Detection of pineapple bacilliform virus using the polymerase chain reaction

Abstract

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.

Novel Approaches for the Management of Mealybug Pests

“…Several mealybug species are vectors of viral diseases of various crops: banana (Kubiriba et al 2001;Thomson et al 1996;Watson and Kubiriba 2005), black pepper (Bhat et al 2003), cocoa (Dufour 1991;Entwistle and Longworth 1963;Hall 1945), grapevine (Cabaleiro and Segura 1997a, b;Cid et al 2007;Garau et al 1995;Minafra and Hadidi 1994;Petersen and Charles 1997;Sforza et al 2003;Tsai et al 2008;Zorloni et al 2006), pineapple (Sether and Hu 2002a, b;Sether et al 1998Sether et al , 2005, rice (Abo and Sy 1998), and sugarcane (Lockhart et al 1992). …”

Optimised cetyltrimethylammonium bromide (CTAB) DNA extraction method of plant leaf with high polysaccharide and polyphenolic compounds for downstream reliable molecular analyses

“…In this study, the PCR-based diagnostic assay used for the detection of badnavirus sequences was similar to those used successfully for detection of many badnavirus sequences in different tropical food crops such as banana (Geering et al, 2000), cacao (Muller et al, 2001), pineapple (Thomson et al, 1996), sugarcane (Braithwaite et al, 1995), taro (Yang et al, 2003) and yam (Eni et al, 2008;Kenyon et al, 2008;Lima et al, 2013). All the PCR tested samples collected from IITA were badnavirus positive.…”

The genetic diversity of ampeloviruses in Australian pineapples and their association with mealybug wilt disease

“…Wakman et al (1995) also observed two distinct serotypes of closterovirid from Australian pineapples, which were named Pineapple closterovirus A and B, but it is not known how these viruses relate to the viruses described from Hawaii. During this study, a bacilliform-shaped virus was also detected in pineapples in Australia (Wakman et al 1995), which from particle size and morphology, and later, sequence analysis (Thomson et al 1996), was most likely a member of the genus Badnavirus. PMWaV-1 was detected in a small number of samples of Australian pineapples but PMWaV-2, -3 and -4 have not previously been reported.…”