美洲斑潜蝇SS-PCR检测技术研究

【背景】美洲斑潜蝇是一种严重威胁瓜果蔬菜、烟草、棉花等经济作物和花卉生产的入侵性害虫。由于潜叶蝇类害虫体型较小、生活方式隐蔽、形态相似,本文针对其难以快速准确地进行形态鉴别的问题,以美洲斑潜蝇为研究对象,以菜田常见的4种潜叶蝇类害虫为参照,采用种特异性PCR方法（species-specific PCR,SS-PCR）,研究其快速分子检测鉴定技术。【方法】调用GenBank中一段936bp的美洲斑潜蝇线粒体DNA（mtDNA）细胞色素氧化酶亚基Ⅰ基因（COⅠ）的序列（Gen-Bank登录号为EU219613）,并根据此基因片段的碱基序列设计引物1对,其扩增片段大小为294bp。【结果】种特异性检验结果显示,该引物只对美洲斑潜蝇的COⅠ基因具有扩增能力,对其他种类如南美斑潜蝇、三叶斑潜蝇、葱斑潜蝇、豌豆潜叶蝇等没有扩增能力。该引物不仅对成虫具有良好的扩增效果,对蛹、幼虫以及单粒卵也具有同样的扩增效果,其最低检出阈值为1/3840头成虫。【结论与意义】SS-PCR技术体系可用于美洲斑潜蝇的鉴定识别与检测监测,对阻止其进一步扩散蔓延具有重要意义。     

重大潜在入侵害虫番茄潜叶蛾的SS-CoI快速检测技术

【背景】番茄潜叶蛾是源自南美洲的一种最具毁灭性的世界性入侵害虫,其适生区范围广,且与其他潜叶类昆虫难以准确、快速识别。【方法】以田间常见的其他6种潜叶类害虫为对照,采用基于mtDNACOl基因的种特异性SS—COI方法,研究番茄潜叶蛾快速分子检测技术。【结果】种特异性检测结果显示,SS-COl引物只对番茄潜叶蛾mtDNA具有扩增能力,对美洲斑潜蝇、南美斑潜蝇、三叶草斑潜蝇、葱斑潜蝇、豌豆潜叶蝇、桃潜叶蛾等其他潜叶类害虫不具有扩增效果。该引物不仅对成虫和单粒卵具有很好的扩增效能,对单根触角、头部、胸部、腹部以及翅和足等成虫残体亦具有同样的扩增能力,其最低检出阈值为312．5Pg·μL^-1。【结论与意义】该方法在有效防范番茄潜叶蛾侵入我国,保障农业生产安全和生态环境安全中意义重大。     

Polymerase chain reaction-restriction fragment-length polymorphism method to distinguish Liriomyza huidobrensis from L. Langei (Diptera: Agromyzidae) applied to three recent leafminer invasions.

A molecular method is presented for differentiating the morphologically cryptic leafminers Liriomyza langei Frick and L. huidobrensis (Blanchard). This method requires polymerase chain reaction (PCR) amplification of a 1031-bp region of mitochondrial cytochrome oxidase DNA followed by restriction fragment analysis using the restriction enzymes SpeI and EcoRV. Spel cuts the mitochondrial fragment of L. langei into two fragments, but does not cut the L. huidobrensis fragment. EcoRV cuts the L. huidobrensis fragment into two fragments, but does not cut the L. langei fragment. This PCR-restriction fragment-length polymorphism (RFLP) method is faster and less costly than DNA sequencing,which is currently the only other way to differentiate these two species. We apply the method to samples from recently introduced leafminer populations in Sri Lanka, Canada, and South Africa and find that the invasive leafminer in all three locations is L. huidobrensis.     

Mitochondrial DNA Sequence Divergence among Lycopersicon and Related Solanum Species

Sequence divergence among the mitochondrial (mt) DNA of nine Lycopersicon and two closely related Solanum species was estimated using the shared fragment method. A portion of each mt genome was highlighted by probing total DNA with a series of plasmid clones containing mt-specific DNA fragments from Lycopersicon pennellii. A total of 660 fragments were compared. As calculated by the shared fragment method, sequence divergence among the mtDNAs ranged from 0.4% for the L. esculentum-L. esculentum var. cerasiforme pair to 2.7% for the Solanum rickii-L. pimpinellifolium and L. cheesmanii-L. chilense pairs. The mtDNA divergence is higher than that reported for Lycopersicon chloroplast (cp) DNA, which indicates that the DNAs of the two plant organelles are evolving at different rates. The percentages of shared fragments were used to construct a phenogram that illustrates the present-day relationships of the mtDNAs. The mtDNA-derived phenogram places L. hirsutum closer to L. esculentum than taxonomic and cpDNA comparisons. Further, the recent assignment of L. pennellii to the genus Lycopersicon is supported by the mtDNA analysis.     

