山东省枣疯病植原体的鉴定及分子变异分析

 本研究对山东省11个地区的枣疯病样品进行了鉴定和分子变异分析。以样品总DNA为模板,经扩增和序列测定,分别得到16S rRNA (1 432 bp)、核糖体蛋白基因rp (1 196 bp)、转运蛋白基因secA (836 bp) 和secY (1 421 bp) 的序列,secA基因序列是首次从枣疯病植原体中扩增获得。对获得的序列与NCBI数据库中相关植原体序列进行聚类和核苷酸变异分析,结果显示山东省枣疯病植原体属于16SrⅤ-B、rpⅤ-C、secYⅤ-C亚组,相对于16S rRNA基因,rp,secA和secY变异更大,非同义突变更多,更利于对国内不同来源的枣疯病植原体的精细系统进化分析。     

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。     

来源于湖北省枣疯病植原体分离物16S rDNA和rp基因的序列分析

以田间采集的来源于我国湖北省枣树产业主产区随州市随县种植的表现为"枣疯病"症状的枣树分离株为试材,对其16S rDNA和核糖体蛋白(ribosomal protein,rp)基因采用Nested-PCR进行扩增以及序列分析。结果表明,湖北JWB-Hubei植原体分离物16S rDNA基因的核苷酸序列与我国山东、河南等地的分离株一致率均为99%以上,在进化树中位于同一亚组的不同进化分支;虚拟RFLP图谱分析表明,JWB-Hubei属于16SrV-B亚组一个成员,与其进化树分组结果一致。JWBHubei分离株rp基因的核苷酸序列也与我国山东、陕西等地区的分离株一致率均为99%以上,在进化树中聚为同一亚组,与报道的基于RFLP分类属于rpV-C亚组的中国枣疯病分离物(JWB)聚集于同一亚组不同分支。该研究结果明确了湖北省枣疯病植原体的分类地位以及与来源于我国不同地区枣疯病分离株之间的遗传进化关系,为进一步研究植原体的株系划分、基因遗传变异研究提供了理论基础。     

杏褪绿卷叶植原体新疆分离物16S rDNA基因克隆与序列分析

利用植原体16S rDNA基因通用引物对新疆轮台县疑似杏褪绿卷叶病植株总DNA进行巢氏PCR检测,扩增出大小约1.2 kb的特异性条带。对扩增产物克隆和测序,确定特异片段大小为1248 bp。序列同源性比较和系统进化分析表明,新疆杏褪绿卷叶植原体不同分离株16S rDNA基因序列同源性极高,达到99.8%~100%。与16SrⅤ组成员的同源性达到98.2%以上,其中与16SrⅤ-B亚组的枣疯病植原体山东宝山分离株,甜樱桃绿化植原体山东分离株同源性最高,达到99.4%~99.6%。进一步虚拟RFLP分析,结果表明该植原体属于榆树黄化组(16SrⅤ)的一个新的亚组,与其相似性最高的是16SrⅤ-B亚组,相似系数为0.94。本研究首次报道了新疆杏褪绿卷叶植原体16S rDNA的序列,确定了其分类地位,为杏褪绿卷叶病的早期诊断和检测提供了基础。     

海南省木豆丛枝病植原体的分子检测及鉴定

 利用植原体通用引物R16mF2/R16mR1和rp (Ⅱ) F1/rp (Ⅱ) R1对海南木豆丛枝病植原体16S rDNA和部分核糖体蛋白(ribosomal protein,rp)基因序列进行PCR扩增、克隆和测序。获得海南木豆丛枝病植原体16S rDNA基因片段为1430bp,rp基因片段为1170bp。核苷酸同源性比较和系统进化树构建表明,引起海南木豆丛枝病的植原体应属于16SrⅡ组中的亚组ⅲ。本研究首次从分子水平确定了引起我国海南木豆丛枝病的病原物为植原体,明确了其分类地位,为该病害流行学研究和防治提供了理论依据。     

