基于线粒体COⅠ基因及形态特征的喜树丛枝植原体主要媒介昆虫种类鉴定

植原体病害是喜树上危害最严重的病害之一,传播喜树植原体病害的主要媒介昆虫是喜树上的一种小绿叶蝉,为进一步分析媒介昆虫传播植原体机制以及植原体在昆虫体内的侵染循环过程,为植原体病害的科学防控提供参考,媒介昆虫的种类及分类地位必须先行明确。通过网捕法采集云南文山喜树上的小绿叶蝉标本,在体视显微镜下根据各龄期外部形态以及雄性外生殖器特征进行种类鉴定,结合PCR扩增线粒体COⅠ基因序列片段进行测序和比对,利用Kimura 2-parameter模型计算出平均遗传距离并用最大似然法构建系统发育树,进一步明确其分类地位,再根据前人制作的属及亚属分类检索表明确其分类地位。共检视并解剖小绿叶蝉雄虫标本100余头,提取20余头DNA进行测序,得到16条709 bp大小的COⅠ基因序列片段,结合NCBI上相关的叶蝉COⅠ序列,建立基因进化树。综合分析其外部形态、雄性外生殖器特征以及COⅠ序列比对,明确了喜树丛枝植原体主要媒介昆虫是拟帕小绿叶蝉Empoasca(Matsumurasca)paraparvipenis Zhanget Liu-CA,分类地位为小绿叶蝉属Empoasca Walsh, 1862,...     

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。     

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。     

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。     

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。     

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。     

应用昆虫介体细胞单层快速检测水稻矮缩病毒的侵染

水稻病毒,诸如水稻矮缩病毒,(Rice-gall dwarf virus)水稻瘤矮病毒(Ric-egall dwarf virus)和水稻短暂性黄化病毒(Rice transitory virus),已知都由其叶蝉介体传播到奇主植物上,而不能用其它方法进行机械传播。这些病毒的侵染力只能通过人工注射少量病毒悬浮液到介体叶蝉腹腔内进行测定。用这种方法而获得     

我国植物病毒病介体昆虫研究的进展

自1939年我国开始报道蚜虫传播蚕豆病毒病以来,至今已知虫传植物病毒和类菌原体有68个,引起100多种病害,其中属禾本科作物的近30种;十字花科、葫芦科、豆科和茄科等果菜类作物的病害60多种;果树及木本植物等病害20种。经接种证实的介体昆虫有52种,其中蚜虫22种,飞虱、叶蝉20种,其它10种。 近年来有关植物病毒介体昆虫的文献报道数量显著增加,从全国省级以上农业及生物科技刊物上发表的,与植物病毒和介体昆虫两者均有关的论文,在四十年代以前仅4篇,五十年代为17篇,六十年代74篇,七十年代42篇,1980—1984年增到130多篇。有     

水稻橙叶病分子检测及其在华南地区的发生与分布研究

对华南地区水稻橙叶病(rice orange leaf disease)进行病原菌分子检测和病情发生与分布调查。结果表明,该病是一种由电光叶蝉(Inazuma dorsalis)和黑尾叶蝉(Nephotettix cinticeps)传播的植原体(Phytoplasma)引起的病害,早期感病植株在分蘖期全株叶片表现为橙黄色,不久干枯死亡,对水稻生产造成严重为害。电镜下观察,病株幼叶叶脉筛管细胞中存在大量植原体。巢式PCR可从来源于水稻病株及介体电光叶蝉和黑尾叶蝉DNA抽提物中获得单一扩增产物,PCR产物核苷酸序列与文献报道的水稻橙叶植原体16S r DNA序列(Gen Bank登录号:KR061356)同一率均为99.8%以上。2015年广东各主要稻区及海南中部和广西东南部均有该病发生与分布。     

Possible vector insect of mulberry dwarf phytoplasma, Tautoneura mori Matsumura

Mulberry dwarf (MD) phytoplasma was surveyed for its presence in presumably nonvector insects (two species of leafhoppers, thrips, and spider mites) collected from MD-diseased trees. MD phytoplasma was found in all species by nested polymerase chain reaction using specific primers for the 16Sr I-group phytoplasma. By electron microscopy, MD phytoplasma was observed in the salivary glands of the leafhopper Tautoneura mori Matsumura. In addition, the vector ability of T. mori was verified through bioassay; 25% of seedlings inoculated with infective leafhoppers had MD symptoms, suggesting that T. mori may act as a vector of MD phytoplasma in fields in some cases.     

