Species-specific PCR-based marker for rapid detection of Aspidiotus rigidus Reyne (Hemiptera: Diaspididae)

Abstract

• A marker was designed from InDels of Aspidiotus rigidus 28 s rDNA sequences. • Species-specific delineation of A. rigidus works in singleplex and multiplex PCR. • A. rigidus multiplex-based detection still works with parasitized samples. The Philippine coconut production has been greatly affected by the recent devastating infestation of Aspidiotus spp. However, identification of the outbreak species, Aspidotus rigidus , has been a challenge using morphological approaches. Molecular identification via PCR sequencing of insect barcoding genes has been implemented, but the overall process is time-consuming and costly. Thus, we developed and optimized a species-specific PCR-based molecular marker for rapid, efficient and cost-effective molecular identification of A. rigidus . The molecular marker was designed based on the sequences of the partial 28S ribosomal RNA gene from species of Aspidiotus that feed on coconut in the Philippines, A. rigidus, A. destructor and A. excisus . Multiple alignment of nucleotide sequences revealed a conserved 16-bp insertion-deletion (InDel) site common to all A. rigidus specimens identified from which the A. rigidus- specific oligonucleotide (RIG1) primer targeting an approximately 570 bp fragment size was designed. Results showed that the species-specific DNA marker technology consistently delineated laboratory-reared and field-collected A. rigidus samples from A. destructor and A. excisus . The protocol offers a rapid and reliable method for the early detection of A. rigidus infestation in high-risk areas planted with coconut in the country.