土霉素对桑树黄化型萎缩病治疗机理的探讨

为探讨土霉素(OTC)对植物类菌原体病害的治疗机理,本课题综合考察了OTC对桑树黄化型萎缩病及其病原类菌原体的影响。结果发现:(1)OTC对病害的治疗效果与进入树体的OTC浓度呈显著正相关;(2)在植物体内处理的情况下,病原类菌原体(MLO)抽提物的蛋白质含量降低,电泳图谱上蛋白带减少;MLO的形态结构发生了改变,并随进入树体的OTC含量的增加而加剧;(3)离体处理,MLO的形态结构无明显变化。认为干扰和破坏MLO的代谢作用是OTC的治病机理之一。     

小麦蓝矮病植原体16S rDNA序列分析研究

 小麦蓝矮病是我国西北地区冬小麦上一种重要病害。本研究利用植原体16S rDNA通用引物对小麦蓝矮病患病植株全DNA进行nest-PCR扩增,获得1.2 kb的特异片段,并对扩增产物进行核苷酸序列测定,从分子水平证明了小麦蓝矮病的病原是植原体。利用最大简约法构建了16S rDNA系统演化树,系统演化关系分析表明:小麦蓝矮病植原体应该归属于翠菊植原体(Candidatus Phytoplasma asteris);小麦蓝矮病植原体与三叶草变叶病植原体(CPh)关系密切,被聚类为同一亚组(16Sr I-C),但是它们在寄主范围和传播介体等生物学性状方面差异很大。     

小麦蓝矮病植原体转运蛋白secY基因片段序列分析

 Wheat blue dwarf(WBD) is a disease caused by phytoplasma and only reported from China. A fragment about 1.3 kb in protein translocation gene, secY was amplified by PCR from the total DNA of di-seased wheat sample with primer pair secYF/secYR, which was designed based on secY gene sequence of known 16SrI group members. Nucleotide acid sequence analysis of amplified fragment indicated that the length was 1 240 bp. A phylogenetic tree based on secY gene sequences was constructed and showed that wheat blue dwarf phytoplasma was clustered into the Candidatus Phytoplasma asteris, subgroup 16SrI-C. Wheat blue dwarf phytoplasma showed high homology with clover phyllody phytoplasma strains based on sequence comparison and phylogenetic analysis.     

滇朴丛芽病植原体的分子鉴定

滇朴Celtis kunmingensis Cheng et Hong是云南的乡土树种,适宜全国大部分地区种植,极具观赏价值,是近年来最热门的绿化首选树种—绿化行道树,云南部分地区滇朴近年常表现丛芽的症状.本研究采用形态学与分子生物学结合的方法,对染病的幼嫩枝条进行扫描电镜(SEM)观察;利用16S rDNA植原体通用...     

竹丛枝植原体16SrDNA片段克隆与序列分析

利用植原体16SrRNA基因序列设计合成的引物,对表现丛枝的竹子植株总DNA进行直接PCR及巢式PCR扩增,得到长1.2kb的目的片段。将此片段与pGEMTEasy载体连接并转化到大肠杆菌DH5α感受态细胞中。通过酶切、PCR鉴定,对筛选得到的重组阳性克隆进行核酸序列测定及同源性比较分析,结果表明其与植原体16SrⅠ组中的西方翠菊黄化植原体(SAY)同源率为99%。依据16SrDNA序列建立了竹子丛枝病植原体株系的系统进化树。对云南竹子丛枝病植原体株系分类鉴定与已报道的结果相似。     

桃红叶植原体检测及鉴定

对表现红叶的桃植株进行植原体16SrRNA基因PCR扩增,得到1.2kb的特异片段.将此片段与pGEM T Easy载体连接并转化到大肠杆茵DH5α感受态细胞中.通过酶切、PCR鉴定,对筛选得到的重组阳性克隆进行序列测定及同源性比较分析,确定该株系属于翠菊黄化植原体组(Aster yellows group,16SrI).在国内首次报道了翠菊黄化组中的植原体侵染桃树.     

