Genetic diversity of Spiroplasma citri strains from different regions, hosts, and isolation dates

Spiroplasma citri, a phloem-limited pathogen, causes citrus stubborn disease (CSD). Losses due to CSD in California orchards have grown over the past decade. To investigate the possibility of introduction or emergence of a new strain, a study of genetic diversity among S. citri strains from various locations was conducted using random amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR) of 35 strains cultured from 1980 to 1993, and of 35 strains cultured from 2005 to 2006. Analysis using 20 primer pairs revealed considerable diversity among strains. However, no unique genetic signatures were associated with recently collected strains compared with those collected 15 to 28 years ago, and no geographically associated pattern was distinguishable. S. citri strains from carrot and daikon radish contain some unique DNA fragments, suggesting some host plant influence. Multiple strains from single trees also showed genetic diversity. Sequencing of five RAPD bands that differed among strains showed that diversity-related gene sequences include virus fragments, and fragments potentially encoding a membrane lipoprotein, a DNA modification enzyme, and a mobilization element. No differences in colony morphology were observed among the strains. The lack of correlation between PCR patterns and isolation date or collection site is inconsistent with the hypothesis that recent infections are due to the introduction or emergence of novel pathogen strains.     

Detection of Spiroplasma citri in plants and insect hosts by ELISA

Enzyme-linked immunosorbent assay (ELISA) was shown to be a sensitive method for the detection of Spiroplasma citri in plants and insect hosts. S. citri was detected in Vinca rosea less than 1 week after infection by grafting, and reached a peak litre of up to 10⁹ spiroplasmas per gram of tissue in infected shoots and root tips. Multiplication of S. citri in the leafhopper Euscelidius variegatus was also monitored by ELISA. S. citri could be detected in a single insect, showing that this technique is suitable for screening insect populations in the field for potential vectors of Spiroplasma diseases. A method is described for raising a pathogen-specific antiserum from V. rosea infected with S. citri which reacted with cultured S. citri and also with S. citri in plant tissue.     

柑橘溃疡病菌抗铜相关基因copA和 copB的克隆及原核表达

 以柑橘溃疡病菌DNA为模板,对抗铜相关基因copA和copB进行了PCR扩增和克隆,获得了大小分别为1 782 bp 和1 095 bp的目标片段。构建了这2个基因的原核表达载体 (pET-copA和pET-copB),并在大肠杆菌 [Escherichia coli BL21(DE3)] 中成功诱导表达。利用原核表达的融合蛋白免疫大耳白兔,制备了抗copA和copB原核表达蛋白的多克隆抗体。用间接ELISA法测定了所制备的多克隆抗体的效价均为1∶6 400。用所制备的抗体分别对copA和copB的原核表达产物进行Western blot分析的结果显示,在相应位置产生了较强的免疫反应条带,表明所制备抗体能特异性地与相应的抗原发生免疫反应。     

Spiroplasma citri Surface Protein P89 Implicated in Adhesion to Cells of the Vector Circulifer tenellus

ABSTRACT Two microtiter plate assays were developed to study the adherence of the plant-pathogenic mollicute Spiroplasma citri to a monolayer of cultured cells of its leafhopper vector, Circulifer tenellus. Adherence was significantly reduced by prior treatment of the spiroplasmas with proteinase K or pronase. Electrophoresis and western blotting of spiroplasma membrane proteins, before and after exposure of intact spiroplasmas to proteases, revealed the concomitant reduction in intensity of a major membrane protein (P89) and a new polypeptide of approximately 46 kDa in protease-treated preparations (P46). Triton X-114 phase partitioning demonstrated that P89 and P46 are amphiphilic, and labeling of the new polypeptide P46 with anti-P89 serum suggested that this molecule may be a breakdown product of P89. Regeneration of P89 after proteinase K treatment of spiroplasmas was directly associated with restoration of the pathogen's attachment capability. Treatment of spiroplasmas with any of several carbohydrates and glycoconjugates or with tetramethyl-urea, a compound that interferes with hydrophobic associations, had a negligible effect on attachment. These results suggest that a spiroplasma surface protein, P89, has a role in S. citri adherence to C. tenellus cells.     

