不同生物型烟粉虱体内Wolbachia共生菌的检测及其系统树分析

Wolbachia是广泛分布于节肢动物体内一类共生细菌,它能够通过多种机制调控宿主的生殖方式。近年来的研究表明,Wolbachia与许多外来生物的成功入侵相关。本文利用长PCR的方法特异扩增了不同生物型烟粉虱（共24个种群）体内Wolbachia的wsp基因,结果发现B型和Q型烟粉虱入侵种群体内均未检测到Wolbachia,而在非B/Q型的浙江种群和肯尼亚种群体内检测到了Wolbachia。对该wsp基因进行测序并和已知序列进行同源性分析发现,浙江烟粉虱种群的Wolbachia属于B组Con/Rug亚种群,而肯尼亚种群属于B组Btab1亚种群。Wolbachia的存在与否可能与烟粉虱的成功入侵有一定的关系。图2表2参23     

中国部分地区柑桔木虱体内感染Wolbachia的检测及其系统发育分析

Wolbachia是一种广泛分布于节肢动物体内的共生细菌, 该共生菌经寄主卵的细胞质传播并参与多种寄主生殖活动调控, 对节肢动物的物种进化有着重要意义并可能应用于害虫的生物防治。本研究以柑桔黄龙病（Huanglongbing, HLB）的主要传播媒介——柑桔木虱Diaphorina citri为研究对象, 通过Wolbachia的16S rDNA、 细胞分裂基因（ftsZ）、 表面蛋白基因（wsp）的特异引物对来自于中国广西北海、 广西柳州、 广东深圳、 广东阳春、 福建福州5个地区的柑桔木虱进行PCR扩增, 并对扩增产物进行克隆测序, 与GenBank中相关序列进行比对分析, 建立了柑桔木虱共生Wolbachia的系统发育树。结果显示上述5个地区的柑桔木虱均存在Wolbachia感染, 通过Wolbachia的16S rDNA, ftsZ基因和wsp基因DNA序列的系统发育分析表明, 5个地区的柑桔木虱体内的Wolbachia均属于B组; 基于wsp基因的系统发育分析进一步表明5个地区的亚洲柑桔木虱体内的Wolbachia属于B组的Con亚组, 且不同地区柑桔木虱体内的Wolbachia的16S rDNA, ftsZ基因和wsp基因序列差异不明显。研究结果为认识柑桔黄龙病传播媒介昆虫的进化及其综合防治提供了重要的参考依据。     

福建省烟粉虱自然种群Wolbachia感染特点

Wolbachia是专性的细胞内细菌,广泛存在于节肢动物生殖组织。已有的研究结果表明,节肢动物中存在A组和B组Wolbachia,而烟粉虱Bemisia tabaci中主要检测到了B组Wolbachia。本研究从福建省采集到17个不同烟粉虱地理种群,首先通过rDNA-ITS1克隆测序鉴定了不同烟粉虱地理种群的生物型,然后采用Wolbachia 16S rDNA的特异引物,并通过PCR-RFLP技术分析了不同烟粉虱地理种群中Wolbachia的感染特点。结果表明：从福建省闽侯、平潭、南平、来舟、漳平和沙县采集到的烟粉虱自然种群属于非B型,而非B型烟粉虱种群中存在广泛的超感染现象,即单个非B型烟粉虱个体中同时感染了不同型Wolbachia。相反,B型烟粉虱自然种群的个体中只感染A组Wolbachia。该研究依据密集采样的数据进一步证实了Wolbachia在烟粉虱自然种群中的分布确实与宿主的生物型密切相关,提示Wolbachia可能在烟粉虱的种群分化中发挥作用。     

