植原体病害研究概况

植原体原名类菌原体,是一类重要的植物病原物,归属于细菌,无细胞壁,专性寄生于植物韧皮部。在菌体大小、结构以及遗传进化上与菌原体、螺原体十分相似。世界范围内植原体已引起千余种植物病害,主要表现为丛枝、黄化、节间缩短等。植原体病原主要依靠吸食植物韧皮部的昆虫介体传播,如叶蝉、木虱等。本文主要对植原体的病原学、遗传进化与基因组、致病机理以及防控进行综述。     

植原体分类鉴定研究进展

植原体(Phytoplasma)是一类重要的植物病原细菌。植原体的分类鉴定是植原体研究和病害防控的基础。本文介绍植原体病原发现的历史,植原体属的系统分类地位以及种、组、亚组分类鉴定的规则,现今已正式命名的植原体种和组,植原体分类鉴定的注意事项,并对分类鉴定研究的发展趋势进行了展望。     

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。     

云南芒果变叶病植原体的分子鉴定

 植原体（phytoplasma）是一类没有细胞壁,不能离体培养的原核生物,对四环素敏感,主要存在于植物筛管细胞中。植原体主要通过叶蝉、飞虱等取食植物韧皮部的昆虫传播,也可通过菟丝子寄生和嫁接等方式传播。目前,全世界已发现1 000多种由植原体引起的植物病害,我国大陆已报道100余种与之相关的病害\[1\]。由植原体引起的病害症状主要表现为植株花器病态、小叶、丛枝、黄化等,从而导致植物产量和品质明显下降。     

植原体基因组学研究进展

植原体（phytoplasma）是一类重要的植物病原细菌, 可经叶蝉、飞虱等昆虫介体传播, 感染1 000多种植物, 产生丛枝、黄化、韧皮部黑斑坏死等症状, 给农业、林业生产带来巨大的损失。本文对植原体基因组学研究的历史、现状及当前植原体效应蛋白等方面的研究进展进行了综述。     

桃黄化病植原体昆虫介体的验证及其带毒部位PCR检测

 利用植原体16S rDNA通用引物对采集的北京和天津黄化病桃树总DNA进行巢式PCR检测,证明发病样本的病原为桃黄化病植原体。经过检测昆虫总DNA和经取食过的人工培养液DNA中桃黄化病植原体的16S rDNA,结果表明桃黄化病植原体的有效传播媒介昆虫为桃一点叶蝉。将带毒桃一点叶蝉个体的头部、胸部以及腹部分离,分别在这些部位检测到桃黄化病植原体的16S rDNA,说明桃一点叶蝉的头部、胸部以及腹部都可带毒,表明植原体可从植物汁液进入叶蝉的口针、食道和肠道。     

植原体TaqMan探针实时荧光PCR检测鉴定方法的建立

 本研究成功建立了植原体分类鉴定和检测的TaqMan探针实时荧光PCR方法,该方法根据植原体16S rDNA保守区设计了1个TaqMan广谱探针和3个植原体组间点突变特异性探针,并对9种植原体和5种细菌以及3个植物样本进行实时荧光PCR。结果表明,用广谱探针可检测到所有植原体产生荧光信号,而细菌不产生荧光信号。当用植原体组间特异性探针检测时,仅能检测到该组植原体产生荧光信号,检测的敏感性比常规的PCR-电泳检测高约100倍、检测速度有较大提高。由于PCR产物是荧光探针检测,本方法特异性强,并可以用组特异探针直接确定植原体种类。实验采用完全闭管检测,降低了污染机会。本研究为其它原核生物、特别是不能培养菌、难培养菌的检测鉴定和分类提供了新方法。     