三种强启动子和egt基因缺陷在重组核型多角体病毒杀虫剂研发中的意义

核型多角体病毒 (nuclearpolyhedrosisvirus ,NPV)是鳞翅目昆虫的重要病原微生物。采用构建重组高效表达外源毒蛋白的 p10、ph、ocu基因的强启动子 ,以提高野生型NPV的毒力 ;探讨删除杆状病毒中的蜕皮甾体尿苷二磷酸葡糖转移酶 (ecdysteroidUDP-glucosyltraansferase ,EGT)基因对改良病毒杀虫剂的杀虫效果 ,为进一步完善重组NPV杀虫剂的研发和安全生产提供了理论基础     

Genetic analysis of Grapevine leafroll‐associated virus 3 population from Galicia,Spain

Grapevine leafroll‐associated virus 3 (GLRaV‐3; Ampelovirus, Closteroviridae) isolates from Galicia in northwestern Spain were selected to characterize their genetic diversity according to different factors (age, origin, location, variety, etc.). The vines belonged either to local white and red varieties autochthonous to Galicia or to varieties from other Spanish regions but widely used in Galicia. These GLRaV‐3 isolates came from different vineyards in Galicia located in coastal or inner areas. Multiplex RT‐PCR allowed the detection of isolates belonging to groups I, II, III–V and VI. Two genomic regions were studied in the isolates, the HSP70h and the capsid protein, using specific primers that allow the detection of variants from groups I to V. Some possible recombinants could be detected; however, multiple infections with different variants indicated that they were not genuine recombinants. No differences were found in the population structure considering variety or geographical factors. Isolates belonging to four groups were found in the distinct areas surveyed: groups I and II were the most common, followed by groups VI and III, as is the case in the rest of the world. In the same surveys, the presence of insect vectors for GLRaV‐3 was investigated and found lacking in inland areas but present in those with milder climate. Genetic analysis did not support isolation of the GLRaV‐3 isolates in Galicia, suggesting that the uncontrolled exchange of infected vines and/or rootstocks has been a major agent of virus spread.     

侵染假酸浆的泰国番茄黄化曲叶病毒全基因组结构特征

为明确假酸浆Nicandra physalodes叶片黄化、皱缩症状是否由菜豆金色花叶病毒属病毒侵染引起,本研究利用分子检测方法和生物信息学技术鉴定了假酸浆样品中的病毒种类。从采集的病样中克隆并获得了2条菜豆金色花叶病毒属病毒DNA-A全序列和1条beta卫星全序列,经全序列分析发现,该双生病毒的两条DNA-A全序列与泰国番茄黄化曲叶病毒(tomato yellow leaf curl Thailand virus, TYLCTHV)云南分离物TYLCTHV-YN1732一致性最高,达99.3%,亲缘关系较近;beta卫星的全序列与云南番茄曲叶beta卫星(tomato leaf curl Yunnan betasatellite, TLCYnB)的分离物YN5230一致性最高,达99.3%,亲缘关系较近。重组分析显示,假酸浆上分离的TYLCTHV-YN5735-12是一个重组病毒,有两个重组事件,一个主要发生在AV1的编码区,由中国番茄黄化曲叶病毒(tomato yellow leaf curl China virus, TYLCCNV)和广西大戟曲叶病毒(euphorbia lea...     

Glomus mosseae bioprotection against aster yellows phytoplasma (16srI-B) and Spiroplasma citri infection in Madagascar periwinkle

