Detection of pineapple bacilliform virus using the polymerase chain reaction

Abstract

Summary.A polymerase chain reaction (PCR) assay has been developed to detect pineapple bacilliform virus (PBV) in extracts from infected plants. Initially, degenerate primers were designed from conserved badnavirus amino acid sequences and used in PCR with partially purified PBV preparations. A 448 bp DNA fragment was amplified from a region in the reverse transcriptase and ribonuclease H genes. The nucleotide sequence of the cloned PCR product indicated that PBV was related to, but distinct from, other badnaviruses. Specific primers designed from the PBV sequence yielded a 403 bp fragment when used in PCR with extracts from infected pineapple plants, but not from plants infected with sugarcane bacilliform or banana streak viruses (genus Badnavirus). The specificity of the PCR product was confirmed by Southern hybridisation using a digoxigenin labelled DNA probe. PBV appears to be present in all pineapple growing areas along the east coast of Australia. PBV was detected in plants grown from seeds, plants propagated through meristem tip culture and in mealybugs which were collected from infected plants. PBV was detected in crown, leaf and root tissue from infected pineapple plants. PCR results of all field‐infected samples were confirmed by immunoelectron microscopy.