Molecular Identification Of A Phytoplasma Associated With Podocarpus Nagi Fasciation In Hainan

The fasciation disease of Podocarpus nagi (Thunb.) Zoll. et Mor ex Zoll was found recently in the Tropical Botanical Garden, Wanning, Hainan. The major symptom associated with fasciation of P. nagi was the shoot apical meristem broadened with flat strip in cristante., The symptom of this fasciation disease was similar to the symptom that caused by phytoplasma. The objective of this study was to classification and identification of the pathogen associated with fasciation of P. nagi using histochemistry detection, transmission electron microscope observation and molecular methods. The main results were as follows:The tender stem of P. nagi with fasciation disease symptoms were observed under laser scanning confocal microscope(LSM) after stained by DAPI. The specific phytoplasma DNA fluorescent spots were observed in the sieve of phloem of fasciation diseased plants, while there was no specific phytoplasma fluorescence in the healthy P. nagi. The initially outcome showed that fasciation disease in P. nagi maybe caused by phytoplasma. Typical phytoplasma-like bodies were observed under transmission electron microscope in the phloem cells within the tender stem of P. nagi with fasciation disease symptoms, which was corresponded with the histochemistry detection result.Universal primers of P1/P7and R16F2n/R16R2for phytoplasmal16SrRNA gene were used to detect phytoplasma by nested PCR respectively. The1.2kb DNA fragments were amplified from the total DNA of P. nagi with fasciation disease symptoms, which were collected from the Tropical Botanical Garden, Wanning, Hainan. Sequence analysis determined that the P. nagi with fasciation disease was caused by a phytoplasma (GenBank accession:KP027298), which shared99.4%similarity of the 'Candidatus Phytoplasma australasiae'reference strain (GenBank accession:Y10097). Furthermore, virtual RFLP analysis showed that the phytoplasma belongs to a new subgroup within the peanut witches'-broom (16Sr ?) group, the most similar is the reference pattern of the16Sr group II, subgroup A (GenBank accession:L33765), with a similarity coefficient of0.97. Primer ftufu/rtufu for phytoplasmal tuf gene was used for PCR amplification. The product of tuf gene fragment was about845bp (GenBank accession:KR633146). This fragment was more similar with the16Sr group ? with Christmas cactus witches'-broom phytoplasma (GenBank accession:GU369818) after DNA sequence comparative, phylogenetic, which shared86.86%similarity,93.24%amino acid homology,respectively. This is the first report to identify the classification status of a phytoplasma in fasciation disease of P. nagi, which is important for effective prevention of the disease.