| Phytoplasmas are kinds of cell wall-less prokaryotic microorganisms that cannot be maintained in axenic culture, causing many plant diseases with various host plants, widely geographical distribution and adverse impact on economics and environments. Those destructive diseases associated with phytoplasma are paulownia witches’-broom, jujube witches’-broom, chinaberry witches’-broom, mulberry dwarf, lettuce yellows. Therefore, it was of comparatively important ecological significance and economic value in phytoplasma mechanism of growth, propagation, genetic variation and the relationships that their interaction with hosts from the aspects of pathogen biodiversity, phylogeny, key gene regulation and control and analysis of disease-resistant substances of host plant. The results will be helpful to solve the basic theoretical problem of relevant diseases, to enhance disease control level, to guarantee sound development of agriculture and forestry.In the study, ten housekeeping gene(rp, tuf, secA, secY, ipt, dnaK, fusA, gyrB, pyrG and rpoB) fragments as compared with 16 S rDNA sequences of phytoplasma were employed to analyze the genetic variation and phylogenetic relationships of 18 phytoplasma strains infecting chinaberry, lettuce, mulberry, paulownia and periwinkle from ten provinces in China. Five phytoplasma strains, including onion yellows phytoplasma(OY-M), aster yellows witches’-broom phytoplasma(AYWB), Candidatus Phytoplasma australiense(CPA), strawberry lethal yellows phytoplasma(SLY) and Candidatus Phytoplasma mali(CPM), of which whole genomes have been sequenced, were used as references. Sequence polymorphism and variation levels of different gene fragments were analyzed by multiple sequence alignment. The results in the study showed that the nucleotide site polymorphisms of rp, tuf, secA, secY, ipt, dnaK, fusA, gyrB, pyrG and rpoB gene fragments resolved all strains into 15 sequence types(STs), demonstrating extensive genetic diversity among the 16 SrI group strain population. All the phytoplama strains, classified into 16SrI-B and-D subgroup by 16 S rDNA analysis were clustered into one clade and clearly differentiated into discrete subclades by phylogenetic analysis of the concatenated gene sequences. Ten CWB phytoplasma strains that were most closely related to two mulberry dwarf strains and hardly distinguished with 16 S rDNA, were definitely split into four distinct clusters and 8 STs apparently relative to their geographical origins. Two LY phytoplasma strains collected in Sanming, Fujian province, China, were more closely related to the onion yellows OY-M strain in Japan than the periwinkle virescence and paulownia witches’-broom strains in China. The levels of variation in dnaK locus were higher than those in 16 S rDNA and other genes tested.The unknown upstream sequence adjoining tuf gene in jujube witches’-broom phytoplasma(JWB) was amplified by thermal asymmetric interlaced PCR(TAIL-PCR). The upstream sequences adjoining tuf gene from paulownia witches’-broom phytoplasma(PaWB) and JWB was recombined with promoter-probe vector pSUPV4. The same methods were used to analyzed and identified genetic variation and promoter activity of tuf gene upstream sequences from 16 SrI group phytoplasma strains including Pa WB, chinaberry witches’-broom(CWB), lettuce yellows(LY), mulberry dwarf(MD) and periwinkle virescence(PeV) phytoplasma strains and 16 SrV group strains including JWB, cherry lethal yellow(CLY), Bischofia polycarpa witches’-broom(BiWB), Sophora japonica witches’-broom(SoWB) phytoplasma strains. The results in the study showed that the sequence length of intergenic region between tuf and its upstream gene fus A in 16 SrI group strains was 129 to 130 bp, with putative complete promoter conservative structure. 130 bp upstream sequence of tuf gene in PaWB-sdyz, LY-fjya1, PaWB-fjfz strains and 129 bp upstream sequence of tuf gene in CWB-hnsy1 strain had promoter activity. There were four variation types in the intergenic region sequences from five kinds of 16 SrI group phytoplasmas including 35 strains; the sequence length of intergenic region between fusA and tuf in 16 SrV group strains was 53 to 54 bp, with predicted incomplete promoter structure. No promoter activity was found in 144 bp and 346 bp upstream sequence of tuf gene in JWB-jsnj strain, 145 bp and 347 bp upstream sequence of tuf gene in JWB-hnpy strain. There were two variation types in the intergenic region sequences from four kinds of 16 Sr V group phytoplasmas including 21 strains. The fusA-tuf intergenic region sequences were comparatively conservative, and the phytoplasma strains belonging to different groups were distinguished clearly by the phylogenetic tree constructed based on the sequences.The long DNA fragments including tuf gene sequences and their upstream six genes, such as rplL, rpoB, rpoC, rps12, rps7 and fusA from Pa WB-sdyz, PaWB-fjfz and LY-fjya1 phytoplasma strains were further amplified by long fragment PCR perimers. Sequence characteristic of conserved regions of phytoplasma gene