小麦蓝矮病植原体16S rDNA基因片段的比较分析

 小麦蓝矮病是陕西乃至西北冬麦麦区的一个重要病害,由介体条沙叶蝉专化性传播。对小麦蓝矮病株叶片和带毒条沙叶蝉进行超薄切片及电镜观察,在叶片韧皮部和叶蝉后肠中均观察到大量典型植原体。利用植原体16S rDNA基因保守序列通用引物对Rm16F2/Rm16R1,应用PCR技术从小麦蓝矮病株叶片中扩增到1.4 kb的特异片段。通过对16S rDNA基因片段序列同源性比较,结果表明小麦蓝矮病病原与三叶草绿变、翠菊黄化、绣球花绿变、草莓矮化和番茄巨芽植原体亲缘关系较近,其同源率为99.2%~99.9%。据此可以判定小麦蓝矮病植原体是属于植原体16SrⅠ组,确定了其分类地位。     

云南泡桐丛枝病植原体核糖体蛋白基因片段序列分析

 应用植原体核糖体蛋白基因通用引物对rpF1/rpR1,对采自云南省曲靖市的泡桐丛枝病植原体DNA (PaWB-QJ)进行PCR扩增,得到1.3 kb的特异片段,证明此病株中存在植原体。将此片段与pGEM-T Easy载体连接并转化大肠杆菌JM109感受态细胞,进行PCR鉴定、核糖体蛋白基因部分核苷酸序列测定及分析。结果表明,该株系(PaWB-QJ)核糖体蛋白基因片段长1 244 bp,包含rps19、rpl22和rps3基因。对PaWB-QJ株系的核糖体蛋白基因序列的同源性比较结果显示与16S rI-B亚组的翠菊黄化(Aster yellows,AY)、长春花黄化(Periwinkle yellows,PY)和泡桐丛枝德国株系(Paulownia witches'-broom,PaWB-German)的亲缘关系最近,达到99.0%以上,而与其它组中的株系明显低于97.0%,所以认为该植原体株系属于翠菊黄化组B亚组(16SrI-B)。     

竹小叶病植原体的分子鉴定

 2008年在杨凌采集到具有典型植原体侵染症状的菲白竹,应用植原体16S rRNA基因的通用引物对R16mF2／R16mR1和R16F2n／R16R2对其进行检测,巢式PCR得到约1.2 kb的特异性片段。对扩增片段进行测序并进行系统进化树分析,结果表明,该病原属于翠菊黄化组(Candidatus Phytoplasma asteris),与该组成员同源性均在98%以上。随后用16Sr Ⅰ组和Ⅴ组特异引物对R16（Ⅰ）F1／R16（Ⅰ）R1和R16（Ⅴ）F1／R16（Ⅴ）R1也证明其属于翠菊黄化组,RFLP 分析表明该植原体属于16SrⅠ-B亚组。植原体侵染菲白竹在中国属首次报道。     

金莲花绿变植原体的分子鉴定

本研究对河北省大面积发生的金莲花绿变病的病原进行检测和鉴定。以金莲花叶片的总DNA为模板,使用植原体16S rDNA和核糖体蛋白(ribosomal protein)基因rp的特异性引物进行PCR扩增,在感病金莲花样品中扩增到植原体的16S rDNA(1 432 bp)片段和rp基因(1 240 bp)片段。序列分析发现,获得的16S rDNA和rp基因片段与洋葱黄化植原体Onion yellows phytoplasma(GenBank登录号:AP006628)的相似度最高,分别为99.9%和99.3%,确定金莲花绿变病的病原为植原体,暂命名为金莲花绿变植原体Trollius chinensis virescence phytoplasma。对金莲花绿变植原体的16S rDNA进行虚拟RFLP分析,发现其酶切图谱与16SrⅠ-B亚组的洋葱黄化植原体的参照图谱完全一致,相似系数1.00。16S rDNA和rp基因的系统发育进化树显示,金莲花绿变植原体与16SrⅠ-B亚组的植原体聚为一支,属于植原体16S rⅠ-B亚组。     

