Detection And Identification Of Woody Plant Yellows Phytoplasmas And Molecular Characterization Of One Plasmid From Chinaberry Witches'-broom Phytoplasma

Lots of plants were infected by phytoplasma in China. Phytoplasmal diseases have caused devastating losses in wood production and forestry economy. Some plant diseases symptomed yellows which were thought to be physiological abnormalities were found to be caused by phytoplasma. There was lower concentration of phytoplasma in tissues of woody plants symptomed yellows than in those symptomed witches'-broom. Therefore, it became considerably difficult to detect phytoplasmas in woody yellowing plants. How to detect and identify phytoplasma as an agent of yellows diseases was very importment for us to effectively control phytoplasma diseases.Much more purer DNA was extracted from yellowing peach and other yellowing woody plants collected from Beijing and Chengdu with modified DNA extraction method. 1.4kb fragments of 16S rDNA could not amplified by direct PCR with primer pair R16mF2/R16mR1. Subsequently, 1.2kb fragments of 16S rDNA were amplified by nested PCR with primer pair R16F2/R16R2 from yellowing peaches collected from Summer Palace, Huairou and yellowing cascara from Beijing Botanical Garden. Sequence similarity alignments of 16SrDNA indicated that phytoplasmas from yellowing peach and cascara were blong to 16SrI group and have 99.2-99.5% homology with 16S rDNA of paulownia witches'-broom phytoplasma. With primer pair R16mF2/R16mR1, an unclear 1.4kb band of 16SrDNA was detected from DNA extracted from enriched phytoplasma of yellowing peachs which were obtained from Huairou by differential centrifugarion. When healthy peach stock was grafted with yellowing peach as scions, it did not show yellows symptom and phytoplasmas could not be detected by nested PCR in grafted stock after 35 and 90 days. Cherry lethal yellows phytoplasmas were successfully transmitted to healthy cherry stock from tissue culture seedling scions. Phytoplasmal distribution in grafted cherry stock of different period and position was studied by PCR. Additionally, concentration of cherry lethal yellows phytoplasmas in grafted cherry stock, roots and leaves of tissue culture plantlet as well as field plantlet were measured by taqman realtime quantitative PCR. The results showed that high titer phytoplasma existed in new axillary bud nearest to grafted position, roots and leaves of tissue culture plantlet, and low titer phytoplasma existed in lateral roots of grafted postion of cherry stock. Tissue culture method was a good way to preserve CLY phytoplasmas.We investigated peach yellows disease in peach orchards of Donggancheng and Xiaowanggu in Puyang, Henan province. Based on plant nutrients, tetracycline copharlan treatment, PCR detection, fertilization and application of paclobutrazol, we concluded that peach yellows disease in Donggancheng was likely caused by lack of some nutritional elements in stead of phytoplasmas. Although phytoplasmas were detected in some yellowing peach trees in Xiaowanggu , major etiology of peach yellows diseases was that the body of tree lacked of nutritional elements.Several phytoplasmas from different plants were detected with antiserum against to pPaWBNy-1 ORF4 protein by western blot. A 34KDa protein band in paulownia witches'-broom phytoplasma Nanyang strain (PaWBNy) was detected and a 90 protein KDa nonspecfic protein band was detected both in PaWBNy-infected and healthy paulownia. 49 KDa and 45 KDa protein bands were detected in periwinkle virescent phytoplasma-Hainan strain (PeVHn) and 45 KDa protein bands was in jujube witches'-broom phytoplasma-Beijing strain (JWBBj), while no protein band was showed in Chinese chestnut yellow crinkle, cherry leathal yellows phytoplasma-Xichang strain (CLYXc) and other healthy plants.One complete circle plasmid designated as pCWBFq from chinaberry witches'-broom phytoplasmas was determined. Analyses of protein characterization indicated that four proteins were predicted to be secretory proteins or membrane proteins with hydrophobic structure except for RepA and SSB proteins relevant to plasmid replication. Homologous comparison among 25 phytoplasmal plasmids showed that pCWBFq have highest homology 71.02% with pPaWBNy-1. Six proteins encoded by pCWBFq had high homology with proteins encoded by other phytoplasmal plasmids. Exceptionally, pCWBFq P2 had 91.9% homology with AYWB 404 protein encoded by chromosomal DNA of aster yellows witches'-broom phytoplasmas. pCWBFq clustered together with other plasmids from 16SrI group in phylogenetic tree based on complete sequence of all determined plasmids. The same phylogenetic tree was constructed based on 16S rDNA sequences. Phylogenetic trees based on DNA and proteins sequences of six ORFs indicated that RepA could not be used to differentiate 16Sr group because phylogenetic tree based on RepA was not in accordance with those based on 16S rDNA and plasmid complete sequences. Phylogenetic tree based on ORF3 was identical with phylogenetic tree based on plasmid complete sequences and phylogenetic trees based on ORF2-ORF6 were identical to that based on 16SrDNA.Southern blotting with pCWBFq repA probe confirmed the existence of the plasmids in CWBFq. The hybridizations occurred with paulownia witches'-broom phytoplasma-Nanyang strain (PaWBNy), periwinkle virescence phytoplasma-Hainan stanin (PeVHn), chinaberry witches'-broom phytoplasma-Fuzhou strain (CWBFz) and mulberry dwarf phytoplasma–Puyang strain (MDPy). No hybridizarions occurred with jujube witches'-broom phytoplasma-Beijing strain (JWBBj), cherry lethal yellows phytoplasma-Xichang strain (CLYXc) and Bischofia polycarpa witches'-broom phytoplasma-Nanchang strain (BiWBNc). So we concluded that CWBFq, PaWBNy, PeVHn, CWBFz and MDPy phytoplasmas contained distinct plasmids in terms of number and size. The number and size of plasmids among different phytoplasmas or different strains of the same phytoplasma were distinct. Plasmid was not detected in JWBBj, CLYXc and BiWBNc, perhaps as a result of low homology among repA genes in plasmids of JWBBj, CLYXc and BiWBNc.