植原体16S rDNA RFLP指纹图谱分析软件研究

 植原体是一类寄生于植物韧皮部的原核生物。目前国际上主要根据植原体16S rDNA RFLP图谱的比对进行分类及鉴定。传统的PCR-RFLP试验步骤繁琐、重复性差,误差大,尤其在检测鉴定大量植原体病原时,耗时、耗力。通过对目前已公布的全部植原体16S rDNA序列进行比对整理,并生成16S rDNA RFLP电子指纹图谱库。在此基础上开发了指纹图谱分析软件DNA-FP Cluster1.0。该软件可基于序列或图谱形式进行比对,并自动输出比对结果。结果呈现形式为3种:即一种16S rDNA RFLP图谱与多种图谱间相似性分析;多种16S rDNA RFLP图谱间相似性分析;16S rDNA RFLP图谱间相似度树状图。该软件在不需要酶切试验操作的情况下实现了对植原体的快速分类与鉴定,并解决了因植原体材料不全而无法进行植原体酶切后比对的难题,为今后植原体候选种内的细化分类提供了方法与工具。     

用荧光显微镜检测类菌原体的快速压片技术

自从1967年首次在植物上发现类菌原体(Mycoplasmalike Organisms简称MLO是植物的病原菌以来,多种技术已应用于显现和证明感病植物中的类菌原体和螺原体(Spiroplasma)。电子显微镜观察是目前最重要和最普遍使用的类菌原体病害诊断方法,但此法昂贵且费时。近些年来,一些植病学家运用能与DNA结合的荧光染料     

介体条沙叶蝉传播小麦蓝矮病植原体特性研究

小麦蓝矮病植原体(wheat blue dwarf,WBD)属于翠菊黄化组三叶草绿变亚组植原体(16SrⅠ-C),由介体条沙叶蝉(Psammotettix striatus L.)专化性传播。通过电镜超微结构观察,在接种小麦、长春花和带毒条沙叶蝉体内有大量植原体,而在健康植物组织、无毒条沙叶蝉和带毒条沙叶蝉所产卵中未见植原体的存在。通过介体传毒试验和PCR检测发现,条沙叶蝉最适获毒期为7 d,植原体在虫体内的潜育期为1 5～1 7 d,接毒期为2～3 d。条沙叶蝉一旦获毒可终生持毒和传毒。不同虫态的条沙叶蝉带毒率没有明显的差异,但寄生植物的种类影响其带毒率。     

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)。     

Detection and identification of the maize bushy stunt phytoplasma in corn plants in Brazil using PCR and RFLP

Plants of corn (Zea mays L.) exhibiting symptoms of stunting and leaf reddening were assayed for the presence of phytoplasma gene sequences through the use of phytoplasma rRNA and ribosomal protein gene and maize bushy stunt (MBS) phytoplasma-specific oligonucleotide primers in polymerase chain reactions (PCR). Polymorphisms in 16S rDNA amplified from diseased plants were those characteristic of phytoplasmas classified in the16S rRNA gene group 16SrI, subgroup IB, of which MBS phytoplasma is a member. Amplification of ribosomal protein (rp) gene sequences in PCR primed by phytoplasma-specific primers confirmed presence of a phytoplasma in the diseased plants. Restriction fragment length polymorphism (RFLP) patterns of the amplified phytoplasma rp gene sequences were similar or identical to those observed for a known strain of MBS phytoplasma. In separate PCR, an MBS-specific oligonucleotide pair primed amplification of a MBS-characteristic DNA from templates derived from the diseased corn. Our data provide the first firm evidence for the presence of maize bushy stunt phytoplasma in corn in Brazil.     