两种外来斑潜蝇实验种群生物学特征比较

美洲斑潜蝇LiriomyzasativaeBlanchard和南美斑潜蝇Liriomyzahuidobrensis(Blanchard)是我国的2种重要入侵性害虫。本实验室在标准化流程下饲养了48(L.huidobrensis)-160(L.sativae)个世代的稳定实验种群。美洲斑潜蝇的实验种群需要较大的繁殖规模(>100对)才能保持群体的稳定性。2种斑潜蝇4个发育阶段的形态特征和幼虫为害特征差别明显。南美斑潜蝇的发育历期比美洲斑潜蝇长,总产卵量低于美洲斑潜蝇,但产卵历期明显长于美洲斑潜蝇。这可能是南美斑潜蝇较之容易繁殖的重要原因。该实验种群的建立为物种驯化、寄主选择性、抗性筛选及其它生理学、生态学等诸多研究打下基础。     

Non-target effects of three biorationale insecticides on two endolarval parasitoids of Liriomyza sativae (Dipt., Agromyzidae)

Abstract:  Side effects of two azadirachtin formulations [NeemAzal-U (17% azadirachtin) and NeemAzal^®-T/S (1% azadirachtin)] and two biorationale pesticides – Success^® (Spinosad) and Abamectin (Avermectin) – on two endoparasitoids Opius ( Opiothorax ) chromatomyiae and Neochrysocharis formosa of Liriomyza sativae were investigated under laboratory conditions. The eggs of O. chromatomyiae , and the eggs, larvae and pupae of N. formosa within the host or within the plant/host complex were exposed to NeemAzal, Success and Abamectin at different dose rates. Adult emergence of O. chromatomyiae from parasitized L. sativae in NeemAzal-U (0.75, 1.5, 2.25 and 3 g/l water) drenched soil was only slightly lower than from untreated control hosts. In contrast, adult emergence of unparasitized L. sativae was almost completely inhibited by NeemAzal-U, indicating a high, direct toxicity. Development of O. chromatomyiae within L3 of L. sativae was very much affected from topical applications of NeemAzal^®-T/S, Success^® and Abamectin at particular dose rates. Spraying of tomato leaves with NeemAzal^®-T/S revealed no detrimental effect on the adult emergence of N. formosa developing in mining L2 of L. sativae . This was in contrast to Success^® and Abamectin which strongly affected N. formosa adult emergence when applied at different immature developmental stages of N. formosa .     

Evaluation of the Extent of Homologous Chloroplast DNA Sequences in the Mitochondrial Genome of Cowpea (Vigna unguiculata L.)

Southern blot hybridization techniques were used to estimate the extent of chloroplast DNA sequences present in the mitochondrial genome of cowpea (Vigna unguiculata L.) The entire mitochondrial chromosome was homogeneously labeled and used to probe blotted DNA fragments obtained by extensive restriction of the tobacco chloroplast genome. The strongest cross-homologies were obtained with fragments derived from the inverted repeat and the atpBE cluster regions, although most of the clones tested (spanning 85% of the tobacco plastid genome) hybridized to mitochondrial DNA. Homologous chloroplast DNA restriction fragments represent a total of 30 to 68 kilobase pairs, depending upon the presence or absence of tRNA-encoding fragments. Plastid genes showing homology with mitochondrial DNA include those encoding ribosomal proteins, RNA polymerase, subunits of photosynthetic complexes, and the two major rRNAs.     

Molecular identification of Liriomyza trifolii (Burgess) (Dipt., Agromyzidae) based on real-time PCR

Abstract:  Rapidly identifying juvenile individuals of Liriomyza trifolii (Burgess) from Liriomyza sativae Blanchard is crucial in plant quarantine. We report a molecular method to identify L . trifolii based on real-time polymerase chain reaction (PCR). By comparing partial DNA sequences of mitochondrial COI genes of L . trifolii samples collected from Guangdong and Taiwan provinces in China, Japan, Philippine, Israel, Germany, the USA, Mexico and Honduras sequenced by authors, and those of related species recorded in GenBank, a L . trifolii -specific probe was developed. There was no difference in individuals of different stages tested by this probe. The total time for real-time PCR assay system was 2 h, and it would save 3–7 h compared with conventional PCR.     

