三叶草斑潜蝇和美洲斑潜蝇的分子鉴定

以三叶草斑潜蝇Liriomyzatrifolii(Burgess)与美洲斑潜蝇LiriomyzasativaeBlanchand为材料,通过特异引物18SP1和5.8SP2,分别扩增出它们的rDNA非编码区ITS1的片段,根据序列分析和比较结果,重新设计出2条新的特异性引物Fly3和Flyus,并筛选到2个特异性内切酶,通过PCR和PCR产物的酶切分析,即可将这2种近缘昆虫区分开来。     

Rapid identification of Bactrocera latifrons (Dipt., Tephritidae) by real-time PCR using SYBR Green chemistry

Abstract:  The solanum fruit fly, Bactrocera latifrons (Hendel), is a major agricultural pest in Asia and Hawaii, and it is important to prevent its widespread invasion in plant quarantine. In this study we introduced a real-time polymerase chain reaction (PCR) essay, using SYBR Green I dye, to rapidly identify B. latifrons on an ABI PRISM 7700 sequence detection system. A latifron-specific PCR primer set was obtained based on mtDNA COI gene of B. latifrons . Nine Bactrocera fruit flies, B. latifrons , Bactrocera dorsalis , Bactrocera papayae , Bactrocera carambolae , Bactrocera philippinensis , Bactrocera occipitalis , Bactrocera correcta , Bactrocera cucurbitae and Bactrocera tau , were used to determine the specificity of primers lati1 and lati2. A series of genomic DNA dilutions of B. latifrons (0.01, 0.1, 1, 10, 20, 40 and 100 ng) were used to assess the sensitivity of the SYBR Green PCR. Template DNA concentration was one of the sources of variability in cycle threshold values (CT) and the optimum DNA concentration was between 1 and 20 ng. Genomic DNA isolated from larvae, pupae and adult specimens of B. latifrons were used to assess the specificity of the SYBR Green PCR. Melting curve analysis and agarose gel electrophoresis was employed to check the specificity of PCR products. Similar amplification plots were obtained using DNA from the three different stages of B. latifrons with primer set lati1/lati2. The melting temperature ( T _m) of PCR products was 77.5 ± 0.1°C, and the length of the amplified fragment 366 bp. Given the specificity and sensitivity of the assay, combined with high speed, low cost and the possibility of automating, SYBR Green PCR can be used as a rapid and specific technique for pest species identification in plant quarantine.     

三叶草斑潜蝇的入侵、鉴定及在中国适生区分析

介绍了2005年12月首次发现入侵中国大陆的三叶草斑潜蝇Liriomyzatrifolii(Burgess)的形态特征及其与近缘种的形态区别、入侵历史与危害性;利用GARP生态位模型对三叶草斑潜蝇在中国的潜在适生区进行了分析预测,结果表明该虫在中国的潜在适生区涉及范围广,以东部地区的最适宜潜在适生区为主,它的潜在适生区涵盖了热带、亚热带和暖温带等3种气候类型;依据中国大陆目前的发生情况提出对该种入侵害虫的监测方法与防控建议。     

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。     

Interspecific interactions between Bemisia tabaci (Hem., Aleyrodidae) and Liriomyza sativae (Dipt., Agromyzidae)

Abstract:  The study examines interspecific interactions between Bemisia tabaci (Hem., Aleyrodidae) and Liriomyza sativae (Dipt., Agromyzidae) feeding on pumpkins and cucumbers. L. sativae density on control pumpkins and cucumbers peaked 3 and 2 weeks after adults were seen, respectively. Compared with controls, L. sativae densities observed for weeks 3–6 and 2–6 in the B. tabaci -pre-infested plants were significantly lower, respectively ( F _2.00 = 22.66, P < 0.01). The survival of L. sativae larvae transplanted to B. tabaci -pre-infested pumpkins and cucumbers was 16.06%, 10.02% and 13.09%, 9.21%, whereas those transplanted to control plants were 52.20%, 36.06% and 48.25%, 28.22% after 5 and 10 days, respectively ( χ ² = 6.9, d.f. = 1, P < 0.01). Pupation rates of L. sativae feeding on control plants were 20.25% and 16.45%, whereas those feeding on the B. tabaci -pre-infested plants only 4.08% and 4.21%, respectively ( χ ² = 7.0, d.f. = 1, P < 0.01). The length and number of mines of L. sativae on the control pumpkins and cucumbers were 54.05%, 54.10% and 89.06%, 76.22% longer or higher than those on B. tabaci -pre-infested plants, respectively. Leaf positions of L. sativae feeding on the plants were also affected by B. tabaci -pre-infested. These results showed that B. tabaci infestations had a significantly negative effect on L. sativae when they were feeding simultaneously on the same host plant.     