Phytoplasmas infecting sour cherry and lilac represent two distinct lineages having close evolutionary affinities with clover phyllody phytoplasma

Phytoplasmas infecting sour cherry and lilac in Lithuania were found to represent two lineages related to clover phyllody phytoplasma (CPh), a subgroup 16SrI-(R/S)C (formerly 16SrI-C) strain exhibiting rRNA interoperon sequence heterogeneity. 16S rDNAs amplified from the cherry bunchy leaf (ChBL) and lilac little leaf (LcLL) phytoplasmas were identical or nearly identical to those of operon rrnA and operon rrnB, respectively, of CPh. There was no evidence of 16S rRNA interoperon sequence heterogeneity in either LcLL or ChBL phytoplasma. Based on collective RFLP patterns of 16S rDNA, ChBL was classified in subgroup 16SrI-R, and LcLL was classified in new subgroup 16SrI-S. The ribosomal protein (rp) gene sequences from LcLL phytoplasma were identical to those of CPh, and strain LcLL was classified in rp subgroup rpI-C. By contrast, rp gene sequences from ChBL phytoplasma differed from those of subgroup rpI-C; based on RFLP patterns of rp gene sequences, ChBL was classified in new rp subgroup rpI-O. Single nucleotide polymorphisms (SNPs), designated here by a new SNP convention, marked members of rp subgroup rpI-C, and distinguished LcLL and CPh from ChBL and other non-rpI-C phytoplasmas in group 16SrI. The results raise questions concerning phytoplasma biodiversity assessment based on rRNA genes alone and encourage the supplemental use of a single copy gene in phytoplasma identification and classification, while drawing attention to a possible role of horizontal gene transfer in the evolutionary history of these lineages.     

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。     

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。     

臭矢菜丛枝病植原体的分子鉴定研究

 本实验采用DAPI荧光显微镜、PCR、克隆和测序等技术,对海南臭矢菜丛枝病样进行了检测和鉴定。以染病臭矢菜总DNA为模板应用3对植原体特异性引物进行PCR扩增,获得PCR产物为16S rDNA(1 430 bp)、16S-23S rDNA(358bp)、rp DNA(1 294 bp)。应用DNA回收试剂盒获得了3个PCR扩增片断的纯化产物,并克隆到DH5α大肠杆菌中测序。应用DNAMAN和MEGA软件对获得的序列与NCBI数据库中植原体序列进行同源性分析和构建系统发育树。结果显示臭矢菜丛枝病植原体与花生丛枝病植原体序列同源性最高,16S rDNA的序列同源性为99.9%,16S-23S rDNA高达100%,rp为99.7%,因而将臭矢菜丛枝病植原体归为花生丛枝组(16SrⅡ),根据16S rDNA的RFLP分析,将其归为16SrⅡ-A亚组。     

Ecological implications from a molecular analysis of phytoplasmas involved in an aster yellows epidemic in various crops in Texas

ABSTRACT In the spring of 2000, an aster yellows (AY) epidemic occurred in carrot crops in the Winter Garden region of southwestern Texas. A survey revealed that vegetable crops, including cabbage, onion, parsley, and dill, and some weeds also were infected by AY phytoplasmas. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis of PCR-amplified phytoplasma 16S rDNA were employed for the detection and identification of phytoplasmas associated with these crops and weeds. Phytoplasmas belonging to two subgroups, 16SrI-A and 16SrI-B, in the AY group (16SrI), were predominantly detected in infected plants. Carrot, parsley, and dill were infected with both subgroups. Onion and three species of weeds (prickly lettuce, lazy daisy, and false ragweed) were predominantly or exclusively infected by subgroup 16SrI-A phytoplasma strains, while cabbage was infected by subgroup 16SrI-B phytoplasmas. Both types of phytoplasmas were detected in three leafhopper species, Macrosteles fascifrons, Scaphytopius irroratus, and Ceratagallia abrupta, commonly present in this region during the period of the epidemic. Mixed infections were very common in individual carrot, parsley, and dill plants and in individual leafhoppers. Sequence and phylogenetic analyses of 16S rDNA and ribosomal protein (rp) gene sequences indicated that phytoplasma strains within subgroup 16SrI-A or subgroup 16SrI-B, detected in various plant species and putative insect vectors, were highly homogeneous. However, based on rp sequences, two rpI subgroups were identified within the subgroup 16SrI-A strain cluster. The majority of subgroup 16SrI-A phytoplasma strains were classified as rp subgroup rpI-A, but phytoplasma strains detected in one onion sample and two leafhoppers (M. fascifrons and C. abrupta) were different and classified as a new rp subgroup, rpI-N. The degree of genetic homogeneity of the phytoplasmas involved in the epidemic suggested that the phytoplasmas came from the same pool and that all three leafhopper species may have been involved in the epidemic. The different phytoplasma population profiles present in various crops may be attributed to the ecological constraints as a result of the vector-phytoplasma-plant three-way interaction.     