桃蚜自然种群初级和次级共生菌的分子鉴定

蚜虫不仅带有专性初级内共生菌,还常常带有不同类群的兼性次级共生菌。本研究采用真细菌16S rDNA的通用引物,从桃蚜Myzus persicae (Sulzer)烟草种群体内扩增出1.5 kb的共迁移目的条带,然后将其克隆,并在阳性克隆筛选时进行了RFLP分析。当采用EcoRⅠ进行单酶切分析时,目的片段被切割成～650 bp和～850 bp两个小片段;当采用EcoRⅠ和HindⅢ进行双酶切分析时,该蚜群共生菌酶切谱带被分成2组,其中1组(GroupⅠ)只具有一个EcoRⅠ酶切位点,而另1组(Group Ⅱ)不仅具有一个EcoRⅠ酶切位点,还具有一个HindⅢ酶切位点,而且GroupⅠ为优势共生菌群。在此基础上分别对2组共生菌的16S rDNA全序列（～1.5 kb）进行了测定,结果表明：GroupⅠ属于泛菌属Pantoea,与成团泛菌Pantoea agglomerans亲缘关系最近（同源性达99.70%）,而Group Ⅱ属于蚜虫的专性内共生菌,即Buchnera aphidicola（同源性达99.50%）。这是在蚜虫体内存在泛菌次级共生菌的首次报道。     

浙江B型与非B型（China-ZHJ-1）烟粉虱种群共生细菌的检测及系统发育分析

烟粉虱体内存在共生细菌,包括初生共生细菌（primary endosymbiont）和次生共生细菌（secondary endosymbiont）。本项研究应用PCR技术检测了烟粉虱浙江B型和非B型China-ZHJ-1种群中共生细菌的分布。结果表明,烟粉虱B型和非B型体内均存在初生共生细菌,而两者次生共生细菌的组成存在差异。一种肠杆菌科次生共生细菌仅在B型烟粉虱中发现,而另两种次生共生细菌Wolbachia和杀雄菌Arsenophonus仅在非B型中发现。初生共生细菌的系统发育分析表明,B型是入侵生物型,而浙江非B型是本地生物型。     

Wolbachia在玉米螟赤眼蜂内的三重感染

Wolbachia是一类广泛存在于节肢动物体内的共生菌。玉米螟赤眼蜂Trichogramma ostriniae是我国玉米田间的优势赤眼蜂种, 据报道, 赤眼蜂种内有Wolbachia感染。本文利用Wolbachia的16s rDNA和wsp基因引物通过PCR方法对玉米螟赤眼蜂的野生种群进行了调查, 发现以wsp基因为鉴定依据, 检测的所有个体都感染了3种Wolbachia ［wOstGDAa (GenBank accession no. EU157103), wOstGDAb (GenBank accession no. EU157104) 和 wOstGDB (GenBank accession no. EU157105)］。本文首次报道了野生赤眼蜂种群内Wolbachia的三重感染率几乎为100%。根据本研究的结果, 可以推测当不同种赤眼蜂寄生同一寄主时, Wolbachia可能会在不同赤眼蜂种间进行横向传播。     

沃尔巴克氏体在中国三种稻飞虱中的感染

用PCR方法检测了采集于不同地域稻田的3种稻飞虱共生菌沃尔巴克氏体(Wolbachia)的感染,发现灰飞虱Laodelphax striatellus、褐飞虱Nilaparvata lugens、白背飞虱Sogatella furcifera为沃尔巴克氏体所感染。克隆了编码沃尔巴克氏体外膜蛋白质的wsp基因并进行了序列测定。对wsp的RFLP分析证实了这些飞虱为单一沃尔巴克氏体感染。研究了灰飞虱中沃尔巴克氏体所诱导的胞质不相容性及其在不同地域灰飞虱中的分布。还发现能寄生于上述3种飞虱的稻虱红螯蜂也受同种沃尔巴克氏体感染。沃尔巴克氏体可能通过这种寄生蜂在不同昆虫间横向传播。     

