北京市区泡桐丛枝病发生特点研究

对北京市区泡桐丛枝病调查结果表明，２ａ以上树龄的泡桐树平均病株率为１９．２３％，病情指数达１０．２７，病害随树龄增大而加重。小片纯林受害最重，行道树次之，散植株最轻。周围高建筑物和稠密的植被可有效地阻止病害通过介体传播。在城市特殊生态环境中，种苗带毒是病害发生和发展的关键因子。在植株休眠期，病树丛枝的筛管中类菌原体（ＭＬＯ）浓度降低，但仍有一定数量的ＭＬＯ存活     

几种植物类菌原体(MLOs)的分子检测及其遗传相关性比较

 以密西根翠菊黄化病MLO 16S rDNA序列为依据,参照有关引物序列,改进合成寡核苷酸R₁和L₁作为引物对,应用聚合酶链式反应(PCR)技术,以甘薯丛枝病(SPWB),泡桐丛枝病(PWB),枣疯病(JWB)和枣疯病长春花(JPWB)罹病植株中提取的DNA为模板,扩增出了1.2 Kb的MLO 16S rDNA片段。该方法所需罹病植株的总DNA量分别为:甘薯丛枝病2×10^-2 ng/μl,泡桐丛枝病0.4×10^-2 ng/μl,枣疯病0.8×10^-2 ng/μl,感染枣疯病的长春花0.3×10^-3 ng/μl。对1.2kb 16S rDNA进行限制性片段长度多态性(RFLP)分析,首次确证枣疯病的罹病植株中存在枣疯病MLO和泡桐丛枝病MLO混合侵染的现象。     

仙人掌丛枝病植原体检测和16S rDNA片段序列分析

 对仙人掌丛生幼嫩组织进行超薄切片电镜观察,在韧皮部筛管中存在大量植原体;根据植原体16S rRNA基因保守序列设计的通用引物对R16 F₂/R₂,应用PCR技术对仙人掌丛枝病进行分子检测,结果扩增到约1.2 kb的特异性片段,而在健康组织中却没有此特异片段;通过16S rDNA片段核酸序列同源性比较,结果表明仙人掌丛枝病植原体与花生丛枝病植原体亲缘关系最近,据此可初步判断仙人掌丛枝病植原体是一种属于16Sr Ⅱ组的植原体,基本确定了其分类地位。     

海南岛几种绿肥作物丛枝病病原体的电子显微镜研究

 在广东省海南岛发现几种绿肥植物:巴西苜蓿、卵叶山蚂蝗、暗紫莱豆、爪哇大豆罹患了植物黄化病害。病株表现症状:植株矮化、枝叶丛生、叶变小、普遍退绿或黄化。电子显微镜观察发现,在病株叶柄韧皮部筛管细胞超薄切片中,存在大量的类菌原体(MLO)。这些菌体形态多样,有些可见菌体内纤维状样核酸物质,有的菌体正处于二分裂、或发芽状态。这些MLO大小分别为170~800nm、170~700nm、250~650nm、375~770nm。由于在病株中发现有MLO存在,而健株中则没有。可以初步认为四种绿肥植物丛枝病是由MLO所致。     

泡桐丛枝病病原MLO在寄主离体培养组织中的保存与增殖

 感染丛枝病病原MLO的泡桐幼苗茎梢,经表面灭菌后,置于附加0~3mg/1 6-BA的MS培养基中进行离体培养,由腋芽或顶芽直接伸长生长所形成的试管苗或经不定芽发生途径所形成的试管苗在1个月内均表现出典型的丛枝症状。在高浓度6-BA(2~3mg/1)处理培养基中,丛枝试管苗1月左右病死。当外植体为0.5mm长的微茎尖时,试管苗的丛枝症状有所减轻。DAPI荧光染色镜检结果表明,这种已表现出丛枝的试管苗的根、茎、叶柄的韧皮部筛管,以及已具维管束分化的愈伤组织的筛管部位发出很强的MLO特异荧光。超薄切片电镜观察发现,离体培养组织中的MLO形态具有较多处于活跃增殖期的哑铃形和芽殖形。以30天为一个继代周期,在无激素培养基或低浓度6-BA(lmg/1)与无激素培养基交替继代培养1年之后,来源于染病愈伤组织和试管苗中的MLO深度一直维持在相当高的水平。试验表明,利用寄王组织进行离体培养是保存、繁殖并进一步深入研究泡桐丛枝病病原MLO的一条良好途径。     