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)。     

云南花生丛枝植原体16S rRNA和核糖体蛋白基因序列分析

 利用植原体16S rRNA基因及核糖体蛋白基因(ribosomal protein, rp)通用引物对发生在云南元谋的花生丛枝病病株DNA进行PCR扩增,并对扩增片段进行序列测定。扩增获得的云南元谋花生丛枝植原体(PnWB-YNym)16S rDNA、16S-23S rDNA和23S DNA片段总长1 806 bp,rp基因扩增片段长1 171 bp。云南株系与来源于台湾和海南的花生丛枝植原体均有较高同源性。比较16S rDNA片段,发现云南株系在5个位点上与来自台湾或海南的株系存在碱基差异,其中有1个位点的差异是云南元谋株系特异的;再分别比较核糖体蛋白rplV-rpsC 2个基因所编码的氨基酸序列,发现云南株系rpsC编码的第194位氨基酸与台湾和海南的株系存在差异。经16S rDNA片段系统进化及iPhyClassifier在线分析,表明PnWB-YNym在分类上属于16SrII-A亚组成员,与候选种‘Candidatus Phytoplasma australasiae’相关;基于rp基因构建的系统进化树表明,PnWB-YNym与16SrII-A亚组各成员聚为同一亚进化支(iii)。     

一种引起香石竹黄化病植原体的初步鉴定

植原体(phytoplasma)(原称类菌原体Myco- plasma-like Organism,简称MLO)是一类无细胞壁,存在于植物筛管细胞内的原核生物。迄今为止,世界上报道的植物植原体病害多达300余种, 且有不断增加的趋势。其主要症状包括丛枝、黄化、花变叶、花器褪化等。由于植原体至今未能分离培养成功,其培养性状、生化特性、营养需求等都无从得知,所以长期以来对其检测及鉴定技术的发展都没有突破性进展。近十多年来,PCR技术使植原体检测、鉴定与分类取得了令人瞩目的进展,对植原体16S rRNA基因的分析使人们从遗传本质上认识到了植原体与其它原核微生物间的差异,也使人们对植原体的检测达到了单拷贝基因水平。目前, 根据植原体16S rRNA和核糖体蛋白(rp)基因的序列分析形成了植原体分类和鉴定的基本框架。     

宁夏马铃薯僵顶植原体的分子鉴定

通过透射电子显微镜,在从宁夏回族自治区固原市彭阳县红河镇采集的表现叶片上卷、红叶、气生薯症状的马铃薯样品叶脉韧皮部筛管细胞内观察到大量直径为500~700 nm的球形植原体粒子。以提取的感病和健康马铃薯叶片总DNA为模板,应用植原体16S rRNA基因和rp基因通用引物进行PCR扩增,从感病样品中扩增得到了长度均约为1.2 kb的片段。对获得基因核酸一致性比较分析表明,马铃薯僵顶植原体宁夏株系16S rRNA基因与‘Candidatus Phytoplasma fragariae’槭树株系(MK501642)16S rRNA基因核酸一致性最高,为99.7%,rp基因与‘Ca.P.fragariae’云南马铃薯YN-2G株系(KJ144889)rp基因核酸一致性最高,为100%;基于16S rRNA基因和rp基因构建系统进化树发现,马铃薯僵顶植原体宁夏株系与16SrⅫ-E亚组成员聚在一起。基于透射电镜观察和基因序列比较分析,证明宁夏发生的马铃薯僵顶病与植原体侵染相关,该植原体在分类地位上属于植原体16SrⅫ-E亚组。     

野生酸枣疯病与栽培大枣疯病发生的关系

为了明确野生酸枣疯病与栽培大枣疯病发生和流行的关系,采用随机徒步调查、挖根和接穗嫁接法对我国野生酸枣、栽培大枣及大枣接穗嫁接野生酸枣的枣疯病进行了田间调查,并取样检测病菌及比较不同菌株的保守基因序列.结果显示,我国野生酸枣疯病发生范围广,且地区间自然发病率差异很大,在0～40%之间;病株呈明显的团簇状分布,病菌在团簇中的根蘖苗与母株间传播或通过介体昆虫传播到后代种子苗上.在枣疯病流行区,栽培大枣发病与枣园周围分布的野生酸枣发病程度有关;用感病品系的接穗或带菌接穗嫁接到野生酸枣砧木上易导致嫁接苗发病和病害流行,而采用抗病的壶瓶枣和婆枣抗病品系接穗嫁接野生酸枣则发病率明显下降.用巢式PCR进行的病菌检测结果显示,在病害流行区酸枣或大枣无症状枝叶样品的带菌检出率为10%～32%.不同地区栽培大枣和野生酸枣上植原体的16S rDNA、16S-23S rDNA间区(SR)及核糖体蛋白基因(rp)序列比较鉴定结果显示,侵染酸枣的植原体与栽培大枣疯病植原体应为相互传染的同种致病菌.     

