不同症状矮牵牛植株植原体16SrDNA 片段的克隆及序列分析

应用植原体16SrRNA基因通用引物,分别对表现矮化和扁茎症状的矮牵牛植株进行巢式PCR检测,均得到约1．2kb的特异片段。将此特异片段与pGEM-T Easy载体连接并转化到大肠杆菌JM109感受态细胞中,通过PCR鉴定,序列测定及同源性比较分析,结果表明：这2个植原体株系16SrDNA片段G C含量分别为47．1％和47．0％,具有其他柔膜菌纲原核生物16SrRNA基因碱基组成特征;与16Sr组中的代表株系(西方翠菊黄化,SAY)同源率达到最高,分别为99．1和98．6％,故认为这2个株系为该组成员之一。     

Role of <Emphasis Type="Italic">Cacopsylla pyri</Emphasis> in the epidemiology of pear decline in Spain

The frequency of pear decline-positive insects and transmission of pear decline (PD) phytoplasma by Cacopsylla pyri in Spain has been studied. Psyllids were used for experiments on phytoplasma transmission both to healthy Pyrus communis trees and to an artificial feeding medium. Over a period of 1 year, about 100 psyllids were collected monthly from pear trees, cv. Williams, using the beating tray method, and tested for the presence of PD phytoplasma. Results indicate that the frequency of PD positive psyllids changes through the year and that C. pyri transmits the pear decline associated disease agent. Phytoplasma transmission was also effective under laboratory conditions using a feeding medium. The relationship between PD positive Cacopsylla pyri, Pear decline phytoplasma transmission and the sex of the vector was also evaluated. Although the percentage of PD positive psyllids was similar in both genders, PD phytoplasma transmission by females was significantly higher than by males. Since the sex ratio (male/female) was less than 1:1 for most of the year, these results should be taken into consideration for controlling Pear decline in Mediterranean climates.     

Detection and molecular characterisation of an alfalfa phytoplasma in Argentina that represents a new subgroup in the 16S rDNA Ash Yellows group(‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma fraxini’)

A phytoplasma infecting alfalfa crops was detected and characterised in Argentina, the Argentinean Alfalfa witches′-broom (ArAWB) phytoplasma. Typical witches′-broom symptoms were observed in diseased alfalfa plants from fields of the ‘Cuyo’ region in the Andean midwest. Pleomorphic bodies were observed by electron microscopy in sieve tubes of the diseased plants. The results obtained from the sequence homology, similarity coefficients derived from RFLP of the 16S rDNA and phylogenetic analysis led us to include this phytoplasma in the 16Sr VII (Ash Yellows) group. However, the ArAWB phytoplasma showed several differences when compared to other members of group 16Sr VII. The RFLP analysis of partial 16S rRNA gene of two ArAWB isolates, digested with 16 restriction enzymes, showed differences between the ArAWB and the reference strain (AshY1^T) in six enzyme patterns. Restriction patterns unique for the group and an exclusive HinfI restriction site were found in the ArAWB phytoplasma rDNA. Moreover, the similarity coefficients (0.92–0.86) were lower than those obtained among other group members. The significant differences detected suggested that this phytoplasma belonged to a subgroup different from those described so far. We propose therefore, that the ArAWB phytoplasma should be included in a new VII-C subgroup, closely related to the EriWB phytoplasma (VII-B) described in Brazil.     

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.     

Transmission of a sugarcane yellow leaf phytoplasma by the delphacid planthopper Saccharosydne saccharivora, a new vector of sugarcane yellow leaf syndrome

During surveys of sugarcane fields in western and central Cuba from December 2001 to March 2003, the delphacid planthopper Saccharosydne saccharivora was the most prevalent of the Auchenorrhyncha fauna surveyed. Individuals of S. saccharivora collected tested positive for the sugarcane yellow leaf phytoplasma (SCYLP). Saccharosydne saccharivora were reared in cages and used for experimental transmission studies of SCYLP. The S. saccharivora were given acquisition-access feeds of 72 h on SCYLP-infected canes collected from the field followed by an inoculation-access period of 15 days on healthy sugarcane seedlings. Symptoms of yellow leaf syndrome developed on 24 out of 36 plants, 7–12 months postinoculation. None of the 36 healthy seedlings that were inoculated with S. saccharivora fed on phytoplasma-free sugarcane developed symptoms. All phytoplasma-positive sugarcane and S. saccharivora samples showed identical RFLP patterns and had 99·89% similarity in their 16S/23S spacer-region sequences, but only 92·6–93·6% similarity with other phytoplasmas. Sequences were deposited with GenBank [accession numbers: AY725237 ( S. saccharivora ) and AY257548 (sugarcane)]. Phylogenetic analysis suggested that the phytoplasmas from sugarcane and S. saccharivora are putative members of a new 16Sr phytoplasma group. This is the first report of vector transmission of a phytoplasma associated with sugarcane yellow leaf syndrome and the first time that S. saccharivora has been shown to vector a phytoplasma.     