水稻褐飞虱内生共生细菌Arsenophonus的鉴定和系统分析

利用16S rDNA通用引物扩增了水稻褐飞虱Nilaparvata lugens（Stål）体内共生细菌的序列,经克隆、测序和NCBI数据库比对,发现褐飞虱体内存在杀雄菌属Arsenophonus类共生细菌,系统发育上与粉虱科和木虱科体内的Arsenophonus属亲源关系较近。在褐飞虱体内该共生细菌具有两种长度不同的16S rDNA序列,分别为1 504 bp和547 bp,其中后者为前者中间缺失了957 bp,其余序列相同。通过重新设计两对引物进行扩增,进一步确认不同褐飞虱地理种群及寄主种群均存在两种片段。Arsenophonus特异的 23S rDNA引物的扩增结果表明,Arsenophonus存在于所有检测的褐飞虱种群中,但不存在于水稻寄主中。荧光定量PCR检测发现3个褐飞虱室内寄主种群Arsenophonus属共生细菌含量不同,其中TN1种群明显高于Mudgo种群和ASD7种群。此为水稻褐飞虱体内存在Arsenophonus属共生细菌的首次报道。     

山东不同地区韭菜迟眼蕈蚊共生菌Wolbachia 的检测及鉴定

【目的】为了揭示山东省韭菜迟眼蕈蚊Bradysia odoriphaga Yang et Zhang种群共生菌 Wolbachia 的感染率及其分类地位,探讨该共生菌对韭菜迟眼蕈蚊的潜在影响。【方法】利用线粒体细胞色素氧化酶I（mtCOI）基因引物（LCO1490/HCO2198）,通过扩增测序和序列比对对采自山东省12个地区的根蛆种群进行了分类鉴定。在上述基础上,利用 Wolbachia 的16S rDNA和 wsp 基因特异引物（分别为16S-F/16S-R和81F/691R）对鉴别出的11个韭菜迟眼蕈蚊种群体内Wolbachia 感染情况进行了PCR检测;对感染个体体内 Wolbachia 依据16S rDNA基因片段序列进行分类鉴定。【结果】山东省12个根蛆种群中,11个种群为韭菜迟眼蕈蚊种群。基于 Wolbachia 的16S rDNA基因特异引物检测结果发现,这些韭菜迟眼蕈蚊种群广泛感染 Wolbachia （感染率为6.67%~93.33%）,而利用wsp 基因特异引物检测的感染率（0.00%~40.00%）相对较低些。基于 Wolbachia 的16S rDNA基因构建系统发育树表明,这些韭菜迟眼蕈蚊种群感染的Wolbachia 全部属于A组。【结论】确定了 Wolbachia 在山东省韭菜迟眼蕈蚊体内的感染率及其分类地位,为研究 Wolbachia 对韭菜迟眼蕈蚊生物学及生态学的影响奠定了基础。     

我国烟粉虱自然种群中存在广泛的Wolbachia感染现象

Wolbachia是广泛分布于节肢动物生殖组织中的细胞质遗传的一类共生菌,可以引起宿主生殖行为的改变.烟粉虱是重要的农业害虫,已有的研究表明烟粉虱中存在Wolbachia的感染,但所检测到的感染率不高,并且迄今在烟粉虱中所检出的Wolbachia均属于B组群.该研究从我国河北、新疆、北京、山东、浙江、广西、海南、广州和福建采集到18个烟粉虱地理种群,首先基于ITS1 rDNA克隆测序鉴定了烟粉虱的生物型,然后采用自行设计的Wolbachia 16S rDNA及wsp基因的专用引物对所采集到的烟粉虱种群进行了分子检测.结果表明几乎所有的烟粉虱自然种群中都存在Wolbachia感染,同时还发现B/Q生物型烟粉虱中主要感染A组群Wolbachia,而非B/Q生物型烟粉虱中存在大量的超感染现象.该研究显示烟粉虱自然种群中Wolbachia的感染率比预想的要高得多,而分子检测方法的灵敏度可能是影响Wolbachia感染率研究的关键因素之一.     