草莓丛枝病的发现及初步研究

 在沈阳,从表现丛枝症的草莓、春香和宝交早生品种上,分离到新发生的类菌原体(MLO)。用小叶嫁接法测定指示植物草莓EMC和UC-10均能表现出典型的丛枝症(witchetbroom),病叶变小,颜色变浅。将病叶经固定和包埋后,制成超薄切片,于电镜下观察,可见到在韧皮部组织内有大量球形、椭圆形及哑铃形,并具有单位膜结构的类菌原体。用四环素处理病株可减轻症状。     

Characterization of a phytoplasma associated with witches' broom disease of potatoes in Korea

 Symptoms of witches' broom disease caused by phytoplasma, including general stunting and yellowing, were observed in potatoes (Solanum tuberosum L.) in a storehouse on Jeju Island, Korea in 1998. Based on sequence analysis of DNA products from polymerase chain reaction (PCR)-amplified 16S ribosomal DNA and 16S–23S spacer region using universal phytoplasma primers, the phytoplasma associated with potato witches' broom disease (PWB) was identified as a member of 16S-group VIII. It was most closely related to elm AH phytoplasma (99.7% similarity, accession no. AF268895), which is in the clover proliferation (CP) subgroup. This report is the first from the East Asian continent of a plant pathogenic phytoplasma belonging to the CP subgroup and includes the nucleotide sequence of most of the potato phytoplasma 16S rDNA. Received: May 1, 2002 / Accepted: July 1, 2002     

组织化学技术快速检测泡桐丛枝病研究

 迪纳氏染色和萤光显微镜技术——苯胺蓝染色法,和DAPI染色法,可从感染泡桐丛枝病的嫩茎和叶柄及将MLO转接到长春花上,表现典型症状的嫩茎和叶柄中诊断MLO的侵染。病株切片的迪纳氏染色阳性,健株切片阴性。苯胺蓝染色病株韧皮部产生的特异性萤光区域比健株大。DAPI染色可直接检测韧皮部组织中的MLO,而且韧皮部萤光强度与MLO的密度和外部症状严重度呈正相关。三种染色方法比较结果说明,DAPI染色法是一种灵敏度高,特异性更强的泡桐丛枝病类菌原体检测方法。     

Monoclonal antibody for the detection and identification of a phytoplasma associated with rice yellow dwarf

One stable hybridoma clone, 247B11, secreting specific monoclonal antibody (MA) against the mycoplasmalike organism (MLO), newly be termed phytoplasma, associated with rice yellow dwarf (RYD) was produced by employing an immunization scheme for inducing the immunological tolerance of mice to rice antigens prior to the administration of RYD-phytoplasma immunogens. Neonatal BALB/c mice were first injected with nontarget rice antigens present in the immunogen preparation and were immunized intrasplenically with RYD-phytoplasmaenriched antigens prepared by Percoll density-gradient fraction 6 wk later. The MA was of the IgG1 class. With this MA, RYD-phytoplasma in diseased rice was specifically detected by indirect enzyme-linked immunosorbent assay (ELISA), immunofluorescent staining and tissue-blotting techniques. Antibody titer determined by indirect ELISA for hybridoma-culture supernatant was 5120. The antibody recognized two polypeptides, 16 kDa and 41 kDa of RYD-phytoplasma determined by western blotting. RYD-phytoplasma was differentiated serologically from the phytoplasmas associated with sweetpotato, peanut, loofah, paulownia, andIpomoea obscura witches' broom, aster yellows (NJ strain), elm yellows, and sugarcane white leaf both in indirect ELISA and immunofluorescent staining.     

枣疯病的研究

 对枣疯丛枝与花、叶畸变的症状作了比较系统的观察和描述。病树根部的超薄切片和疯枝韧皮匀浆的部分提纯物在电镜下都检查到类菌原体(MLO)。健树的相应对照样品中未见任何病原物。试验表明:四环素族抗菌素对枣疯病有一定的疗效,少数病树经治疗后,外观康复,正常开花结果已维持两年之久。认为枣疯病是由MLO感染所致。     

C/A值与甘薯丛枝病症状发生的关系

 采用高效液相色谱(HPLC),对比测定了由植物类菌原体(MLO)引起的甘薯丛枝病病株和甘薯健株中细胞分裂素和生长素含量在整个生长周期内的变化。结果显示:甘薯中细胞分裂素以玉米素为主。病株中玉米素含量一直处于较高水平,而吲哚乙酸含量仅在芽萌发时期病、健材料间存在明显差异,随着甘薯的生长,这种差异逐渐消失。结合甘薯丛枝病发病特点,讨论了植物激素C/A值的改变对病状发生的作用。     

Partial characterization of phytoplasmas associated with lettuce and wild lettuce phyllodies in Iran

