Development of LAMP-based molecular method to identify two diamondback moth parasitoids, Diadegma fenestrale and Diadegma semiclausum for application in biological control

The diamondback moth, Plutella xylostella L., is a lepidopteran pest that seriously affecting Brassica vegetables worldwide. Various biological controls, such as parasitoid wasps Diadegma semiclausum and Diadegma fenestrale, have been widely used to control this pest. The taxonomy of these two species based on morphological characteristics is very similar, and they are both parasitic on P. xylostella larvae. In this study, we developed a molecular diagnostic method using loop-mediated isothermal amplification (LAMP) to identify these species accurately. The mitochondrial genome (MN599978) of D. fenestrale and partial nucleotide sequences (OM572528) including ITS region of D. semiclausum were analyzed for use as species diagnosis markers. D. fenestrale species-specific primers for LAMP were designed in COX3, and the optimal diagnostic conditions for the four primers (F3, B3, FIP, BIP) was at 63 °C for 35 min. A species-specific primer capable of identifying D. semiclausum was developed from the ITS2 region, and the optimal condition for diagnosis was 40 min at 63 °C. The diagnostic limit concentration was up to 10 pg under both optimal conditions, which it was able to detect even very low concentrations. In the case of D. fenestrale, it was attainable to diagnose the parasitoid on P. xylostella larvae. Therefore, the developed LAMP diagnostic method can be used in a variety of ways to diagnose whether P. xylostella has been parasitized in the process of field research and mass breeding. This LAMP-based diagnostic method can be applied to various parasitoid identification and for the biological control of P. xylostella.