A PCR–RFLP Assay for Identifying the Cryptic Australian Endemic <i>Plutella australiana</i> From Invasive <i>Plutella xylostella</i>

ABSTRACT Plutella xylostella , diamondback moth, is a major invasive worldwide pest. In Australia, a local Plutella known as P. australiana is almost morphologically indistinguishable from P. xylostella . Therefore, we developed a PCR–restriction fragment length polymorphism (PCR–RFLP) assay targeting the mitochondrial COI gene to differentiate the two species. COI amplicons were digested with BfaI , producing two fragments (~490 and ~270 bp) for P. australiana , whereas P. xylostella remained undigested (~658 bp). In total, 71 and 15 COI sequences were generated for P. xylostella and P. australiana , respectively. We found seven unique haplotypes for P. xylostella and two unique haplotypes for P. australiana . In all cases, P. australiana samples were consistently digested, whereas P. xylostella samples remained undigested. Our assay therefore produced consistent, species‐specific banding patterns across multiple individuals. This provides a simple, rapid, reliable and cost‐effective method for distinguishing these cryptic species without having to sequence COI. This will aid experimental and survey work where the cryptic Plutella species need to be identified to provide information on their distribution, host use and pest impact.