Phytoplasmas associated with lettuce phyllody (LP) and wild lettuce phyllody (WLP) in southern Iran were partially characterized by molecular analyses and host-range studies. Agents of both diseases were transmitted by Neoaliturus fenestratus , a leafhopper colonizing lettuce and wild lettuce, to lettuce, wild lettuce, sowthistle and periwinkle, but not to safflower, sunflower, calendula and sesame. Both phytoplasmas induced bud proliferation, virescence, phyllody and witches' broom in infected plants. Total DNA extracted from infected lettuce and wild lettuce or from vector tissues was subjected to PCR using phytoplasma-specific primer pair P1/P7 or nested PCR using P1/P7 followed by R16F2n/R16R2. PCR product of nested PCR (1·2 kbp) was subjected to restriction fragment length polymorphism (RFLP). RFLP analysis of nested PCR product identified the LP, WLP and N. fenestratus -associated phytoplasmas as members of the pigeon pea witches' broom group, 16SrIX. Phylogenetic analysis of the 16S rRNA gene sequence also clustered LP and WLP phytoplasmas with other known members of the 16SrIX group. While no significant differences could be detected between LP and WLP phytoplasmas, both isolates differed from Lebanese wild lettuce phyllody in molecular properties.     

木麻黄黄化丛枝病的研究

 通过电镜观察、传毒试验及抗菌素处理,首次摸清了木麻黄黄化丛枝病的病原为类菌原体(MLO)和类细菌(BLO)复合侵染;传病虫媒为菱纹叶蝉(传播MLO)和短头叶蝉(传播BLO)。聚丙烯酰胺圆盘凝胶电泳的结果,病、健植株内的过氧化物酶同工酶的谱型及活性差异显著,病植株中都缺少AIII兀区谱带。
经土壤分析发现,健病苗植土中的Mg、Cu、Fe、Mn、Zn的含量相差极显著,施相应元素盆栽活疗试验,处理3个月后病菌有明显好转。
以畜用土霉素液淋根结合施元素、碳肥和杀虫媒的综合防治有较好效果,相对防效为55.62%     

Changes in morphology and measurement of cytokinin levels during the development of witches' brooms on cocoa

The anatomy of diseased stems from brooms induced on cocoa ( Theobroma cacao ) by the fungus Crinipellis perniciosa was examined using the scanning electron microscope, and the amounts of selected cytokinins were measured by immunoassay.
Although the diseased stems were wider in diameter with a lower dry weight:fresh weight ratio than stems of healthy plants, the overall internal organization of tissues remained unchanged, and all the cell types were present. However, the vascular tissue was less differentiated in diseased stems since xylem vessels were absent and the ratio of phloem fibres and sieve tubes to phloem parenchyma was reduced. Cell sizes in diseased stems were larger for all tissues but cell numbers were unchanged except for xylem where fewer cells were seen.
Cytokinins (zeatin, zeatin riboside, isopentenyl adenine and isopentenyl adenosine) were measured in healthy plants and diseased stems at specific stages of disease development. Of these cytokinins only zeatin riboside was present in significantly greater amounts in diseased tissue. The likely role of plant growth regulator balance in stem enlargement and proliferation in witches' broom disease of cocoa is discussed.     

Molecular detection and identification of phytoplasma associated with pepper witches’ broom in China

Pepper witches’ broom (PWB) disease was observed in a field in Yangling, Shaanxi Province, China. The result of mechanical inoculation test for this disease was negative. Phytoplasma-like bodies were observed in ultrathin sections of petiole tissues of symptomatic samples. 16S rRNA gene and tuf gene of phytoplasma were amplified from the total DNA of symptomatic samples. Phylogeny analysis of the 16S rRNA gene and tuf gene suggested that the pepper witches’ broom associated phytoplasma belongs to the subgroup 16SrI-B, which was confirmed by the RFLP analysis of the 16S rRNA gene. The phytoplasma subgroup 16SrI-B was also detected in the vector Cicadella viridis trapped from the infected field. To our knowledge, this is the first report of 16SrI-B phytoplasma causing pepper witches’ broom in China.     

A novel phytoplasma associated with witches’ broom disease of Ligustrum ovalifolium in Turkey

California privet (Ligustrum ovalifolium Hassk.) plants exhibiting leaf yellowing, witches’ broom, dieback and decline symptoms were observed for two years (2010–2011) in three gardens at Adana region (Turkey). DNA isolated from symptomatic and healthy plants was used to amplify 16S rDNA fragments by direct and nested-PCR. Phytoplasmas were detected in 21 symptomatic plants, out of 30 samples collected, whilst no PCR amplifications were obtained from asymptomatic plants. BLAST analysis of the 16S rDNA showed that the phytoplasma found in L. ovalifolium from Turkey, denoted as Turkish Ligustrum witches’ broom phytoplasma (TuLiWB), most closely resembled members of group 16SrII (peanut witches’ broom group) and shared up to 92 % sequence identity. Based on in silico 16S rDNA RFLP analysis and automated calculation of the pattern similarity coefficient, TuLiWB showed molecular characteristics different from all previously described phytoplasma species to represent a new taxon. Similar indication also emerged from the phylogenetic tree which allocated it in a novel discrete subclade within the phytoplasma clade. This is the first report on the presence of a phytoplasma affecting L. ovalifolium and whether this novel phytoplasma is the same agent reported as a mycoplasma-like organism (MLO) and associated with witches’ broom disease of Ligustrum in Korea (1989) is yet to be determined.     