Polymerase chain reaction amplification and restriction fragment length polymorphism analysis of 16S rRNA genes from methanogens

For restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes, the rDNA fragments of 1.5 kb were amplified by polymerase chain reaction (PCR) from crude cell lysates of various methanogenic species which were prepared by a combined technique of ultrasonic treatment and protease digestion. The PCR products were purified by the polyethylene glycol precipitation method and treated with various restriction enzymes. The 16S rDNA fragments digested with HaeIII or HhaI gave species-specific RFLP profiles on simplified agarose gel electrophoresis. 16S rDNA gragments of 0.4 kb from the bulk DNA extracted from mixed populations of anaerobic sludge were also amplified by PCR with a pair of methanogen-specific primers and cloned directly by the T-A cloning technique. The cloned 16S rDNAs from recombinants were reamplified by PCR, and RFLP pattern analysis was performed following digestion with HhaI. The PCR-RFLP analysis of 16S rDNA with the present protocol can be completed within one day, provided that sufficient amounts of test cells are available, and has great promise as a simple and rapid technique for identification of methanogens. A combined method consisting of PCR amplification, direc cloning with T vectors, and RFLP analysis of 16S rDNA is also useful for rapid estimation of the mixed population structure of methanogens without the need for cultivation and isolation.     

烟粉虱复合种内共生菌多样性及其生物学意义

烟粉虱复合种Bemisia tabaci（Gennadius） complex内共生菌的种类可分为5类：变形菌门（Proteobacteria）γ变形菌纲的初级内共生菌Candidatus Portiera aleyrodidarum和次级内共生菌,变形菌门α变形菌纲Wolbachia属细菌,衣原体门（Chlamydiae）衣原体纲细菌CandidatusFritschea bemisiae以及拟杆菌门（Bacteroidetes）类噬胞细菌（Cytophaga-like-organism, CLO）。系统发育分析表明,烟粉虱初级内共生菌分为2支,一些初级内共生菌和宿主系统发育并不严格一致; 次级内共生菌分为3支,一些次级内共生菌可能存在水平传播或多次侵入的现象。上述5类内共生菌在烟粉虱不同生物型/地理种群内的分布存在差异。B型烟粉虱可能具有独特的初级、次级内共生菌,并且不含有或很少含有Wolbachia属细菌、衣原体纲细菌Candidatus Fritschea bemisiae以及类噬胞细菌（CLO）。最后探讨了不同内共生菌对烟粉虱宿主的生物学意义及其研究方向。     

Wolbachia infections of the whitefly Bemisia tabaci

We report the first systematic survey for the presence of Wolbachia endosymbionts in aphids and whiteflies, particularly different populations and biotypes of Bemisia tabaci. Additional agriculturally important species included were predator species, leafhoppers, and lepidopterans. We used a polymerase chain reaction (PCR)-based detection assay with ribosomal 16S rDNA and Wolbachia cell surface protein (wsp) gene primers. Wolbachia were detected in a number of whitefly populations and species, whitefly predators, and one leafhopper species; however, none of the aphid species tested were found infected. Single, double, and triple infections were detected in some of the B. tabaci populations. PCR and phylogenetic analysis of wsp gene sequences indicated that all Wolbachia strains found belong to group B. Topologies of the optimal tree derived by maximum likelihood (ML) and a ML tree in which Wolbachia sequences from B. tabaci are constrained to be monophyletic are significantly different. Our results indicate that there have been at least four independent Wolbachia infection events in B. tabaci. The importance of the presence of Wolbachia infections in B. tabaci is discussed.     

Prevalence of Wolbachia supergroups A and B in Bemisia tabaci (Hemiptera: Aleyrodidae) and some of its natural enemies

Wolbachia, a bacterial symbiont, is maternally transmitted in arthropods and nematodes. We report a systematic survey of Wolbachia taxonomy in the sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), and in some of its natural enemies. For the first time, Wolbachia infections in B. tabaci are correlated with various whitefly genetic groups, host plants, and natural enemies as well as with geographical regions. Polymerase chain reaction using 16S rDNA and fisZ genes revealed two Wolbachia supergroups, A and B, exist as single or double infections in B. tabaci as well as in some of its aphelinid parasitoids and predatory beetles. Approximately 89% of B. tabaci sampled were infected by Wolbachia, among which 34% were infected by A, 51% were infected by B, and 5% were infected by both A and B supergroups. These infection frequencies differed among B. tabaci genetic groups and locations. The invasive B. tabaci genetic group from the Middle East Asia Minor 1 (also referred as B biotype) and Mediterranean (also referred as Q biotype) was more likely to harbor A than B, whereas native genetic groups in AsiaI and AsiaII were more likely to harbor B than A. Although 60% of aphelinid parasitoids and 72% of coccinellid beetles also were infected by Wolbachia, they were more likely to host B than A. Furthermore, for the first time we report Wolbachia in B biotype from specimens collected outside of China. Construction of a phylogenetic tree clearly indicated that the Wolbachia sequences from different genetic groups of B. tabaci were not only similar to each other but also to sequences from beetles and parasitoids, which may provide evidence of coevolution and horizontal transmission of Wolbachia populations.     

