Detection of Mycoplasma-like Organisms in Infected Potato by Electron Microscopy

By using electron microscopy pleomorphic mycoplasma-like bodies were observed only in the phloem cells of the field infected potato plants showing purple top roll symptoms. The bodies surrounded by unit membranes were 50–300 nm in diameter.     

Mycoplasmalike Organisms (MLOs) Associated With the Witches' Broom Disease of Poplar

The witches' broom disease has been recently observed on poplars in Paris and its suburbs. The incidence of the disease appeared to be considerably high along main roads. The electron microscopic examination of 350 nm thick sieve tube sections revealed the presence of wall-less mycoplasmalike organisms (MLOs) in diseased samples of Populus alba var. nivea Wesm. They could not be found in their healthy counterparts.     

Morphological Changes of Paulownia Seedlings Infected Phytoplasmas Reveal the Genes Associated with Witches' Broom through AFLP and MSAP

Paulownia witches'' broom (PaWB) caused by phytoplasma might result in devastating damage to the growth and wood production of Paulownia. To study the effect of phytoplasma on DNA sequence and to discover the genes related to PaWB occurrence, DNA polymorphisms and DNA methylation levels and patterns in PaWB seedlings, the ones treated with various concentration of methyl methane sulfonate (MMS) and healthy seedlings were investigated with amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP). Our results indicated that PaWB seedlings recovered a normal morphology, similar to healthy seedlings, after treatment with more than 20 mg·L⁻¹ MMS; Phytoplasma infection did not change the Paulownia genomic DNA sequence at AFLP level, but changed the global DNA methylation levels and patterns; Genes related to PaWB were discovered through MSAP and validated using quantitative real-time PCR (qRT-PCR). These results implied that changes of DNA methylation levels and patterns were closely related to the morphological changes of seedlings infected with phytoplasmas.     

Occurrence of Anomalous Mycoplasma-like Organisms in Grapevine Yellows-diseased Phloem

Pathological changes in Vitis vinifera cv. Caveccia phloem from leaves showing symptoms of a flavescence doreé-like disease consisted of obliteration, necrosis and collapse of the sieve elements and associated companion cells, and excessive callose accumulation in lateral sieve areas and sieve plates of apparently normal mature sieve elements. Unusual structures, also found in degenerate sieve elements of diseased leaf vein specimens, were strongly electron-dense and bounded by a unit membrane or an electron-transparent border, and considered to be senescent forms of mycoplasma-like organisms. The significance of these findings in relation to possible host responses to the yellows pathogen is discussed.     

Mycoplasma-like Organisms in Phloem Elements of Ammobium alatum

Using transmission electron and fluorescent microscopes, Mycoplasma-like organisms (MLOs) were found in phloem cells of stems and leaves of Ammobium alatum. The diameter of these organisms ranged from 0.12–0.73 μm and averaged 0.4 μm. Symptoms induced by MLOs included chlorosis and reddening of leaves and winged stems, plant stunting and flower phyllody. This is the first report of MLOs associated with disease symptoms in A. alatum.     

Occurrence of Anomalous Mycoplasma-like Organisms in Grapevine Yellows-diseased Phloem

Pathological changes in Vitis vinifera cv. Caveccia phloem from leaves showing symptoms of a flavescence doreé-like disease consisted of obliteration, necrosis and collapse of the sieve elements and associated companion cells, and excessive callose accumulation in lateral sieve areas and sieve plates of apparently normal mature sieve elements. Unusual structures, also found in degenerate sieve elements of diseased leaf vein specimens, were strongly electron-dense and bounded by a unit membrane or an electron-transparent border, and considered to be senescent forms of mycoplasma-like organisms. The significance of these findings in relation to possible host responses to the yellows pathogen is discussed.     

Ethylene Production in Sweet Potato Root Tissue Infected by Ceratocystis fimbriata

The rate of ethylene production by sweet potato (Ipomoea batatasLam. cv. Norin No. 1) root tissue infected with Ceratocystisfimbriata Ell. & Halst. increased markedly during incubationat 29?C under high relative humidity. During incubation thefungus progressively invaded root tissue. The rate of ethyleneproduction reached a peak two days after inoculation when thebrowning region that contained the penetrating mycelia had expandedinward about 0.3 mm from the surface, followed by a declinein ethylene production. Apparently, the 1-aminocyclopropane-1-carboxylicacid (ACC) synthase activity was not high enough, and the amountof ACC in the infected tissue was too low to account for thehigh rate of ethylene production throughout the incubation period.Ethylene production by the infected tissue showed scarcely anyinhibition by amino-ethoxyvinylglycine, a specific inhibitorof ACC synthase. These findings suggest that the pathway ofethylene biosynthesis that operates in infected sweet potatoroot tissue may differ from the methionine pathway in whichACC serves as an intermediate. (Received March 24, 1984; Accepted June 27, 1984)     

Production and Preliminary Application of Monoclonal Antibodies Against Paulownia Witches' Broom MLO

Monoclonal antibodies against mycoplasma-like organisms (MLO) associated with paulownia witches’broom (PWB) were produced by using partially purified preparations from diseased paulownia. Splenic cells from immunized mice were fused with sp2/0murine myeloma cells. Screened by indirect ELISA using partially purified PWB-MLO and healthy paulownia extracts as detecting antigens, two hybridoma clones that stably secreted specific antibodies against PWB-MLO were obtained from 459 clones of four successful fusions. The monoclonal antibodies were isotyped and determined to be immunoglubin classes IgG2a and IgG3. Antibody titers of ascitic fluids were both over 1.6 × 104 assayed by indirect ELISA. Priliminary application on several specimens proved that they were the monoclonal antibodies against PWB-MLO.     