A Leafhopper (Hishimonus sellatus) Transmits Phylogenetically Distant Phytoplasmas: Rhus Yellows and Hovenia Witches' Broom Phytoplasma

Phytoplasmas causing a severe decline of three tree species, i.e., Rhus javanica, Hovenia tomentella and Zizyphus jujuba, in Japan were examined for their transmissibility by a leafhopper species Hishimonus sellatus, and for their phylogenetic relatedness. By H. sellatus, Rhus yellows (RhY) phytoplasma was transmissible to white clover and periwinkle seedlings, causing typical symptoms in these plants. Jujube witches' broom (JWB) phytoplasma was also transferred to the host plant, Z. jujuba, by the leafhopper. Because JWB phytoplasma was transmitted to Hovenia tomentella and caused the same symptoms as Hovenia witches' broom (HWB), JWB phytoplasma may be very closely related to HWB phytoplasma. RFLP analysis of the PCR products of 16S rDNA revealed that RhY phytoplasma belongs to the Aster yellows (AY) group, and JWB and HWB phytoplasmas belong to a different group (possibly Elm yellows group). Thus, we found that one species of leafhopper can carry phylogenetically distant phytoplasmas. Received 23 April 2001/ Accepted in revised form 29 October 2001     

Detection and Indentification of European Stone Fruit Yellows and Other Phytoplasmas in Wild Plants in the Surroundings of Apricot Chlorotic Leaf Roll-affected Orchards in Southern France

Between 1994 and 1998 a field study was conducted to identify plant hosts of the European stone fruit yellows (ESFY) phytoplasma in two apricot growing regions in southern and southwestern France where the incidence of apricot chlorotic leaf roll was high. A total of 431 samples from 51 different plant species were tested for the presence of phytoplasmas by PCR using universal and ESFY-specific primers. ESFY phytoplasma was detected in six different wild growing Prunus species exhibiting typical ESFY symptoms as well as in symptomless dog rose bushes (Rosa canina), ash trees (Fraxinus excelsior) and a declining hackberry (Celtis australis). The possible role of these plant species in the spread of ESFY phytoplasma is discussed. PCR-RFLP analysis of ribosomal DNA amplified with the universal primers was carried out to characterize the other phytoplasmas found. Thus, elm yellows phytoplasma, alder yellows phytoplasma and rubus stunt phytoplasma were detected in declining European field elm trees (Ulmus carpinifolia Gled), in declining European alder trees (Alnus glutinosa) and in proliferating Rubus spp. respectively. The presence of rubus stunt phytoplasma in great mallow (Malva sylvestris) and dog rose was demonstrated for the first time. Furthermore, the stolbur phytoplasma was detected in proliferating field bindweed (Convolvulus arvensis) and a previously undescribed phytoplasma type was detected in red dogwood (Cornus sanguinea). According to the 16S rDNA-RFLP pattern this new phytoplasma belongs to the stolbur phytoplasmas group.     

Differential detection and genetic relatedness of phytoplasmas in papaya

The genetic relatedness of phytoplasmas associated with dieback (PDB), yellow crinkle (PYC) and mosaic (PM) diseases in papaya was studied by restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene and 16S rRNA/23S rRNA spacer region (SR). RFLP and SR sequence comparisons indicated that PYC and PM phytoplasmas were identical and most closely related to members of the faba bean phyllody strain cluster. By comparison the PDB phytoplasma was most closely related to Phormium yellow leaf (PYL) phytoplasma from New Zealand and the Australian grapevine yellows (AGY) phytoplasma from Australia. These three phytoplasmas cluster with the stolbur and German grapevine yellows (VK) phytoplasmas within the aster yellows strain cluster. Primers based on the phytoplasma tuf gene, which amplify gene products from members of the AY strain cluster, also amplified a DNA product from the PDB phytoplasma but not from either the PYC or PM phytoplasmas. Primers deduced from the 16S rRNA/SR selectively amplified rDNA sequences from the PDB and AGY phytoplasmas but not from other members of the stolbur strain cluster. Similarly, primers designed from 16S rRNA/SR amplified rDNA from the PYC and PM phytoplasmas but not from the PDB phytoplasma. These primers may provide for more specific detection of these pathogens in epidemiological studies.     