安徽桑黄花型萎缩病植原体16S rDNA序列分析及分子检测

 Mulberry yellow dwarf(MYD)disease is an quarantine disease and the causal agent is a phytoplasma.Two pairs of published universal primer, P1/P7 and Rm16F2/Rm16R1, based on the 16S-23S rDNA sequence of phytoplasma and total DNA extracted from infected mulberry tissues were employed for PCR and nested-PCR detection.The results revealed that a phytoplasma-specific 1 830 bp fragment with a G+C content of 46.01% was sequenced(GenBank accession No.GQ249410).The sequence shared 99.7% and 99.8% identity with aster yellows, the representatiive phytoplasma in 16SrI group, and mulberry dwarf phytoplasma classified into subgroup B in 16SrI group and named as the MYD phytoplasma strain Anhui(MYD-Anh).A phylogenetic tree based on 16S rDNA sequences was constructed and showed that MYD-Anh was clustered into 16SrI group.Identity of 16S rDNA sequence between MYD-Anh and mulberry yellow dwarf phytoplasma strain Zhenjiang(MD-zj) was nearly 100%, and they might belong to the same strain.Nested-PCR was used to detect the pathogenic phytoplasma from the differential tissues of mulberry infected with MYD-Anh.The results showed that a phytoplasma-specific 1.4 kb fragment was amplified with total DNA extracted from bark and vein.Nested-PCR was more sensitive than PCR for detecting MYD phytoplasma.     

Genetic and serological analyses of elongation factor EF‐Tu of paulownia witches’‐broom phytoplasma (16SrI‐D)

This study determined the tuf gene sequence of the phytoplasma specific to paulownia witches’‐broom from Nanyang, China (hereby designated PaWB‐Ny). The PaWB‐Ny tuf gene was 1185 nucleotides in length and confirmed that the phytoplasma belongs to subgroup 16SrI‐D of aster yellows. Three characteristic GTP‐binding protein motifs were identified based on the peptide deduced from the tuf gene sequence. Results suggested that the elongation factor EF‐Tu was localized in the cytoplasm and lacked hydrophobic transmembrane domains. Antibodies against PaWB‐Ny EF‐Tu were prepared by rabbit immunization with glutathione‐S‐transferase (GST)‐tagged EF‐Tu fusion protein expressed in Escherichia coli. EF‐Tu exhibited a molecular weight of ～43 kDa and was detected in PaWB‐infected paulownia plants by western blot analysis. Indirect enzyme‐linked immunosorbent assays (ELISA) and dot blotting analyses were performed with freezing and thawing treatments during antigen preparation. Dilution of extracts to an appropriate scale significantly reduced non‐specific reactions. The resultant PaWB EF‐Tu antibody reacted with antigens from plants infected with periwinkle virescence and chinaberry tree witches’‐broom phytoplasmas, but not those infected with jujube witches’‐broom or bishopwood witches’‐broom phytoplasma. The EF‐Tu was characteristically localized within the phytoplasmal cytoplasm of infected plant phloem tissues.     

Distribution patterns of mulberry dwarf phytoplasma in reproductive organs,winter buds,and roots of mulberry trees

Distribution patterns of mulberry dwarf (MD) phytoplasma were investigated in several organs of dwarf-diseased mulberry trees using direct and nested polymerase chain reactions and electron microscopy. MD phytoplasma was detected in the root of all MD-diseased trees collected from overwintering to before sprouting in the cold district; however, it was not always found in winter buds of the same trees. On the other hand, MD phytoplasma was also detected frequently in reproductive organs, such as flowers (female and bisexual flowerets), fruits (catkins), and seed coats, from a single diseased tree, although the distribution patterns were not uniform. In addition, MD phytoplasma was verified to move into roots from ground plant organs after an artificial inoculation test using the leafhopper Hishimonoides sellatiformis as a vector.     