Molecular characterization of Pisolithus spp. isolates by rDNA PCR-RFLP

 Variation within ribosomal DNA (rDNA) genes of 19 isolates of Pisolithus from different geographic origins and hosts was examined by polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP) analysis. The primers utilized amplify rDNA regions in a wide range of fungi. One amplified region includes the internal transcribed spacer (ITS), which has a low degree of conservation. The ITS amplification products (640–750 bp) were digested with a variety of restriction endonucleases. Cluster analysis based on the restriction fragments grouped the isolates into three distinct groups: group I contained isolates collected in the northern hemisphere, except Pt 1, group II contained those collected in Brazil and group III contained isolate Pt 1. Additional analysis of other rDNA regions, IGS, 17 S and 25 S rDNA, resulted in similar groups. The data suggest that the taxonomy and systematics of this ectomycorrhizal fungus should be revised. Accepted: 16 September 1998     

The groEL gene as an additional marker for finer differentiation of ‘Candidatus Phytoplasma asteris'‐related strains

Phytoplasma classification established using 16S ribosomal groups and ‘Candidatus Phytoplasma’ taxon are mainly based on the 16S rDNA properties and do not always provide molecular distinction of the closely related strains such as those in the aster yellows group (16SrI or ‘Candidatus Phytoplasma asteris'‐related strains). Moreover, because of the highly conserved nature of the 16S rRNA gene, and of the not uncommon presence of 16S rDNA interoperon sequence heterogeneity, more variable single copy genes, such as ribosomal protein (rp), secY and tuf, were shown to be suitable for differentiation of closely related phytoplasma strains. Specific amplification of fragments containing phytoplasma groEL allowed studying its variability in 27 ‘Candidatus Phytoplasma asteris'‐related strains belonging to different 16SrI subgroups, of which 11 strains were not studied before and 8 more were not studied on other genes than 16S rDNA. The restriction fragment length polymorphism (RFLP) analyses of the amplified fragments confirmed differentiation among 16SrI‐A, I‐B, I‐C, I‐F and I‐P subgroups, and showed further differentiation in strains assigned to 16SrI‐A, 16SrI‐B and 16SrI‐C subgroups. However, analyses of groEL gene failed to discriminate strains in subgroups 16SrI‐L and 16SrI‐M (described on the basis of 16S rDNA interoperon sequence heterogeneity) from strains in subgroup 16SrI‐B. On the contrary, the 16SrI unclassified strain ca2006/5 from carrot (showing interoperon sequence heterogeneity) was differentiable on both rp and groEL genes from the strains in subgroup 16SrI‐B. These results indicate that interoperon sequence heterogeneity of strains AY2192, PRIVA (16SrI‐L), AVUT (16SrI‐M) and ca2006/5 resulted in multigenic changes – one evolutionary step further – only in the latter case. Phylogenetic analyses carried out on groEL are in agreement with 16Sr, rp and secY based phylogenies, and confirmed the differentiation obtained by RFLP analyses on groEL amplicons.     

香蕉束顶病植原体16S rDNA片段的RFLP和序列分析

香蕉束顶病（ＢＢＴ）是一种发生在蕉类作物的严重病害。从带有典型香蕉束顶病症状的香蕉植株中按照检测植原体的方法提取ＤＮＡ,扩增患病植株中植原体１６ＳｒＤＮＡ片段,证明香蕉束顶病中有植原体存在。对此扩增片段进行限制性酶切片段长度多态性（ＲＦＬＰ）分析和核酸序列分析,并与已知植原体的序列进行同源性比较,构建进化树。结果显示该片段与Ｇｒ１的亲缘关系最近。