Long PCR Is a Sensitive Method for Detecting Liberobacter asiaticum in Parasitoids Undergoing Risk Assessment in Quarantine

l c t a The accidental importation of plant pathogens in or on the bodies of parasitoids imported as natural enemies has been raised as a potential risk of classical biological control projects involving insects that serve as vectors of plant diseases. During quarantine evaluation of two parasitoids, Tamarixia radiata Waterston and Diaphorencyrtus aligarhensis (Shafee, Alam and Agarwal), imported for classical biological control of Asian citrus psylla Diaphorina citri (Kuwayama) in lorida, we were asked to determine whether these arasitoids were free of the causal agent of Asian reening disease, the bacterium Liberobacter asiatium. Preliminary tests with allele-specific polymerase hain reaction (Standard PCR) suggested that the asays were prone to false negatives. Another PCR proocol, Long PCR, previously was shown to be more eliable than Standard PCR for screening of insects or another bacterium (Wolbachia). The sensitivity of ong and Standard PCR protocols was compared by se of plasmid DNA containing two DNA fragments rom the greening disease agent or plasmid mixed ith DNA extracted from host plants, psyllids, or paraitoids. Results indicated that inhibitors of the PCR ere present in both plant and insect DNA, making he Standard PCR relatively insensitive and allowing igh levels of false negatives. Long PCR, which incororates a second DNA polymerase with proof-reading ctivity, yielded consistent results and was orders of agnitude more sensitive than the Standard PCR. As ew as 100 copies of plasmid mixed with either plant or nsect DNA consistently could be detected. Long PCR ssays conducted on pooled and individual T. radiata nd D. aligarhensis, their psyllid hosts, or their host lants over a period of 6 months failed to produce any ositives, indicating that release of these two parasioids should elicit little concern that greening bacteia would be introduced accidentally into Florida hrough this classical biological control program.