Molecular Detection And Identification Of Phytoplasmas Associated With Four Ornamental Plant Diseases

Phytoplasma comprise a large group of bacteria that lack a cell wall, belonging to Mollicutes, Procaiyotae. They reside and propagate in the sieve cells of plant phloem tissue and the most phloem-feeding insect vectors such as Leafhoppers and planthoppers. Owing to the difficulty in culturing phytoplasma in cell-free media, and the consequent inaccessibility of measurable phenotypic characteristics that are traditionally used for the differentiation of culturable microbes, molecular biology-based methods have become the most reliable tools for the identification and classification of phytoplasma. Moreover, the highly conserved16S rDNA sequence has been primarily employed for elucidating phytoplasma taxonomy. In particular, Lee and his colleagues provided a reliable method for the differentiation of phytoplasma by constructing the first comprehensive phytoplasma classification scheme based on restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified16S rDNA. After periodic updates and revisions, this16S rDNA PCR-RFLP-based system has become widely accepted for classifying phytoplasma strains. In this study, phytoplasmas associated with tree peony yellows, Chinese tallow tree yellows, camellia yellows and paper flower yellows were detected and identified using molecular methods.Tree peony(Paeonia suffruticosais) plants with yellowing symptoms suggestive of a phytoplasma disease were observed in Shandong Peninsula, China. Typical phytoplasma bodies were detected in the phloem tissue using transmission electron microscopy (TEM). The association of a phytoplasma with the disease was confirmed by PCR using phytoplasma universal primer pair P1/P7followed by R16F2n/R16R2as nested PCR primer pair. The sequence analysis, phylogenetic analysis and RFLP analysis indicated that the phytoplasma associated with tree peony yellows (TPY) was an isolate of’Candidatus. Phytoplasma solani’ belonging to the16SrXII (stolbur) group, and represented a new subgroup. Then we amplified the rp gene and tuf gene, the results of sequence and phylogenetic analyses were consist with that of the16S rRNA gene. This is the first report of a phytoplasma associated with tree peony.Branch and leaf samples of the Chinese tallow tree (Sapium sebiferum) displaying yellowing symptoms were collected from Tai’an, Shandong Province, China. Typical phytoplasma bodies were detected in the phloem tissue using TEM. The association of phytoplasma with yellowing disease was ascertained using nested PCR of the16S rRNA gene by using the phytoplasma-specific universal primer pair P1/P7, followed by R16F2n/R16R2 as nested primers, and rp genes primed using rpL2F3/rp(I)R1A followed by rp(III)-FN/rp(I)R1A. The sequence and phylogenetic analyses of the16S rRNA gene and rp genes revealed that the phytoplasma associated with the Chinese tallow tree belonged to the16SrIII group (the X-disease group). Computer-simulated and gel-based restriction fragment length polymorphism (RFLP) analyses revealed that the RFLP patterns were different from the reference patterns of all previously established16SrIII subgroups, with the similarity coefficient≤0.97. Thus, the phytoplasma associated with the Chinese tallow tree yellowing disease, designated as’CTTY’, represents a new subgroup. This study shows the Chinese tallow tree as a new host of phytoplasma belonging to the16SrIII group in China and worldwide.The camellia yellowing symptom was observed in Tai’an of Shandong province, China. TEM observation showed phytoplasmas in the phloem sieve tube elements of symptomatic plants but not in healthy ones. PCR with universal phytoplasma primers P1/P7followed by R16F2n/R16R2and Restriction RFLP analyses of16S rRNA gene F2n/R2fragment allowed us to classify the detected phytoplasma into the elm yellows (EY) group (16SrV), subgroup16SrV-B. Sequence analyses of the ribosomal protein (rp) gene confirmed the closest relationship with phytoplasmas belonging to the rpV-C subgroup. Thus, the phytoplasma associated with camellia yellows disease, designated as’CY’, is a member within the16SrV-B (rpV-C) subgroup.This is the first report of phytoplasma associated with camellia.Paper flower (Bougainvillea sp.) yellowing disease expressing symptoms of phytoplasma infection were sampled from Tai’an, Shandong province of China. The presence of phytoplasma was confirmed by amplification of16S rRNA gene (1244bp) using phytoplasma universal primer pair P1/P7followed by R16F2n/R16R2as nested PCR primer pair. Sequence analysis indicated that the paper flower yellowing (PFY) phytoplasma is a member of stolbur group (’Ca. Phytoplasma solani’), and belonging to16Sr XII subgroup A by RFLP analysis. Moreover, sequence analyses of the rp gene (1253bp) and tuf gene (946bp) confirmed the result. This is the first report of phytoplasma associated with paper flower.This is the first report of phytoplasma associated with tree peony, Chinese tallow tree, camellia and paper flower in the world.