不同地理种群美洲斑潜蝇及近缘种的rDNA-ITS1序列分析和比较

【目的】通过对美洲斑潜蝇Liriomyza sativae Blanchard 不同地理种群及近缘种间的核糖体DNA第一内转录间隔区（rDNA-ITS1）进行比较,分析美洲斑潜蝇不同地理种群间的遗传分化情况,并为美洲斑潜蝇与近缘种间提供分子鉴别标记。【方法】用PCR产物直接测序法及克隆测序法对我国美洲斑潜蝇8个地理种群的rDNA-ITS1序列进行测序,并调用GenBank中3个近缘种的rDNA-ITS序列,运用软件MEGA3.1对美洲斑潜蝇不同地理种群及近缘种间的rDNA-ITS1序列进行分析。【结果】美洲斑潜蝇8个地理种群间的分化程度较低,只有8个变异位点,遗传距离都在0.02以下,但4个近缘种间的碱基差异显著,遗传距离为0.149～0.390,有126个变异位点,12个美洲斑潜蝇特异性识别位点。【结论】虽然基于rDNA-ITS1序列所显示的美洲斑潜蝇各地理种群之间的遗传分化很小,但是其分化趋势与地理分布基本相吻合;得到的12个特异性识别位点不仅可以作为美洲斑潜蝇与其近缘种间鉴别的分子标记,而且可为今后设计鉴别性PCR引物提供重要的参考依据。     

Mapping and cloning of Neurospora crassa mitochondrial transfer RNA genes.

We have obtained collections of recombinant Escherichia coli plasmids containing restriction fragments of Neurospora crassa mitochondrial DNA cloned into pBR322. By hybridization of 32P end-labeled total mitochondrial tRNAs and seven different purified tRNAs to restriction digests of mitochondrial DNA and of recombinant plasmids carrying specific restriction fragments, we have located the tRNA genes on the mitochondrial DNA. We have found that the mitochondrial tRNA genes are present in two major clusters, one between the two ribosomal RNA genes and the second closely following the large rRNA gene. Only one of the two DNA strands within these clusters codes for tRNAs. All of the genes for the seven specific purified tRNAs examined--those for alanine, formylmethionine, leucine 1, leucine 2, threonine, tyrosine, and valine--lie within these clusters. Interestingly, the formylmethionine tRNA hybridizes to two loci within one of these gene clusters. We have obtained a fairly detailed restriction map of part of this cluster and have shown that the two "putative" genes for formylmethionine tRNA are not arranged in tandem but are separated by more than 900 base pairs and by at least two other tRNA genes, those for alanine and for leucine 1 tRNAs.     

取食经历对美洲斑潜蝇雌成虫寿命及生殖力的影响

在实验室条件下 ,研究了取食经历对美洲斑潜蝇雌Liriomyzasativae成虫寿命和生殖力的影响。结果表明 ,幼虫期在黄瓜取食而成虫期在四季豆上取食时 ,其雌成虫寿命、总产卵量和日平均产卵量均明显小于幼虫期在长豇豆上取食而成虫期在四季豆上取食时 (P <0 0 1 ) ;当幼虫期同在长豇豆上取食时 ,成虫在黄瓜上取食时的雌成虫寿命、总产卵量和日平均产卵量均明显小于成虫在四季豆取食时 (P<0 0 5或P <0 0 1 )。这说明美洲斑潜蝇幼虫和成虫取食经历对其成虫寿命和生殖力均有显著的影响     

Paternal leakage of mitochondrial DNA inPinus

Summary We studied mitochondrial DNA restriction fragment length polymorphism in 11 parents and 125 seedlings of 23 controlled matings within and between jack pine (Pinus banksiana Lamb.) and lodgepole pine (P. contorta Dougl.). A potential mitochondrial distinction between these two conifers was evident in the parental samples. Only maternal mitochondrial restriction fragments were observed in a majority of the seedlings, which is consistent with results from angiosperms and other members of the genusPinus L. However, we detected exclusively paternal mitochondrial DNA in six of the seedlings. These unusual seedlings were not attributable to heteroplasmy or contamination of the experimental material, indicating that mitochondrial inheritance was not strictly maternal. Paternal mitochondrial leakage inPinus may permit novel insights into the transmission genetics and evolution of organellar polymorphisms.     

Species displacements are common to two invasive species of leafminer fly in China, Japan, and the United States

