Phytoplasmas in Australian grapevines - detection, differentiation and associated diseases
Keywords:phytoplasma, Australian grapevine yellows, nested PCRs, RFLP
AbstractPhytoplasmas associated with Australian grapevine yellows (AGY) symptoms were detected using the polymerase chain reaction (PCR). To optimise the diagnostic, nested PCRs were compared with single PCRs using different primer pairs. Grapevine DNA known to be AGY phytoplasma positive was serially diluted and subjected to nested and single round PCR tests to determine which was the most sensitive. Samples taken over two growing seasons were used to determine the optimum sampling time for phytoplasma detection. The specificity of primer pairs was determined using phytoplasmas detected in Australian grapevines and overseas reference grapevine phytoplasmas. DNA extracted from grapevine exhibiting a range of symptoms was screened for phytoplasmas. Two different phytoplasmas were amplified in the PCR and they were identified using specific PCR primers and by restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene and 16S rRNA/23S rRNA spacer region. RFLP analysis confirmed that one phytoplasma was the AGY phytoplasma and the other phytoplasma was indistinguishable from the tomato big bud (TBB) phytoplasma. The AGY phytoplasma was associated with AGY symptoms but was occasionally detected in asymptomatic vines and those with late season leaf curl (LSLC) and restricted growth (RG) symptoms. The TBB phytoplasma was detected in some vines with LSLC symptoms and very occasionally in vines with AGY symptoms. A 'variant' of the AGY phytoplasma was also detected in vines showing typical AGY symptoms.
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