辣椒发生的一种类菌原体新病害

 受害辣椒呈现丛枝小叶症状,叶柄变细伸长,花器变绿。严重时茎杆带化,花序聚生,花器萎落,鳞片发育成小叶片。经多年反复试验,该病原能通过大青叶蝉及嫁接顺利传播,而不能通过汁液摩擦和蚜虫传播。罹病辣椒的叶片中脉切片经甲苯胺兰(TBO)染色后,用荧光显微镜可在韧皮部观察到清晰的枯黄色萤光反应斑,整个维管束组织严重退化变小。用电子显微镜对超薄切片观察,在辣椒病株的筛管中观察到大量的172~311nm的MLO菌体。病株用土霉素(OTC)溶液灌根3周后症状开始缓解,而青霉素处理无效。据此,作者认为引起辣椒丛枝病的病原是一种类菌原体(MLO)。本文在国内首次报道了MLO对辣椒的为害。     

木麻黄丛枝病病原研究

 通过超薄切片的电镜观察,在木麻黄丛枝病病枝的筛管及伴胞中发现类菌原体,在木质部薄壁细胞发现类立克次细菌、类菌原体多态形,大小为45~300nm,单位膜厚约7.5nm;类立克次细菌多态型,大小为114~762nm×1000~1760nm,膜壁厚22~28nm。并观察到菱纹叶蝉的唾腺中存在类菌原体;短头叶蝉唾腺中存在类立克次细菌。在健株组织中没有发现任何病原体。     

枣疯病植原体侵染对枣树叶绿素含量的影响

为明确枣疯病植原体侵染对枣树叶绿素含量的影响,本研究以田间‘赞皇大枣,健树和患枣疯病病树以及‘婆枣’枣疯病组培苗为材料,测定了健叶、病叶以及不同浓度生长调节剂处理后组培苗叶片的叶绿素含量.结果表明,在田间条件下枣疯病病树叶片的叶绿素a、b含量及叶绿素a/b值显著低于健康叶片;在培养基中添加IBA、6-BA可提高枣疯病组培苗叶片叶绿素a、b含量,明显改善患病组培苗的光合作用,但随着继代时间延长,这种影响逐渐消失.此结果表明枣疯病植原体、激素代谢及叶绿素合成之间存在密切联系.     

Induction of resistance in cocoa against Crinipellis perniciosa and Verticillium dahliae by acibenzolar- S-methyl (ASM)

The benzothiadiazole compound acibenzolar- S -methyl (ASM) was assessed as an inducer of resistance against Crinipellis perniciosa , agent of witches' broom, and Verticillium dahliae , agent of vascular wilt, both on cocoa. ASM induced a reduction in incidence of witches' broom of up to 84·5% when sprayed 30 days before inoculation on cocoa seedlings of cv. Catongo. ASM also induced a reduction in severity of Verticillium wilt to 55·4% on cv. Theobahia. For both pathosystems, effects of dose on disease were not clearly observed. The efficacy of the inducer increased with the interval between sprayings and the respective inoculations with the pathogens. In another experiment, the effect of ASM on the control of witches' broom on cocoa seedlings was compared with that of cuprous oxide and tebuconazole, all sprayed 15 days before inoculation. ASM reduced disease incidence by 60·1% compared with the inoculated control. ASM was superior to tebuconazole, and there was also a tendency for ASM to be better than cuprous oxide. To understand the mechanism of action of ASM as an inducer of resistance, alterations in the levels of total phenolics, polyphenol oxidases and peroxidases were evaluated 3, 15 and 30 days after spraying of seedlings of cv. Catongo. Enzyme activities from seedlings of cv. Theobahia were evaluated 30 days after spraying. On cv. Catongo, no significant differences in total phenolic content and polyphenol oxidase activity were detected after spraying. However, an increase in peroxidase activity was detected at all times of evaluation. On cv. Theobahia, significant increases in activities of peroxidase and polyphenol oxidase were detected, indicating that defence responses due to ASM were dependent on host genotype.