Detection of phytoplasma by polymerase chain reaction of insect feeding medium and its use in determining vectoring ability

A polymerase chain reaction (PCR)-based method was developed for the detection of phytoplasma in insect feeding medium (sucrose). A correlation was established between the transmissibility of Flavescence dorée phytoplasma in the experimental leafhopper vector Euscelidius variegatus and its detection by PCR in the insect feeding medium. However, phytoplasma were detected in the insects' bodies 3 weeks before they began to transmit. Hence, PCR assays of the sucrose medium reflected phytoplasma vectoring ability probably by detecting it in the insect saliva, whereas detection of phytoplasma in the insect's body did not identify it as a vector. The assay was applied to two field-collected leafhoppers suspected of being phytoplasma vectors in Israel (Orosius albicinctus and Anaceratagallia laevis). The presence of phytoplasma in the body of specimens of the latter species was assayed by PCR in 1999. Phytoplasmas were detected in insects' bodies throughout the year, with no specific seasonal pattern. In the saliva, however, no phytoplasma could be detected in the autumn. This seasonal pattern supported the validity of the feeding-medium tests and their correlation to the insect's ability to transmit phytoplasma. Transmission assays indicated, to our knowledge for the first time, that O. albicinctus and A. laevis are vectors of phytoplasma in Israel. A simple PCR-based assay is thus provided, circumventing the need for tedious biological assays and enabling epidemiological studies of phytoplasma transmissibility on a large scale.     

Oncopsis alni (Schrank) (Auchenorrhyncha: Cicadellidae) as a Vector of the Alder Yellows Phytoplasma of Alnus glutinosa (L.) Gaertn.

Alder yellows phytoplasma was detected by PCR in Alnus glutinosa trees in the Palatine and Mosel areas of Germany. The restriction profiles obtained by TaqI and AluI digestion of a PCR amplified ribosomal DNA fragment from this phytoplasma and a periwinkle isolate of alder yellows from Italy (ALY) could not be distinguished while elm yellows isolates from Europe and North America led to different fragment patterns. Different restriction profiles for ALY and the German alder phytoplasma were obtained by TruI digestion of a non-ribosomal DNA fragment. Phloem feeding insects were collected from infected alder trees. Phytoplasmas of the elm-yellows group were detected by PCR in psyllids and the leafhopper Oncopsis alni. These pathogens were indistinguishable from the phytoplasma found in alder. Only O. alni was able to transmit the pathogen to healthy alder seedlings. Thus, it is the first insect known to transmit this phytoplasma. This leafhopper could be responsible for the ubiquitous infection of Alnus glutinosa due to its close association with alder and its wide distribution in Europe.     

Macropsis mendax as a vector of elm yellows phytoplasma of Ulmus species

A 3-year study was carried out in north-east Italy, the site of recent elm yellows epidemics, to identify vectors for the elm yellows phytoplasma. Using PCR analysis, Ulmus minor and Ulmus pumila , each with and without symptoms, were positive for the elm yellows phytoplasma. Macropsis mendax , a univoltine and monophagous leafhopper, was shown to be the vector of the elm yellows-associated disease agent. PCR analyses demonstrated that the insect was infected both in natural conditions and in the screenhouse after acquisition-feeding on infected elm plants. Groups of M. mendax , collected from naturally infected elm trees, transmitted elm yellows phytoplasma to elm test plants. In nature, Alnus glutinosa trees affected by alder yellows were found in the surroundings of yellows-affected elm trees; the associated disease agent of alder yellows was transmitted under controlled conditions from alder to elm test plants by grafting.     

Composition,abundance and phytoplasma infection in the hawthorn psyllid fauna of northwestern Italy

Hawthorn (Crataegus monogyna) is one of the natural hosts of Cacopsylla melanoneura, the acknowledged vector of ‘Candidatus Phytoplasma mali’, the causal agent of Apple Proliferation disease, a serious and growing problem for apple production in Europe, particularly in northern Italy. Wild plants could be important sources of both insects and phytoplasmas, but their role in the epidemiology of phytoplasma diseases and their insect vectors has never been thoroughly examined. Cacopsylla melanoneura’s primary host is hawthorn, a plant closely related to apple which often grows wild near orchards. Other psyllid species feed on hawthorn, but no data are available on their possible role as phytoplasma vectors. We investigated the hawthorn’s psyllid fauna in northwestern Italy using yellow sticky traps, beat trays, and molecular analyses from 2003–2005, to study the relationship between hawthorn, the phytoplasma and the insect vector. Population dynamics were monitored, and insects and hawthorn samples were analysed by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and DNA sequencing for the presence of phytoplasmas. Cacopsylla melanoneura was the dominant psyllid species, followed by C. peregrina, C. affinis and C. crataegi. PCR and RFLP analyses revealed the presence of different fruit tree phytoplasmas in hawthorn plants, and in all four psyllid species.     

