Detection of phormium yellow leaf phytoplasma in New Zealand flax (Phormium tenax) using nested PCRs

A reliable diagnostic method was developed for use in studying the relationship between phormium yellow leaf disease of New Zealand flax (Phormium tenax) and its associated phytoplasma (phormium yellow leaf phytoplasma: PYL). Diagnosis involved a nested PCR (polymerase chain reaction) technique targeting the 16S rRNA gene. DNA was extracted from woody rhizome tissues of NZ flax plants using CTAB and a high salt precipitation step. This method effectively eliminated polysaccharides, gum-like material and other compounds inhibitory to PCRs that occur at high concentrations in diseased NZ flax rhizomes. PCR competence of each DNA preparation from both healthy and yellow leaf diseased plants was assessed using the general prokaryotic 16S rRNA gene primers, Gd1/Berg54. These primers amplified DNA from both diseased and healthy plants. PYL 16S rDNA sequences were not detected consistently following amplification by PCR (35 cycles) using the 'universal' phytoplasma-specific primer pairs R16F2/R16R2 or P1/P6. By contrast, PYL was consistently detected in diseased, but not healthy, NZ flax plants, following nested PCR of the products of the above three primer pairs. Nested PCRs involve the primers NGF/NGR, which were designed to hybridize with all phytoplasmas for which published sequences were available. The most sensitive level of detection by nested PCR was achieved using primers R16F2/R16R2, rather than primers P1/P6 or Gd1/Berg54, for the primary amplification step. The consistent association found in this study between yellow leaf disease and PYL further substantiates this phytoplasma as the causal agent. PCR products of the expected size were also amplified by nested PCR using the primers R16F2/R16R2 followed by NGF/NGR from C. roseus tissues infected with five other phytoplasmas representing three distinct phytoplasma groups. Therefore nested PCRs with these Fairs of primers should be useful for detecting other phytoplasmas, in particular those occurring at low concentrations or in recalcitrant tissues.