promoter studied in the work were revealed by statistic analysis. Transcription expression of phytoplasma tuf gene sequences and their upstream gene sequences were explored by the reverse transcription PCR(RT-PCR). The results in the study showed that the sequences, 12745-12748 bp in length, upstream tuf genes were amplified from PaWB-sdyz, PaWB-fjfz, LY-fjya1 strains. Comparative analysis results showed that the gene structure order of tuf gene and its upstream six gene sequences of PaWB-sdyz, Pa WB-fjfz, LY-fjya1, OY-M, AYWB, PAa, SLY, AT phytoplasma strains was 5’-rplL-rpoB-rpoC-rps12-rps7-fusA-tuf-3’. According to sequence characteristic of conserved regions of 52 phytoplasma gene promoters studied in the work, potential sequence pattern of conserved region of phytoplasma gene promoter was deduced, T90T100G92T75G67A85(-35 region); T90A96T92A98T73T90(-10 region). The phytoplasma strains of different plant hosts, or belonging to different groups and subgroups were clearly divided with comparatively high bootstrap values based on MLSA of coding genes, non-coding sequences in intergenic regions, and putative amino acid sequences of rplL-tuf nucleotide sequences. Genetic variation was comparatively higher in non-coding regions of rplL-tuf nucleotide sequences than that in coding sequences of rplL-tuf nucleotide sequences.Active phytoplasma-resistant subtances of resistant jujube leaves against jujube witches’-broom disease were extracted by phytochemistry method and the bacteriostatic activity of the jujube extracting solutions to some bacterium was detected. Disease-resistant relevant substances and their contents of jujube extracting solutions were detected and analyzed by high performance liquid chromatography(HPLC). The effect of different concentration jujube extracting solutions on the growth and symptoms of jujube witches’-broom and paulownia witches’-broom disease tissue culture seedlings was analyzed by treating the tissue culture plantlets with the extracting solutions. The results in the study showed that salicylic acid contents were detected with significant difference(P=0.000<0.05) in 20 jujube extracting solutions that had been extracted from the 20 extracting solutions of jujube with different resistance to JWB before or after grafting inoculation. Jasmonate acid was only detected in jujube leaves extracting solutions of scions 87, 138, moderate resistant lines(IV-V grades) that showed severe witches’-broom symptom, while the compound wasn’t detected in the jujube leaves extracting solutions including susceptible lines showing severe withches’-broom symptom, disease-resistant scion grafted disease stock lines showing light symptom(I-III grades) or renew no symptom(0 grade). Methyl salicylate and methyl jasmonate wasn’t detected in 20 disease-resistant and susceptible jujube extracting solutions. The growth of Bacillus firmus, B. subtilis, B. mycoides were comparatively clearly inhibited by different jujube extracting solutions. Inhibition zone diameter of B. mycoides treated with resistant scions 143, 55, 103 jujube extracting solutions was in 5.75~8.5 mm, with significant difference(P=0.004<0.05). Comparatively high concentration methyl alcohol and jujube extracting solutions in MS medium had lethal phytotoxicity to jujube witches’-broom and paulownia witches’-broom disease tissue culture seedlings. The growth and symptom of jujube witches’-broom and paulownia witches’-broom disease tissue culture seedlings were definitely influenced by treatment using extracts of certain jujube line with high resistance, not lethal phytotoxicity, when there was change of methyl alcohol or jujube extracting solution content in MS medium.It is suggested that the MLSA should potentially be a useful and reliable approach for phytoplasma identification and differentiation as well as for depth examination of strain diversity and wildly study of genetic variation and evolutionary relationships of various 16 Sr groups or subgroups phytoplasma strains in the future. The methods and results obtained from the study about phytoplasma promoter, adjacent gene arrangement and jujube disease-resistant substances would contribute to further investigate and understand the key genes structure arrangement,metabolism regulation and genetic variation of the phytoplasma. And the conclusions of the study were of comparatively important theoretical significance and actual value in providing insight into the problems about the phytplasma growth and propagation, habitat diversity and adaption, interaction with its plant host and insect vector, its pathogenicity mechanism, jujube resistance mechanism, further screening and development of new type cure agentia of jujube witches’-broom and other phytoplasma disease and sufficiently rational utilization jujube resources, strengthening plant disease resistance and reducing the loss of host plant disease resistance caused by pathogen variation, fundamentally improving comprehensive control of the phytoplasma diseases. |
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