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     

Association of 'Candidatus Phytoplasma australiense' with green petal and lethal yellows diseases in strawberry

The identity of phytoplasmas detected in strawberry plants with green petal (SGP) and lethal yellows (SLY) diseases was determined by RFLP analysis of the 16S rRNA gene and adjacent spacer region (SR). RFLP and sequence comparisons indicated that the phytoplasmas associated with SGP and SLY were indistinguishable and were most closely related to ' Candidatus Phytoplasma australiense', the phytoplasma associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases. This taxon lies within the aster yellows strain cluster. Primers based on the phytoplasma tuf gene, which amplify only members of the AY strain cluster, amplified a DNA product from the SGP and SLY phytoplasmas. Primers deduced from the 16S rRNA/SR of P. australiense that amplify only members of this taxon amplified rDNA sequences from the SGP and SLY phytoplasmas. Primers that selectively amplify members of the faba bean phyllody (FBP) phytoplasma group, the most commonly occurring phytoplasma group in Australia, did not amplify rDNA from the SGP and SLY phytoplasmas.     

An Antibody Against the SecA Membrane Protein of One Phytoplasma Reacts with Those of Phylogenetically Different Phytoplasmas

ABSTRACT Antisera raised against phloem-limited phytoplasmas generally react only with the phytoplasma strain used to produce the antigen. There is a need for an antiserum that reacts with a variety of phytoplasmas. Here, we show that an antiserum raised against the SecA membrane protein of onion yellows phytoplasma, which belongs to the aster yellows 16S-group, detected eight phytoplasma strains from four distinct 16S-groups (aster yellows, western X, rice yellow dwarf, and elm yellows). In immunoblots, approximately 96-kDa SecA protein was detected in plants infected with each of the eight phytoplasmas. Immunohistochemical staining of thin sections prepared from infected plants was localized in phloem tissues. This antiserum should be useful in the detection and histopathological analysis of a wide range of phytoplasmas.     

Use of heteroduplex mobility assay for identification and differentiation of phytoplasmas in the aster yellows group and the clover proliferation group

ABSTRACT This paper describes the identification and differentiation of phytoplasmas by a highly sensitive diagnostic technique, DNA heteroduplex mobility assay (HMA). Closely related phytoplasma isolates of clover proliferation (CP), potato witches'-broom (PWB), and alfalfa witches'-broom (AWB) were collected from the field from 1990 to 1999. The entire 16S rRNA gene and 16/23S spacer region were amplified by polymerase chain reaction (PCR) from the field samples and standard CP, PWB, and AWB phytoplasmas and were subjected to restriction fragment length polymorphism (RFLP) analysis and HMA. Two subgroups (I and II) of phytoplasmas in the CP group were identified by HMA but not by RFLP analysis. The results were confirmed by 16/23S spacer region sequence data analysis. After HMA analyses of the PCR-amplified 16/23S spacer region, 14 phytoplasma isolates from field samples were classified into two aster yellows subgroups: subgroup I, phytoplasma isolates from China aster (Callistephus chinensis) yellows, French marigold (Tagetes patula) yellows, cosmos (Cosmos bipinnatus cv. Dazzler) yellows, clarkia (Clarkia unguiculata) yellows, California poppy (Eschscholzia californica cv. Tai Silk) yellows, monarda (Monarda fistulosa) yellows, and strawflower (Helichrysum bracteatum) yellows; and subgroup II, phytoplasma isolates from zinnia (Zinnia elegans cv. Dahlia Flower) yellows, Queen-Annes-Lace (Daucus carota) yellows, scabiosa (Scabiosa atropurpurea cv. Giant Imperial) yellows, Swan River daisy (Brachycombe multifida cv. Misty Pink) yellows, pot marigold (Calendula officinalis) yellows, purple coneflower (Echinacea purpurea) yellows, and feverfew (Chrysanthemum parthenium) yellows. The results indicate that HMA is a simple, rapid, highly sensitive and accurate method not only for identifying and classifying phytoplasmas but also for studying the molecular epidemiology of phytoplasmas.     

棣棠丛枝病相关植原体的分子鉴定

 植原体（Candidatus Phytoplasma）是一种没有细胞壁的原核微生物,主要由取食韧皮部的昆虫（叶蝉、飞虱等）传播, 也可由菟丝子寄生和嫁接等途径传播,常常引起植株黄化、丛枝、花器变态、萎缩等症状。迄今为止,世界上报道的植物植原体病害有1 000余种,仅我国就有100多种,造成巨大损失。