Detection and characterization of the phytoplasma associated with a phyllody disease of black pepper (Piper nigrum L.) in India

Using polymerase chain reaction (PCR), the phytoplasma was detected in black pepper (Piper nigrum) with phyllody symptoms in India. A 1.20 kb DNA fragment encoding the portion of phytoplasma 16S rDNA consistently amplified by nested PCR was cloned and sequenced. The sequenced region contained 1230 nucleotides. Sequence analyses showed that the gene was most closely related to members of aster yellows group (16Sr I) of phytoplasma. The sequence identity with members of aster yellows group (16Sr I) was >98% while that with members of other groups (16Sr II to 16Sr XV and other undesignated groups) ranged from 87 to 96%. On the basis of sequence identity and phylogenetic relationship studies, it is concluded that phytoplasma infecting black pepper in India belongs to aster yellows group. This is the first report of identification of phytoplasma in black pepper.     

云南花生丛枝植原体secY基因序列分析及结构预测

 对采自云南元谋的花生丛枝植株总DNA进行secY基因的巢式PCR扩增,得到约1700bp的特异扩增片段,将该扩增产物克隆后进行序列测定,表明该片段长1709bp,该序列包括部分rpl15基因、全部secY基因序列以及部分的map基因,为植原体部分spc核糖体蛋白操纵子,其中secY基因编码的蛋白包括420个氨基酸,为含有10个明显跨膜区的整合性跨膜蛋白。通过同源性比对及构建的系统进化树,表明该株系与来源于台湾、海南的花生丛枝植原体亲缘关系最近,确定了该株系属于16SrII A亚组成员。该分类结果与基于16SrDNA及rp基因的分析结果一致。     

应用分子生物学方法检测植原体研究进展

 从血清学、核酸杂交技术以及PCR技术等方面综述了国内外植原体检测研究的概况及最新进展。     

泡桐丛枝植原体pPaWBNy-1-ORF5编码蛋白的原核表达和抗体制备

 利用含泡桐丛枝植原体质粒pPaWBNy-1的3.0 kb片段的克隆为模板,PCR扩增pPaWBNy-1-ORF5的亲水性肽段编码区。目的片段连接到原核表达载体pET28a(+),重组质粒pET28a-ORF5-5转化大肠杆菌(Escherichia coli)Rosseta (DE3)感受态细胞,菌液处在对数生长期时(OD₆₀₀=0.52),添加IPTG诱导5 h后收集菌体,提取蛋白并进行聚丙烯酰胺凝胶电泳。用六聚组氨酸标签抗体进行Western blot检测,发现分子量约为15 kDa的含多聚组氨酸标签的目的蛋白得到表达。切胶回收融合蛋白,免疫德国大白兔以制备抗血清,间接ELISA法测定抗血清的效价约为1∶8 100。用制备的ORF5编码蛋白的抗血清进行Western blot分析,结果在带菌的介体昆虫茶翅蝽(Halyomorpha halys)中检测到大小约为17 kDa的特异蛋白条带,但在健康和感病泡桐(Paulownia sp.)及无菌茶翅蝽中均未检测到,由此推测pPaWBNy-1-ORF5蛋白与介体昆虫传播植原体有关。     

骏枣不同株系间的枣疯病抗性多样性

【目的】揭示骏枣品种内枣疯病抗性多样性,筛选高抗枣疯病的骏枣株系。【方法】以骏枣的16个株系为试材,通过田间调查和PCR检测,研究不同株系在嫁接侵染枣疯病后的症状表现和病原侵染情况;利用“合成-合理满意度”和多维价值理论的合并原则,对不同株系果实的相对含水率、单果重、可食率、果形指数、Vc含量、可溶性糖和可滴定酸含量等性状指标进行综合评价;采用AFLP技术对不同株系进行基因组遗传多样性分析。【结果】不同株系对枣疯病抗性表现出显著差异,可分为感病型、延迟感病型、延迟抗病型和抗病型4种类型,T15、J2、J5、J8这4个骏枣株系高抗枣疯病,其中,T15、J5和J8的果实综合性状在骏枣株系中亦居于较高水平;进一步的AFLP分析发现,不同抗性类型间在基因组水平上存在一定差异。【结论】筛选出了4个抗枣疯病骏枣株系,其中3个株系果实综合品质良好。