Under field conditions, species displacements have occurred in different directions between the same invasive species of leafminers (Diptera: Agromyzidae). Liriomyza sativae (Blanchard) was displaced by L. trifolii (Burgess) in the western United States, with evidence suggesting that lower insecticide susceptibility of L. trifolii is a factor. However, in Japan, the opposite has occurred, as L. trifolii was recently displaced by L. sativae. This displacement is probably because of the higher fecundity of L. sativae and differential effects of parasitoids on the two leafminer species. Here, we carried out long-term surveys of these same two invasive leafminer species during January through March in 1999, 2007, and 2011, as well as June through July in 2011, in eight locations (Sanya, Dongfang, Haikou, Leidong, Lingshui, Wuzhisan, Qionghai, and Danzhou) across Hainan Island of southern China. Our results indicate that, between 2007 and 2011, L. trifolii rapidly replaced L. sativae as the predominant leafminer of vegetables on Hainan Island, similar to the situation in the western United States. Further surveys of growers revealed that avermectins and cyromazine are the two most frequently used insecticides against leafminers on Hainan Island. Dose-mortality tests showed that L. trifolii populations from Hainan Island are less susceptible to avermectins and cyromazine compared with L. sativae populations. This lower insecticide susceptibility of L. trifolii may be associated with the displacement of L. sativae by L. trifolii, although additional ecological or environmental factors cannot be ruled out.     

Study of mitochondrial genomes of allopllasmic recombinant wheat lines constructed on the basis of barley-wheat hybrids Hordeum geniculatum all. (= H. marinum subsp. gussoneanum) (2n = 28) x x Triticum aestivum L. (2n = 42) with using of RELP and PCR analyses

Using RELP analysis with three probes homologous to specific regions of mitochondrial DNA genes and PCR analysis of the mitochondrial recombining-repeat-sequence 18S/5S region of cereals, five alloplasmic wheat lines of different origin and fertility expression were studied. These lines are self-pollinated progeny of BC1-BC4 generations of barley-wheat hybrids Hordeum geniculatum All. (2n = 28) x Triticum aestivum L. (2n = 42). It was found that recombinant alloplasmic lines characterized by partial fertility contain either maternal (barley) DNA fragments or maternal and paternal (wheat) DNA fragments simultaneously (heteroplasmy). In lines with stable expression of self-fertility, fragments of only paternal mitochondrial DNA were detected. It is assumed that in alloplasmic lines, there is the interrelation between the presence of definite fragments of the mitochondrial genome belonging to either parental type and fertility expression.     

Discrimination between Atlantic and Pacific Subspecies of Northern Bluefin Tuna (Thunnus thynnus) by Magnetic-Capture Hybridization Using Bacterial Magnetic Particles

The previously developed magnetic-capture hybridization technique employing bacterial magnetic particles was applied to discriminate between Atlantic and Pacific subspecies of the northern bluefin tuna (Thunnus thynnus) using specific DNA sequences. Nucleotide sequences of a 925-bp fragment (ATCO) flanking the mitochondrial ATPase and cytochrome oxidase subunit III genes in these two subspecies were compared. Two regions having single-nucleotide and three-nucleotide differences between the subspecies were adopted to design DNA probes (NR1, 21-mer; NR2, 29-mer), and two internal primer sets were designed to amplify DNA fragments containing these regions. The DNA probes were immobilized on bacterial magnetic particles via streptavidin-biotin conjugation and subjected to magnetic-capture hybridization with the digoxigenin-labeled fragments amplified using the internal primers. The luminescence intensities of DNA on bacterial magnetic particles obtained by hybridization between the probes and the complementary fragments were higher than those obtained by hybridization with noncomplementary fragments. These data suggest that this system employing DNA on bacterial magnetic particles may be useful for discrimination of these two subspecies by recognizing a single-nucleotide difference. Received January 17, 2000; accepted January 28, 2000.     

江苏地区三叶斑潜蝇和美洲斑潜蝇的发生危害及种群动态

【目的】三叶斑潜蝇Liriomyza trifolii和美洲斑潜蝇Liriomyza sativae是一类危害蔬菜、花卉的世界性害虫,也是我国重要的外来入侵害虫。了解其发生危害特征,可为这两种斑潜蝇的防治提供科学依据。【方法】于2015年对江苏地区的两种斑潜蝇的发生分布、危害程度以及在扬州地区的发生动态进行了调查,采集含有潜道的叶片,待蛹羽化为成虫后鉴定斑潜蝇种类,同时统计叶片上的潜道数量划分危害等级。【结果】美洲斑潜蝇主要在苏北地区发生危害,三叶斑潜蝇主要在苏南、苏中地区发生危害;两种斑潜蝇在江苏地区的发生危害程度均未超过3级。在扬州地区的蔬菜上仅调查到三叶斑潜蝇的危害,其危害的寄主植物有6科、12种,主要包括茄科、豆科、葫芦科、十字花科的蔬菜。三叶斑潜蝇的发生危害从3月开始,11月结束,全年有3个发生危害高峰,其中在7月下旬至8月初的发生危害最重。【结论】两种斑潜蝇在江苏地区的种类分布呈现出明显的地理差异,同时扬州地区三叶斑潜蝇的虫口密度随寄主和时间不同呈现的明显,因此生产上需加强对斑潜蝇发生危害进行监测,并指导斑潜蝇防治。