance; acetyl‐CoA carboxylase: A2666V – keto‐enol resistance; AChE: MACE S431F – dimethyl carbamates resistance; nAChR: R81T and T74I linked to CYP6CY3 overexpression – resistance to nAChR competitive modulators). RESULTS Bioassays revealed frequent cross‐resistance cases. Notably, we report the first case of resistance to flonicamid. FliPPER (fatty acids potassium salts) showed the highest resistance frequency (50.0%) and sulfoxaflor the lowest (1.7%). The ddPCR analysis (on 634 aphids) confirmed the presence of six resistance mutations. The mutation T74I was almost fixed across all populations (mean resistant allele frequency of 99.9%). Other key mutations (MACE, kdr / super ‐ kdr and R81T) were present at moderate‐to‐high frequencies, with the M918L super‐kdr variant showing a notable increase. CONCLUSIONS Myzus persicae in Greece exhibits a high level of genetic variability, leading to severe and dynamic resistance problems. The widespread metabolic resistance ( CYP6CY3 overexpression) which has been found to compromise neonicotinoids and fluryradifurone and the emergence of flonicamid resistance complicate the implementation of integrated pest management. The ddPCR panel provides a valid, evidence‐based tool for large‐scale monitoring of resistance and decision‐making, essential for managing this difficult to control pest. However, bioassays are also useful when molecular markers are not available. © 2026 Society of Chemical Industry.