The bio-control potential of arbuscular mycorrhizal fungus Glomus mosseae against two pathogenic microorganisms aster yellows (AY) phytoplasma and Spiroplasma citri has been examined in Madagascar periwinkle (Catharanthus roseus). G. mosseae had a positive influence on healthy C. roseus plants and S. citri infection. It provided bioprotection against S. citri pathogen and induced significant degree of resistance to spiroplasma infection. Besides, symptom expression significantly reduced and shoot height, leaves number, root fresh and dry weight increased in spiroplasma-infected plants treated with mycorrhiza fungus. Although, G. mosseae had no positive effect on phytoplasma disease. The root architectures were affected by the phytoplasma pathogen, and the root surface area dramatically decreased in G. mosseae treated AY-infected periwinkles compared with the control. Nitrogen and Phosphorus concentrations notably increased in spiroplasma + G. mosseae compared with control plants. Potassium concentration did not differ significantly in all mycorrhizal treated and untreated infected plants except in G. mosseae treated healthy plants. The spore density and root colonization rate did not vary in both pathogen treatments G. mosseae + spiroplasma and G. mosseae + phytoplasma. To our knowledge, this is the first report showing the bioprotective effect of G. mosseae on S. citri. The possible mechanisms involved in complex interaction between plants, cell wall-less bacteria and arbuscular mycorrhizal fungi (AMF) are discussed and the underlying mechanisms for the functioning of AMF are hypothesized.     

基于全基因序列的中国北方3省(区)马铃薯Y病毒遗传多样性分析

本研究以马铃薯Y病毒(PVY)全基因组为基础,分析吉林、黑龙江和内蒙古3省(区)PVY群体遗传多样性和群体分化,并评估突变、重组、选择等遗传力所起的作用。根据已报道的PVY全基因序列保守区设计4对引物,采用片段重叠法对来自内蒙古和吉林的24个PVY分离物全基因序列进行测定,并联合NCBI中已登录的9个黑龙江分离物全基因组序列进行遗传多样性参数评估、群体分化检验和分子变异等分析。结果显示,我国北方3省(区)PVY群体遗传多样性高,其中内蒙古和黑龙江PVY群体遗传多样性高于吉林群体,并且3个群体之间呈现一定程度的遗传分化。分子变异分析发现在PVY基因组中存在1 786个变异位点,表明我国北方3省(区)PVY群体变异程度较高,并且这种高变异度有85.54%来自各个马铃薯种植区内PVY个体的遗传变异。重组分析和系统发育分析发现,我国北方3省(区)PVY群体中重组株系占比高达90.3%,并具有明显的株系多样性,表明PVY重组株系已成为我国北方3省(区)马铃薯种植区的流行株系。选择压力分析显示,使用FEL和IFEL法分别检测出501个和315个净化压力选择位点,这表明3省(区)PVY群体受净化选择压力为主。以上结果表明,中国北方3省(区)PVY群体遗传多样性高,突变、重组和自然选择都对遗传多样性和群体分化存在一定影响。     

Genetic variability and molecular evolution of arabis mosaic virus based on the coat protein gene sequence

Arabis mosaic virus (ArMV) is an important pathogen infecting a wide range of plant species worldwide. In the present study, we carried out exhaustive phylogenetic analyses based on the coat protein (CP) sequence of ArMV isolates originating from different host plants and geographic regions. Maximum-likelihood reconstruction revealed the presence of three distinct, well-supported phylogroups. BaTS analyses indicated that the diversity of ArMV isolates may be correlated with both host plant and geographic origin. Moreover, population genetic analyses showed significant differences in the level of genetic diversity among variants within ArMV that originated from rhubarb plants, as expected by quasispecies. Further analysis revealed that both selection and recombination are shaping the population structure of ArMV. In total 14 groups of residues were detected as coevolving for different amino acid properties.     

一种侵染鳢肠的双生病毒基因组特征

菜豆金色花叶病毒属病毒是热带及亚热带地区经济作物的重要病原病毒,该类病毒在田间的杂草寄主范围较为广泛。本研究从云南红河流域采集到叶脉黄化的鳢肠植株,经克隆获得了菜豆金色花叶病毒属病毒分离物YN3306,该分离物核苷酸序列全长2749 nt,具有典型的双生病毒基因组结构特征。进一步分析发现,分离物属于金腰剑曲叶病毒(Synedrella leaf curl virus,SyLCV),RDP软件分析表明,该分离物是由烟草曲茎病毒(Tobacco curly shoot virus,Tb CSV)、云南烟草曲叶病毒(Tobacco leaf curl Yunnan virus,TbLCYnV)和金腰剑曲叶病毒重组产生的病毒。本研究首次报道了GenBank中在印度注册的SyLCV可以侵染鳢肠植株。     