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。     

Molecular identification of a new phytoplasma associated with alfalfa witches'-broom in oman

ABSTRACT Alfalfa (Medicago sativa) plants showing witches'-broom symptoms typical of phytoplasmas were observed from Al-Batinah, Al-Sharqiya, Al-Bureimi, and interior regions of the Sultanate of Oman. Phytoplasmas were detected from all symptomatic samples by the specific amplification of their 16S-23S rRNA gene. Polymerase chain reaction (PCR), utilizing phytoplasma-specific universal primer pairs, consistently amplified a product of expected lengths when DNA extract from symptomatic samples was used as template. Asymptomatic plant samples and the negative control yielded no amplification. Restriction fragment length polymorphism profiles of PCR-amplified 16S-23S rDNA of alfalfa using the P1/P7 primer pair identified phytoplasmas belonging to peanut witches'-broom group (16SrII or faba bean phyllody). Restriction enzyme profiles showed that the phytoplasmas detected in all 300 samples belonged to the same ribosomal group. Extensive comparative analyses on P1/P7 amplimers of 20 phytoplasmas with Tru9I, Tsp509I, HpaII, TaqI, and RsaI clearly indicated that this phytoplasma is different from all the other phytoplasmas employed belonging to subgroup 16SrII, except tomato big bud phytoplasma from Australia, and could be therefore classified in subgroup 16SrII-D. The alfalfa witches'-broom (AlfWB) phytoplasma P1/P7 PCR product was sequenced directly after cloning and yielded a 1,690-bp product. The homology search showed 99% similarity (1,667 of 1,690 base identity) with papaya yellow crinkle (PapayaYC) phytoplasma from New Zealand. A phylogenetic tree based on 16S plus spacer regions sequences of 35 phytoplasmas, mainly from the Southern Hemisphere, showed that AlfWB is a new phytoplasma species, with closest relationships to PapayaYC phytoplasmas from New Zealand and Chinese pigeon pea witches'-broom phytoplasmas from Taiwan but distinguishable from them considering the different associated plant hosts and the extreme geographical isolation.     

广东枣疯病植原体的鉴定

Several jujube plants with witches′ broom, little leaf, and big bud symptoms, which were likely infected by jujube witches′ broom (JWB) phytoplasma, were collected in Guangzhou, Guangdong Province. To identify the pathogen, PCR was performed using phytoplasma 16S rDNA universal primer pairs R16mF2/R1 and P1/P7 and SecA gene primer pair SecAfor1/rev3 with total DNA of the symptomatic plants as templates. Specific fragments, 1.4 kb, 1.8 kb, and 0.8 kb in length, were amplified from one of three symptomatic samples. Phylogenetic analysis based on 16S rDNA verified that the pathogen harming jujube plants in Guangzhou was jujube witches′ broom phytoplasma which belonged to 16SrV-B subgroup. Comparison results also showed that the 16S rDNA sequence of Guangzhou JWB phytoplasma shared the highest nucleotide identity (100%) with the reported jujube witches′ broom phytoplasma Japanese strain (AB442218) and JWB strain (AY197661) and shared the nucleotide identity ranging from 99.74% to 99.80% with the other JWB phytoplasma strains. In addition, phylogenetic analysis based on SecA also showed that Guangzhou jujube witches′ broom phytoplasma belonged to 16SrV-B subgroup and shared 99.28%-99.76% similarity with other phytoplasma strains. All these results suggested that jujube witches′ broom phytoplasma has infected jujube plants in Guangdong Province.