Detection and characterization of a phytoplasma associated with annual blue grass (Poa annua) white leaf disease in southern Italy

A phytoplasma was detected in annual blue grass (Poa annua L. Fienardo), exhibiting white leaf symptoms, that was grown in the fields near Caserta in southern Italy. Based on restriction fragment length polymorphism analysis of PCR-amplified 16S rDNA sequences, the phytoplasma associated with annual blue grass white leaf disease was identified as a new member of phytoplasma 16S rRNA group XI (16SrXI) (type strain, rice yellow dwarf phytoplasma). The annual blue grass white leaf phytoplasma is most closely related to Bermuda grass white leaf phytoplasma found in Asia. Annul blue grass white leaf and Bermuda grass white leaf phytoplasmas were designated as the third subgroup (16SrXI-C) of group XI. This is the first report that a plant pathogenic phytoplasma belonging to group 16SrXI is present on the European continent.     

三叶草绿变病植原体的分子鉴定

三叶草(Trifolium pratense Linn.)为车轴草属,蝶形花科多年生草本植物,原产亚洲南部和欧洲东南部,是一种世界性分布与栽培的优良牧草.因其花叶兼优、草姿美、绿期长而具有较高的观赏价值,近几年作为草坪用草被广泛种植.在自然条件下,三叶草很容易受到不同种植原体的侵染,国外已报道的侵染三叶草的植原体有:三叶草绿变植原体( Clover phyllody phytoplasma,CPh)和三叶草增殖植原体(Clover proliferation phytoplasma,CP)等,这些植原体分别属于16SrI组和16SrⅥ组[1,2].     

Phylogenetic analysis of elongation factor Tu gene of phytoplasmas from Japan

The elongation factor Tu (tuf) gene from nine Japan phytoplasma isolates was amplified with the polymerase chain reaction, and the DNA sequences of the tuf gene were determined. The tuf gene from 14 phytoplasma isolates, including reference isolates and other bacteria, were phylogenetically analyzed. A nucleotide sequence of the tuf gene among seven aster yellows group (16Sr I-B and I-D) phytoplasmas had 97%–100% similarity, and the tuf gene of two phytoplasmas of the X-disease group (16Sr III-B) had 99% similarity. The tuf genes had lower homology than did the 16S rRNA gene in the phytoplasma groups. A phylogenetic tree of amino acid sequences of the tuf gene was nearly equal to that of the 16S rRNA gene but differed somewhat from the tree based on the 16S rRNA gene in that paulownia witches broom (PaW: 16Sr I-D) and American aster yellows (AAY: 16Sr I-B) were in a subclade.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB095495, AB095667, AB095668, AB095669, AB095670, AB095671, AB095672, AB095673 and AB095674     

紫花苜蓿丛枝病植原体的分子检测及鉴定

 利用植原体16S rRNA基因通用引物对云南昆明发生的苜蓿丛枝病感病植株总DNA进行巢式PCR扩增,得到1.2kb的特异片段,从分子水平证实了苜蓿丛枝病的病原是植原体。从PCR产物的RFLP酶切图谱可看出,该植原体株系的酶切图谱与马里兰翠菊黄化植原体(AY1)相同。对扩增片段进行克隆及序列测定后,利用最小进化法做Bootstrap验证的系统进化树,表明苜蓿丛枝病植原体为Candidatus Phytoplasma asteris成员之一,与植原体16SrI-B亚组成员关系密切。     

小麦黄矮病及其抗性育种研究进展

由大麦黄矮病毒(barley yellow dwarf viruses, BYDVs)侵染引起的小麦黄矮病(wheat yellow dwarf disease)是世界小麦上的主要病害之一, 对小麦生产及粮食安全构成了严重威胁。本文综述了BYDVs的生物学特征、分类地位、致病机理及抗性育种等相关研究, 并基于相关研究进展, 提出了进一步扩大抗性基因挖掘、加快优异抗病基因克隆、加强抗病机理研究等方面的建议, 旨在为我国小麦黄矮病抗性育种及科学防治提供一定的参考。