Genetic diversity and molecular epidemiology of the T strain of Plum pox virus

Very limited information is available on the origin, diversity and evolution of Plum pox virus (PPV) ‘Turkey’ (T) strain. Phylogenetic analyses based on partial sequences of 421 isolates and complete genome sequences of 57 isolates, representing the geographical distribution of PPV-T in Turkey, revealed the existence of several monophyletic and, in some cases, geographically limited groups within the PPV-T strain (Ankara-Konya1-Kayseri, Ankara-Balkan, Istanbul, Konya2 and Balkan). PPV-T diversity (0.018%) was found to be greater than that of PPV strains D and Rec but lower than that of the M strain when including the newly described and divergent M-Istanbul isolates, suggesting a long evolutionary history for PPV-T. The European part of Turkey in the Balkans, close to Bulgaria where PPV was identified for the first time, appears as a likely centre of origin for PPV-T isolates. The colonization of various parts of Turkey by diverse isolates from that region, followed by secondary local spread, is the most likely scenario for the diffusion of PPV-T in Turkey.     

两种植物病原黄单胞菌基因组中同义密码子使用的分析

 根据已释放的基因组序列,对野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv. campestris,Xcc)和地毯草黄单胞菌柑桔致病变种(X. axonopodis pv. citri,Xac)的密码子使用进行了分析。相对同义密码子使用值(relative synonymous codon usage,RSCU)的计算表明,它们具有高度相似的密码子使用模式。2个基因组密码子第3位的GC含量(GC3s)平均达0.806±0.077(Xcc)和0.791±0.075(Xac),倾向于使用GC含量较高的密码子。对有效密码子数量和密码子适应指数的分析表明,Xcc与Xac基因组中,高表达基因具有较高的GC含量,倾向于使用少数种类的密码子,而低表达基因具有较高的AT含量,倾向于随机地使用密码子。对密码子使用绝对次数进行的对应分析也证明了上述结论。同时,计算也证明了基因在基因组中的位置不影响密码子使用的模式。因此,基因组的GC含量、基因的表达水平和基因的种类与起源是影响这2个基因组密码子使用的主要因素。     

新疆加工番茄马铃薯Y病毒株系鉴定

 马铃薯Y病毒 (Potato virus Y,PVY) 是世界范围内对茄科属植物造成影响的重要病毒,为了解PVY对新疆加工番茄的危害,本文采集205份加工番茄样品进行血清学检测,结果PVY^O、PVY^O\N\C、PVY^N检出率分别为20%、18.05%和0％,且PVY不同株系总检出率达24.9%。根据血清学检测结果,在苋色藜 (Chenopodium amaranticolor) 上进行单斑分离获得2个PVY分离物S1-10、S2-12,其基因组序列分析结果显示,S1-10分离物是PVY^O与PVY^N的重组体,属于PVY^N∶O株系,S2-12属于PVY^O株系。本研究表明新疆加工番茄上的PVY主要为PVY^N∶O和PVY^O株系。     

基于外壳蛋白基因的湖南烟草黄瓜花叶病毒遗传多样性分析

为探明湖南烟草上发生的黄瓜花叶病毒Cucumber mosaic virus(CMV)的遗传多样性及分子进化特征,对来自湖南烟区的303份疑似感染病毒的烟草样品进行检测,分析CMV系统发育、遗传变异和群体结构等特征。结果表明：部分分离物的外壳蛋白(coat protein, CP)基因与NCBI上登录的CMV分离物的一致性为86.34%～98.42%;系统发育分析发现湖南烟草CMV分离物属ⅠB组,不同组间的分离物地理特征不明显,无重组现象,进化的主要驱动力是负选择;组间遗传变异比较明显,基因交流频率较低,受到遗传漂变影响,遗传多样性高,群体趋于扩张。研究结果为烟草抗CMV育种提供了理论依据,对病害防治具有重要意义。     

Genetic structure of a Pyrenophora teres f. teres population over time in an Australian barley field as revealed by Diversity Arrays Technology markers

