Indentification Of Several Phytoplasmas In Nanjing And Preliminary Study On Pathogenic Mechanism Of Paliurus Hemsleyanus Witches' Broom Phytoplasma

Phytoplasmas(Mycoplasma-like organism),belong to Mollicutes,Procaiyoate.They are obligated to inhabit sieve elements in the phloem of plants and are mainly transmitted between plants by phloem-feeding insects.Owing to the more difficulty in axenic culture in vitro,the detection and identification of phytoplasma are uaually dependent on molecular biology.At present,PCR detection and analysis employed by the highly conserved 16S rDNA are the principal classification methods of phytoplasma.Both PCR sequencing analysis and PCR-RFLP analysis based on 16S rDNA have been widely accepted and applied for the preliminary classification of phytoplasma.Subsequently,several relatively less-conserved genes,such as rp,tuf,secA,secY genes,have also been gradually used in the classification of the phytoplasma subgroups and strains which usually have closer genetic relationship.In this study,several diseases infected suspected-phytoplasma in Nanjing were detected and the pathogens were identified by the molecular methods.Our results provided a theoretical basis for further research on the occurrence,the propagation characteristics and the integrated control of these diseases.Firstly,the distribution characteristics of phytoplasma in plants were evaluted by DAPI staining and the specific fluorescent spots were observed in the vicinity of the phloem within the tender stem of the diseased Violet Orychophragmus.Using the universal primers of phytoplasma,the 16S rDNA,rp,tuf,sec A,secY gene fragments were successfully amplified from the diseased Violet Orychophragmus by nested PCR separately.The resulted sequence alignment and phylogenetic analysis showed that the phytoplasma in the diseased Violet Orychophragmus clustered with the members of the 16SrV-B subgroup phytoplasmas and belongs to the 16SrV-B subgroup,which were in accordance with the virtual RFLP analysis results based on 16S rDNA.Secondly,using the designed primers based on sec A gene and the universal primers of phytoplasma 16S rDNA and rp gene,we successfully amplified the 16S rDNA,rp and secA gene fragments from the diseased Paliurus hemsleyanus.The resulted sequence alignment and phylogenetic analysis also showed that the phytoplasma in the diseased Paliurus hemsleyanus constituted a clearly distinct evolutionary branch with the members of the 16SrV-B subgroup phytoplasmas and belongs to the 16SrV-B subgroup,which were also supported by the virtual RFLP analysis results based on 16S rDNA.In addition,phytoplasma 16S rDNA fragments were also successfully amplified from the diseased Trachycarpus fortunei,Petunia hybrida,Prunus persica,Hosta plantaginea and bamboo through phytoplasma-specific 16S rDNA universal primers separately.The obtained approximately 1.2 kb products were also consistent with the sizes of expected objective genes.The resulted sequence alignment,RFLP analysis and phylogenetic analysis revealed that all of these five phytoplasmas in the diseased Trachycarpus fortunei,Petunia hybrida,Prunus persica,Hosta plantaginea and bamboo clustered with the members of the 16SrV group phytoplasmas and their gene sequences have the relatively high similarity.So they belong to the 16SrV group and were temporarily classified as different phytoplasma strains.Finally,in this research,the potential distribution regions of 16SrV group phytoplasmas in Nanjing were predicted based on the reported data of 16SrV group phytoplasma diseases by using Maxent model and ArcGIS.Both the prediction results and the detection results showed that the central and northern regions in Nanjing were at moderate risk of the pathogen establishment,while the southern regions in Nanjing were more adjacent to the high risk area,and more suitable for the 16SrV group phytoplasma diseases.Studying the pathogenesis of phytoplasma is extremely important for the prevention of phytoplasma.In the disease process,the changes of enzyme activity and metabolite content in plants could reflect the interaction between hosts and pathogens to a certain extent.The enzymes and metabolites are usually involved in stress and antioxidation response in plants and are helpful to understand the pathogenic mechanisms of phytoplasma on the physiological and biochemical aspects.The changes of CAT,POD,PPO,PAL,APX,SOD enzyme activities and the MDA,soluble sugar,soluble protein contents in the healthy and diseased plant leaves were measured in different growth stages of Paliurus hemsleyanus.The results showed that the activities of the aboved enzymes and the contents of MDA and soluble sugars increased on several levels in the diseased plants,comparing with the healthy plants.However,the contents of soluble proteins reduced in the diseased plants.Except of the PPO enzyme,the activities of the other enzymes and the contents of MDA,soluble sugars,soluble proteins in plant leaves exhibited relatively significant changes in different growth stages of Paliurus hemsleyanus.With the growth and reproduction activities of phytoplasma,the severity of plant disease increased gradually and these enzyme activitities and these metabolite contents usually changed more significantly.The thymidylate kinase(TMK),catalysing the transfer of a terminal phosphoryl group from ATP to dTMP to form dTDP,is crucial to the salvage pathways for pyrimidine deoxyribonucleotides in the process of DNA replication in phytoplasma.TMK exists in the various organisms and plays an essential role for the the survival of phytoplasma.In this research,Paliurus hemsleyanus witches' broom(PahWB)phytoplasma,which belongs to the 16SrV-B subgroup,was selected to study its tmk gene and this gene was successfully cloned from PahWB phytoplasma.The resulted sequence alignment also showed that the nucleotide sequences were completely consistent between PahWB phytoplasma tmk gene and JWB phytoplasma tmk-Y gene.The amino acid sequences were aligned and analyzed among PahWB phytoplasma TMK and the other phytoplasma TMKs already reported.The results showed the amino acid sequence of PahWB phytoplasma TMK had the relatively high similarity with those of OY phytoplasma TMK-a,WBD phytoplasma TMK-1,PaWB phytoplasma TMK-a-1 and PaWB phytoplasma TMK-a-2,while the sequence had thelow similarity with those of OY phytoplasma TMK-b,WBD phytoplasma TMK-2 and PaWB phytoplasma TMK-b.At the same time,PahWB phytoplasma TMK was successfully expressed in E.coli and approximately 28 kDa fusion proteins were obtained after purification.The results of TMK catalytic activity using an enzyme-coupled assay showed that the PahWB phytoplasma TMK had almost no activity.The reason was that they might play an important role in other pathways of nucleotide biosynthesis as other kinases of phytoplasma in vivo.These results of the PahWB phytoplasma TMK will contribute to the further study of antiserum preparation,the function of this TMK in the PahWB phytoplasma and the interaction mechanism between PahWB phytoplasma and the infected host plants.