Molecular detection of the Australian grapevine yellows phytoplasma and comparison with grapevine yellows phytoplasmas from Italy

Abstract

A diagnostic test using the polymerase chain reaction is described for the detection of phytoplasma DNA in grapevines collected from South Australia and Victoria. Grapevines with Australian grapevine yellows disease tested positively for a phytoplasma but those with ‘restricted spring growth syndrome’ (formerly called ‘grapevine decline’) tested negatively. Restriction fragment length polymorphism analyses were done to determine the relationships between phytoplasmas of the Australian grapevine yellows and of representatives from both the aster yellows group (which includes phytoplasmas of grapevine yellows from Italy) and the elm yellows group (which includes phytoplasmas of flavescence dorée). Results showed that Australian grapevine yellows is associated with a unique phytoplasma that is more closely related to the phytoplasmas of the aster yellows group than to those of the elm yellows group.

‘Candidatus Phytoplasma’, a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects

“…Oligonucleotides that have been shown to specifically target 'Ca. Phytoplasma' 16S rRNA genes in PCRs have been published (Deng & Hiruki, 1991;Ahrens & Seemüller, 1992;Firrao et al, 1993;Lee et al, 1993b;Namba et al, 1993a;Schneider et al, 1993;Padovan et al, 1995;Gundersen & Lee, 1996). All strains of 'Ca.…”

Plant hosts of the phytoplasmas and rickettsia-like-organisms associated with strawberry lethal yellows and green petal diseases

“…Total DNA was extracted from plant samples according to Doyle and Doyle (1990) using a modified CTAB buffer (Padovan et al 1995). Deoxyribonucleic acid quality was determined by subjecting the samples to electrophoresis in a 1% agarose gel, which was then stained with ethidium bromide and viewed by UV trans-illumination.…”

“…Plant samples were screened for phytoplasmas using the primer pairs fP1 / rP7 (Deng and Hiruki 1991;Schneider et al 1995) and fU5 / m23sr (Lorenz et al 1995;Padovan et al 1995), which amplify the phytoplasma 16S rRNA gene and 16S-23S spacer region. The PCR reactions were prepared according to Schneider et al (1997) and subjected to 35 cycles of 95 • C / 1 min; 55 • C / 1 min and 72 • C / 1.5 min.…”

‘Candidatus Phytoplasma spartii’, ‘Candidatus Phytoplasma rhamni’ and ‘Candidatus Phytoplasma allocasuarinae’, respectively associated with spartium witches'-broom, buckthorn witches'-broom and allocasuarina yellows diseases

“…PCR products were purified by using a QIAquick gel extraction kit (Qiagen), according to the manufacturer's instructions. Sequencing of both strands was performed by using an ABI Prism Big Dye Cycle Sequencing Ready Reaction kit (Applied Biosystems) with the following primers: P1, fP3, fU5, 16R723f and m23SR (Deng & Hiruki, 1991;Lorenz et al, 1995;Padovan et al, 1995). Sequences were then assembled and edited by using MacVector and Assembly Lign (Eastman Kodak) and a consensus sequence was generated.…”

“…AY135523) had been determined previously by Gibb et al (2003), was resequenced in order to obtain the full-length 16S rDNA and 16S-23S rDNA spacer region sequences. For this purpose, rDNA of the AlloY pathogen was amplified by PCR using the universal phytoplasma primer pairs P1/rU3 (Deng & Hiruki, 1991;Lorenz et al, 1995) and fU5/m23SR (Lorenz et al, 1995;Padovan et al, 1995). PCR products were purified by using a QIAquick gel extraction kit (Qiagen), according to the manufacturer's instructions.…”