Quick detection of Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) in chestnut dormant buds by nested PCR

Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) develops in chestnut buds that remain asymptomatic from oviposition (June-July) until budburst; it is, thus, easily spread by plant material used in propagation. Therefore, it is particularly interesting to identify infested plant batches before their movement. Unfortunately, a non-destructive method for checking buds has not yet been developed, and the only technique available is the screening of a bud sample. The visual investigation is long and requires highly skilled and trained staff. The purpose of this work was to set up an effective and fast method able to identify the presence of first instar larvae of D. kuriphilus in a large number of chestnut buds by PCR. Four primer pairs were designed on nuclear and mitochondrial sequences of a set of seven gall wasp taxa and tested on five different cynipid's DNA. Nested diagnostic PCR was carried out on DNA extracted from samples of 2g buds simulating four levels of infestation (larvae were added to uninfested buds); 320bp amplicon of 28S sequence was chosen as a marker to detect one larva out of 2g buds. The method showed a potential efficiency of 5000 to 15,000 buds per week, depending on bud size.