Note: A comparison of molecular diagnostic procedures for the detection of aster yellows phytoplasmas (16Sr-I) in leafhopper vectors

Different molecular procedures were compared for the detection of aster yellows phytoplasmas (AYP) in the leafhopper vectorsMacrosteles quadripunctulatus (Kirschbaum),Euscelidius variegatus (Kirschbaum) andEuscelis incisus (Kirschbaum). Polymerase chain reaction (PCR) with universal and group-specific primers designed on the 16S-rDNA sequence was most sensitive in nested assays. A dot-blot procedure with an oligoprobe designed on the 16S-rDNA was less sensitive and consistent to detect phytoplasmas in total insect DNA, but consistently detected amplicons from direct PCR. The dot-blot assay with a probe based on a phytoplasma plasmid sequence detected AYP in most vector specimens and did not react with DNAs from leafhoppers infected by flavescence dorée and psyllids infected by apple proliferation phytoplasmas. This last assay is almost devoid of contamination risks, faster and cheaper compared to PCR, therefore it has to be preferred for field-scale analysis of leafhopper populations. http://www.phytoparasitica.org posting Feb. 24, 2004.     

Transmission Characteristics of the European Stone Fruit Yellows Phytoplasma and its Vector Cacopsylla Pruni

A study was carried out on the transmission parameters of the European stone fruit yellows phytoplasma by the vector Cacopsylla pruni. In the greenhouse, using groups of psyllids, the minimum acquisition period was 2–4 days, the minimum latent period 2–3 weeks and the minimum inoculation period 1–2 days. The vectors retained infectivity until their death. Under natural conditions retention of infectivity in C. pruni lasts through the winter and the following spring, when the overwintering insects reach the stone fruit trees, they are already infected and infective. The research shows that the vector C. pruni transmits the European stone fruit yellows phytoplasma in a persistent manner.     

New subgroup 16SrIII-Y phytoplasmas associated with false-blossom diseased cranberry (Vaccinium macrocarpon) plants and with known and potential insect vectors in New Jersey

The identity of the presumed phytoplasmal pathogen associated with cranberry false-blossom disease has never been fully clarified. In the present study a molecular-based procedure was employed to determine the identity of the phytoplasma. Tissues of cranberry plants exhibiting cranberry false-blossom symptoms were collected from multiple bogs on each of three randomly selected commercial cranberry farms in New Jersey. Leafhoppers, including the known vector Limotettix vaccinii (Van Duzee) (=Scleroracus vaccinii, Euscellis striatulus) and the sharp-nosed leafhopper Scaphytopius magdalensis (Provancher), a known vector of blueberry stunt disease, were collected from two different farms in New Jersey. Nested PCR assays and RFLP analysis of 16S rRNA gene sequences were employed for the detection and identification of the associated phytoplasmas. All of 20 cranberry plants sampled and five out of 14 batches of leafhoppers tested positive for phytoplasma. Virtual RFLP and sequence analyses revealed that all the associated phytoplasmas were members or variants of a new subgroup, 16SrIII-Y. Phylogenetic analysis of 16S rRNA sequences indicated that cranberry false-blossom phytoplasma strains represented a lineage distinct from other 16SrIII subgroups. This is the first report confirming that a new phytoplasma (designated as a new subgroup 16SrIII-Y) is associated with cranberry false-blossom disease and associated with both leafhopper species in New Jersey.     

Insect injection and artificial feeding bioassays to test the vector specificity of flavescence dorée phytoplasma

ABSTRACT The specificity of vector transmission of Flavescence dorée phytoplasma (FDP) was tested by injecting FDP, extracted from laboratory-reared infective Euscelidius variegatus, into specimens of 15 other hemipteran insect species collected in European vineyards. Concentrations of viable phytoplasma extracts and latency in vectors were monitored by injection of healthy-reared E. variegatus leafhoppers. Based on these preliminary results, insects were injected by using phytoplasma extracts that ensured the highest rate of FDP acquisition and transmission by E. variegatus. Transmission into an artificial diet through a Parafilm membrane about 3 weeks after insect injection was attempted. FDP-injected insects that belonged to 15 hemipteran species were confined in cages and fed through the membrane for a 4- to 5-day inoculation access period. FDP DNA was detected by polymerase chain reaction (PCR) in the feeding buffer fed upon by Anoplotettix fuscovenosus, Aphrodes makarovi,E. variegatus, and Euscelis incisus. PCR amplification with specific primers detected FDP DNA in injected insects of all test insect species. Band intensity was positively correlated with the transmissibility of FDP. Transmission of FDP to plants by feeding was confirmed for Anoplotettix fuscovenosus, E. variegatus, and Euscelis incisus, but not for Aphrodes makarovi. Our results suggest that vector competency of FDP is restricted to specimens belonging to the family Cicadellidae, subfamily Deltocephalinae.