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 rel ationship between phormium yellow leaf disease of New Zealand flax (Ph ormium tenax) and its associated phytoplasma (phormium yellow leaf phy toplasma: PYL). Diagnosis involved a nested PCR (polymerase chain reac tion) technique targeting the 16S rRNA gene. DNA was extracted from wo ody rhizome tissues of NZ flax plants using CTAB and a high salt preci pitation step. This method effectively eliminated polysaccharides, gum -like material and other compounds inhibitory to PCRs that occur at hi gh 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/Ber g54. These primers amplified DNA from both diseased and healthy plants . PYL 16S rDNA sequences were not detected consistently following ampl ification by PCR (35 cycles) using the 'universal' phytoplasma-specifi c primer pairs R16F2/R16R2 or P1/P6. By contrast, PYL was consistently detected in diseased, but not healthy, NZ flax plants, following nest ed PCR of the products of the above three primer pairs. Nested PCRs in volve the primers NGF/NGR, which were designed to hybridize with all p hytoplasmas for which published sequences were available. The most sen sitive level of detection by nested PCR was achieved using primers R16 F2/R16R2, rather than primers P1/P6 or Gd1/Berg54, for the primary amp lification step. The consistent association found in this study betwee n yellow leaf disease and PYL further substantiates this phytoplasma a s the causal agent. PCR products of the expected size were also amplif ied by nested PCR using the primers R16F2/R16R2 followed by NGF/NGR fr om C. roseus tissues infected with five other phytoplasmas representin g 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 t issues.