花生丛枝植原体免疫膜蛋白基因克隆及细菌双杂交诱饵质粒构建

初步分析植原体免疫膜蛋白的结构,以Imp构建诱饵质粒,为研究植原体与寄主互作的分子机理,探讨植原体传播、侵染及在寄主内的运输方式奠定基础。根据本实验室获得的花生丛枝植原体免疫膜蛋白基因序列设计特异性引物Imp-F/Imp-R,通过PCR扩增获得花生丛枝植原体免疫膜蛋白基因,大小519 bp,编码蛋白含有172个氨基酸残基,与SPWB膜蛋白的核苷酸差异一个碱基,氨基酸序列相同。另外与WBDL膜蛋白的核苷酸和氨基酸序列同源性分别为79.8%和70.2%。对其进行系统发育树分析;初步分析imp基因编码蛋白的跨膜区和疏水区。分析结果表明：花生丛枝植原体免疫膜蛋白C端有一跨膜锚定区,N端主要为膜内亲水区,没有前导信号序列。预测花生丛枝植原体免疫膜蛋白是一类C端跨膜的植原体免疫膜蛋白。将imp基因克隆到带有λcI基因的pBT质粒上,构建诱饵载体pBT-Imp,并通过IPTG诱导表达,western blot检测和自激活检验对其进行检测。结果显示：所构建的诱饵载体pBT-Imp可用于细菌双杂交的进一步实验。     

Ultrastructural Visualization of Glucide Determinants in Membranes of Mycoplasma-like Organisms

Periodic acid-thiocarbohydrazide-silver proteinate (PATAg) reaction for poiysaccharide localization was performed on ultrathin sections of Spurr resin-embedded tissues of Physostegia virginina and Catharanthus roseusknown to be infected by mycoplasma-like organisms (MLO). Electron microscopy of PATAg-treated sections revealed heavy silver deposition on hmiting membranes of all MLO. It was not possible to detect differences m silver deposition within the multi stratified membrane structure because of masking by silver-grain deposition. Silver deposition was not found in the cytoplasmic region of the MLO. The results indicate the presence of glucide determinants only in the limiting membrane of the MLO. The significance of this finding is discussed.     

Occurrence of Peroxidases in Sweet Potato Infected by the Black Rot Fungus

Changes in the peroxidase activity and the patterns in sweet potato tissue infected by Ceratocystis fimbriata were investigated, by the method of starch-gel electrophoresis, DEAE-cellulose column chromatography and immunochemical analysis, compared with that in cut and healthy tissue. Time-course analysis of the increment of the total peroxidase activity also showed noticeable differences between diseased and cut tissues. The peroxidases in diseased and cut tissues were composed of four major and several minor components. Though electrophoretic analysis did not show so striking differences in the patterns of peroxidases between diseased and cut tissues, there were distinct differences in the ratio of activities of major peroxidase components between both tissues. Immunochemical works indicated that peroxidase A which showed the most prominent increase in diseased tissue was found to be formed in cut tissue though the amounts were appreciably small. The activity of peroxidase C in diseased tissue was not so high as seen in cut tissue.     

Electrophoretic Study of Protein Metabolism in Sweet Potato Infected with Ceratostomella fimbriata

It was shown in previous papers that in sweet potato infected with Ceratostomella fimbriata, there occurred metabolic activations such as respiratory increase, polyphenols production, protein synthesis and organic phosphate accumulation. In this report on an electrophoretic experiment, forming a part of those studies, it is stated that the pattern of the protein components in the sound part, adjacent to the infected part of sweet potato attacked by the pathogen differs from that of sound sweet potato.     

苯酚对枣疯病病原检测及AFLP分析的影响

苯酚是进行高质量植物DNA提取常用的试剂,但苯酚对患病材料中病原检测及后续分子生物学试验的影响尚未见报道。本试验利用苯酚对健康和患枣疯病的枣树叶片中DNA进行提取,并进行了后续的病原检测和AFLP分析,结果表明(1)苯酚抽提获得的DNA质量及产率明显高于对照;(2)苯酚抽提处理对枣疯病病原的检测有直接影响,当病原含量较低时,加苯酚抽提后的DNA检测不到病原;(3)苯酚抽提处理与否获得的DNA对后续AFLP分析的扩增效果没有明显影响;但两者出现了有规律的差异条带,经分析此差异应该是由材料中有无病原引起的。本试验结果为寄主材料中病原检测及寄主与病原之间的互作等研究提供了有益参考。     

Development of Cloned DNA Probes for Phytoplasma Associated with Loofah Witches' Broom

DNA was isolated from periwinkle ( Catharanihus roseus ) infected with a phytoplasma that originated in loofah witches' broom affected by loofah. Cloned DNA inserts from six LfWB-phytoplasma specific recombinant plasmids were not only labelled with digoxigenin, but also used as probes. Probes hybridized with DNA derived from LfWB-phytoplasma affected periwinkle and loofah, but not with DNA from healthy plants or plants infected with phytoplasmas associated with elm yellows, red bird cactus, peanut witches' broom, paulownia witches' broom, Ipomoea obscura witches' broom, aster yellows (two isolates), and sweet potato witches' broom obtained with DNA from different phytoplasmas experimentally maintained in periwinkle. The probes could detect LfWB-phytoplasma DNA with as little as 16 ng and 32 ng of DNA from periwinkle and loofah, respectively. The method proposed herein provides a means for specifically detecting and identifying of loofah witches' broom phytoplasma, as well as confirming the notion that this phytoplasma represents a distinct strain cluster,