Molecular Characterization And Variability Of Jujube Witches'-broom Phytoplasmas From Different Jujube Cultivars In Various Regions Of China

Zizyphus jujuba Mill,a popular cultivated species, is indigenous to China and have a very long planting history. It has wide distribution ranges and more than 700 cultivars or lines. Jujube witches'-broom caused by phytoplasma is a destructive disease in many jujube cultivation regions, disturbing the development of jujube fruit production and richness of the jujube growing farmers. It is known that there are distinct resistances against phytoplasma in various jujube cultivars growing in different geographic regions. To make clear the kinds of phytoplasma and genetic diversity for the purpose of better application of resistance germplasm resources and disease control, molecular identification and variability of JWB phytoplasma population were studies using different phytoplasma stains from different jujube cultivars of various regions.To understand the conserved gene variability and genetic diversity of jujube witches'-broom phytoplasma population from the different jujube cultivars and regions of China, 32 samples collected from 14 cultivars jujubes or wild sour jujubes (Z. spinosa Hu) in 7 regions were detected by PCR with the primers R16mF2/R16mR1 for phytoplasma 16SrDNA, SR1/SR for16S-23SrRNA space region (SR), FD9f/r for secretion proteins (secY). Then the direct sequencing of PCR products and sequencing by cloned PCR products were used for analysis of sequence polymorphism and phylogenetic analysis by comparison to the databases of known conserved gene sequences. The results showed that the phytoplasmas were easily detected by PCR amplification of 16SrDNA from all the symptomed jujube samples. All the phytoplasma strains infecting various jujube cultivars belonged to subgroup 16SrV-B and were more related to Bischofia polycarpa witches'-broom and cherry lethal yellows phytoplasmas occurred in China than other phytoplasmas of elm yellows group in other countries. The sequence polymorphism at different extent in 16SrDNA, SR and secY gene and genetic diversity were revealed in phytoplasma strain population related to different habitats, among which the dominant strains were always detected by the direct sequencing of PCR products in all the diseased areas of China. SecY gene of collected phytoplasma strains was more various than 16SrDNA and SR sequences, and some base substitutions did not change encoded amino acid, however, certain single base deletions detected in a Shandong and a Beijing strains may have impact on the gene structure or function. Compared with direct sequencing of PCR products, sequencing after PCR products cloned to plasmid vector was showed to be more available for the displaying of variants and phylogeny in phytoplasma strain population. The results from the sequence analysis of EF-TU gene (tuf) of two strains and 23SrDNA of one strain of JWB coincided with those of other conserved gene analysis.Thymidylate kinase (TMK, EC 2.7.4.9), catalyses the transfer of a terminal phosphoryl group from ATP to dTMP, is crucial to both the de novo synthetic and the salvage pathways for pyrimidine deoxyribonucleotides. By using the methods of both direct sequencing of PCR products and sequencing by cloned PCR products, three types of thymidylate kinase genes, tmka, tmkb and tmkc (TMK gene), were identified in jujube witches'-broom phytoplasma strains. There were three main tmk gene forms of both tmka and tmkb detected in different jujube cultivars and various regions, tmka-dominant (AA) distributing in Beijing, Zanhuang county of Hebei province, Jishan county of Shanxi, Ningyang county of Shandong province and Tianjin, tmkb-dominant (BB) in Tang, Xingtang and Fuping Counties of Hebei and Nanyang city of Henan province and tmka-tmkb heterozygous (AB) in Xuanwu and Haidian districts of Beijing, Tang, Pingshan, Xingtang and Fuping counties of Hebei, Puyang and Luoyang cities of Henan,Yongji county of Shanxi and Jian city of Jiangxi province. Eleven strains of AA, four strains of BB and 19 strains of AB were identified in all the 34 jujube cultivars, and the corresponding percentage were 32.3%, 11.7%, 55.9%, respectively. The third kind of tmk gene, tmkc, was cloned and identified only from the sample BJLZ, an unique phytoplasma strain infecting an old protected jujube tree of cultivar"Dragon jujube"in the Beijing City . Type AA seems to be detected mainly in susceptible cultivars, while type AB was likely to be much more related to resistant cultivars. PCR-RFLP analysis of full-length DNA of tmk genes, SSCP analysis of the partial DNA fragments and selective PCR amplification of tmka or tmka gene from various strains using specially designed primers totally verified obtained results about JWB phytoplasma tmk genes.The reseason that determined the formation of genotype diversity and gene status of tmk genes might be jujube cultivars and cultivation environment as well as gene recombination generated by allocation and transportation of germplasm materials among different regions.Both tmka and tmkb genes contain 639 nucleotides with deviant A+T preference, and encode the deductive proteins of 212amino acids. There are differences of 14 different nucleotides ranging from position 303 to 369, a distinguishing variable domain and 5 amino acids between tmka (TMKa) and tmkb (TMKb). Tmka and tmkb shared 97.8% nucleotide acid identity, and and 97.6 % amino acid identity. The cloned tmkc gene fragment, containing 654 nucleotides and encoding 216 amino acids shared 59.8% nucleotide identity and 47.5% amino acid identity with tmka,and share 59.5% nucleotide identity and 47.9% amino acid identity with tmkb. All deduced proteins, TMKa, TMKb and TMKc contain three specific motifs, P-loop domain involved in the binding of ATP and other phosphoryl donors, TMP-binding motif and the LID region, which are conserved in orther thymidylate kinases. Helix and coil were main secondary structure in all three TMKs, and no disulfide bond formed in them. No transmembrane region and signal peptide are found in the JWB TMKs and they are hydrophilic proteins localized in the cytoplasm of phytoplasma. Theoretical pI values of TMKa/TMKb/TMKc are 10.075/9.965/9.765, respectively.Phylogenetic analysis of DNA and deduced amino acid sequences to all the known gene sequences of 34 phytoplasmas tmks and 29 other organism tmk genes revealed that all the JWB tmk genes belonged to cluster I, rather than cluster II and III. Tmka and tmkb have the closest relationship with tmk-a gene(AB010446) in OY-W strain of onion yellows and tmkc have the closest relationship with OY-M-tmk2(AP006628,391588…392202) and AY-tmk3 (NC₀07716, 203228…203860).