Genetic characterization and classification of two phytoplasmas associated with spartium witches'-broom disease

In plants of Spanish broom (Spartium junceum) affected by an etiologically uncertain witches'-broom disease, plant-pathogenic phytoplasmas were detected by fluorescence and scanning electron microscopy. Restriction fragment length polymorphism (RFLP) analysis of ribosomal DNA, polymerase chain reaction (PCR)-amplified by means of universal phytoplasma primers, revealed two different phytoplasmas present in plants with similar symptoms. The less frequently detected phytoplasma had the same RFLP profile as elm yellows phytoplasmas, whereas the prevalent agent had an RFLP profile similar to that of phytoplasmas in the apple proliferation strain cluster. Sequence analysis of 16S rDNA confirmed that the prevalent Spartium witches'-broom phytoplasma is a member of the apple proliferation strain cluster, but this organism is distinctly different from other cluster members. With universal, group- and pathogen-specific rDNA primers, the causal agents were detected in most symptomatic plants. The plants testing negatively in direct PCR yielded an amplification product when the rDNA fragment obtained with universal primers was re amplified with group- or pathogen-specific primers. The nested-PCR assay also revealed that most of the plants examined were doubly infected with the two phytoplasmas detected by direct PCR. In the infected plants, one of these organisms was predominant and readily detectable by direct PCR while the other occurred in low numbers and could only be identified by nested PCR.