Net form of net blotch caused by Pyrenophora teres f. teres (Ptt) is a major foliar disease of barley (Hordeum vulgare) worldwide. Knowledge of the evolution of Ptt pathogen populations is important for development of durable host-plant resistance. This study was conducted to investigate changes in genetic structure of a Ptt population within a barley field during three cropping years. The susceptible barley cultivar Henley was inoculated with Ptt isolate NB050. Leaf samples were collected during the years 2013–15 and 174 single spore Ptt isolates stored. Genotyping using Diversity Arrays Technology markers identified that 25% of isolates were clones of the inoculated isolate and 75% of isolates were multilocus genotypes (MLGs) differing from the original inoculated genotype. The novel genotypes probably originated from a combination of windborne spores from neighbouring fields, infected seed and sexual recombination in the field. The rapid change in the genotypic composition of the Ptt population in this study suggests adaptive potential of novel genotypes and demonstrates the need for barley breeders to use multiple sources of host-plant resistance to safeguard against resistance being overcome.     

Genetic and physiological evidence for the production of N-acyl homoserine lactones by Pseudomonas syringae pv. syringae and other fluorescent plant pathogenic Pseudomonas species

N-acyl homoserine lactones (AHLs) function as cell density (quorum) sensing signals and regulate diverse metabolic processes in several gram negative bacteria. We report that strains of Pseudomonas syringae pvs. syringae (Pss), tabaci and tomato as well as P. corrugata and P. savastanoi produce difussible AHLs that activate the lux operons of Vibrio fischeri or the tra::lacZ fusion of Agrobacterium tumefaciens. In Pss strain B3A, AHL production occurs in cell density dependent manner. Nucleotide sequence and genetic complementation data revealed the presence of ahlI_Pss, a luxI homolog within the Ahl⁺ DNA of Pss strain B3A. The DNA expresses in AHL-deficient strains of P. fluorescens and E. carotovora subsp. carotovora (Ecc), and restores extracellular enzyme production and pathogenicity in the Ecc strain. The derivatives of Pss strains B3A and 301D carrying chromosomal ahlI::lacZ do not produce AHL, but like their wild type parents, produce extracellular protease and the phytotoxin syringomycin as well as elicit the hypersensitive reaction in tobacco leaves. While these strains also produce a basal level of -galactosidase activity, the expression of ahlI::lacZ is substantially stimulated in the presence of multiple copies of the DNA or by the addition of cell-free spent cultures containing AHL. The activation of -galactosidase production occurs with spent cultures of some, but not all Pseudomonas strains which produce AHL as indicated by the Lux and tra::lacZ assays. Pss strains deficient in the global regulatory genes, gacA or lemA, produce very low levels of AHL. Since inactivation of ahlI_Pss eliminates AHL production and since Ahl⁺ Pseudomonas strains carry the homolog of ahlI_Pss, we conclude that ahlI_Pss specifies a key step in AHL biosynthesis and it has been conserved in many plant pathogenic pseudomonads.     

番茄斑驳花叶病毒在广东省的首次报道

番茄斑驳花叶病毒(tomato mottle mosaic virus, ToMMV)是2013年发现的烟草花叶病毒属一个新种,目前在多国(地区)有发生。本文采用小RNA深度测序及RT-PCR检测方法在广东省广州市南沙区辣椒疑似病样中检测到ToMMV,命名为番茄斑驳花叶病毒广东分离物(ToMMV-GD-2020)。采用RT-PCR分段扩增获得了ToMMV-GD-2020的基因组全序列,该分离物基因组全长6 399 nt,包含4个开放阅读框,分别编码4个蛋白。序列相似性分析表明,ToMMV-GD-2020与已登录GenBank的14个ToMMV分离物基因组序列相似性分别为99.0%～99.7%,其中与中国辽宁分离物ToMMV-LN(GenBank登录号：MN853592)的相似性最高(99.7%),与危害我国番茄、同属烟草花叶病毒属的番茄花叶病毒(tomato mosaic virus, ToMV)、番茄褐色皱果病毒(tomato brown rugose fruit virus, ToBRFV)代表分离物的相似性分别为84.6%和81.0%。系统进化分析表明,ToMMV-GD-2020...