江苏泰兴市首次发现三叶草斑潜蝇疫情

2008年7月初泰兴市植物检疫站在泰兴市泰兴镇蔬菜基地发现并采集带有斑潜蝇潜道的药芹叶片,通过对其进行形态特征和分子生物学鉴定,确认为江苏地区首次发现检疫性害虫三叶草斑潜蝇Liriomyza trifolii(Burgess)。通过黄板诱集成虫和田间寄主作物调查,对泰兴市2008-2009年发生特征及年度间、寄主间为害情况差异进行分析,并针对今后加强三叶草斑潜蝇监测与防治工作中存在的问题提出设想与建议。     

The feasibility of using the sterile insect technique against Liriomyza trifolii (Diptera: Agromyzidae) infesting greenhouse chrysanthemum

Liriomyza trifolii is a serious pest of chrysanthemum greenhouses and other crops around the world. The larvae feed within the leaves of the host plants and create serpentine mines. The Sterile Insect Technique (SIT) has been successfully used against some Dipteran species in the field, and it is especially efficient against isolated populations like those on islands. Therefore, SIT against pest populations in confined environments such as greenhouses should have high potential for success. The objectives of this study were to determine the optimum gamma‐irradiation dosage required for the sterilisation of L. trifolii adults, and to determine the quality (emergence percentage, flight ability, longevity, copulatory success, and sperm transfer) of the irradiated males compared with that of non‐irradiated (normal) males. We found that sterility (< 0.7 mine per female) was achieved with the dose of 170 ± 5% Gy. The copulatory success and sperm transfer during copulation of the sterile males were not significantly different from those of normal males. Moreover, the longevity, emergence percentage and flight ability of irradiated males were also not significantly different from normal males. The SIT experiments suggest that the release of sterile L. trifolii can significantly reduce the reproductive capacity of a wild leafminer population. Our studies indicate that sterilisation of L. trifolii flies is feasible and that sterile males are of high quality and competitive with normal males. Based on these data, research on the use of SIT against L. trifolii populations in greenhouses is ongoing.     

Functional response of a parasitoid Ganaspidium utilis （Hymenoptera： Eucoilidae） on the leafminer Liriomyza trifolii （Diptera： Agromyzidae）

Functional response of a solitary, larval-pupal endoparasitoid of Liriomyza leafminers, Ganaspidium utilis Beardsley, was estimated on Liriomyza trifolii Burgess at three temperatures （17℃, 25℃, 29℃ ） and host densities. A type Ⅱ random parasitoid equation （RPE） was used to estimate instantaneous search rate and handling time. The instantaneous search rate increased as temperature increased. All of the RPE regressions obtained for functional response of G. utilis at different temperatures were significant （P〈0.01）. The slope of RPE regression lines was lower across the temperatures. At 29±2℃, the maximum number of larvae parasitized was 7.8 per day. It decreased to 7.2 larvae parasitized at 25±2℃. At 17±2℃, no significant increment of parasitization was observed due to the host density increments. The estimated handling time was lowest at 17±2℃ and highest at 25 ± 2℃, respectively. The ability of G. utilis to find and parasitize L. trifolii over a wide range of temperatures makes them a good candidate for biological control of Liriomyza leafminers.     

基于线粒体COI基因的印度新曲厉螨的分子鉴定

本研究利用线粒体COI基因对印度新曲厉螨进行分子鉴定,以期为研究该螨的生物学习性及对蜜蜂的危害提供技术支持。分别从中华蜜蜂、意大利蜜蜂工蜂体上,以及桉树花序上采集试螨,扩增印度新曲厉螨的COI基因,并将获得的COI基因序列在Genbank中Blast对比;利用Genedoc软件分析基因序列的相似性;利用MEGA 7.0计算厉螨科不同种螨之间的遗传距离,构建COI基因的系统发育树。结果表明:采自中华蜜蜂、意大利蜜蜂及桉树花序上的印度新曲厉螨样本COI基因序列完全一致,均表现出明显的A/T碱基偏向性,与同属Neocypholaelaps apicola的COI基因序列的相似性为88%。基于COI基因的印度新曲厉螨的种内的遗传距离(0.00)显著小于与厉螨科6种螨的遗传距离(0.203～0.403)且不存在重叠区域。构建的COI基因的系统发育树显示,采自印度新曲厉螨样本COI与同属的Neocypholaelaps apicola聚同一进化枝,其它不同属螨则按照属的不同聚集在不同的进化枝。本研究所用的线粒体COI基因序列的差异可应用于印度新曲厉螨的分子鉴定。     

基于通用引物的多重PCR对小圆胸小蠹的分子鉴定

本研究利用通用引物的多重PCR方法开展小圆胸小蠹Euwallacea fornicatus的分子鉴定,以期探究多重PCR在昆虫分子鉴定中的可行性,并为开展小圆胸小蠹的有效、准确鉴定及综合防治等提供重要依据。使用多重PCR方法扩增了小圆胸小蠹的COI、16S和28S的3个分子片段,并将获得的目的序列在GenBank中进行BLAST比对;利用MEGA 7计算方胸小蠹属不同种间的遗传距离,并基于邻接法和最大似然法分别构建单基因系统发育树。结果表明：多重PCR可以用于小圆胸小蠹分子序列的获取;基于COI和16S的遗传距离分析表明了小圆胸小蠹的种内遗传距离均小于2%;基于单个基因构建的系统发育树均显示本研究扩增的小圆胸小蠹COI和16S序列与GenBank中获取的小圆胸小蠹COI和16S序列聚为一支。多重PCR可以应用于小圆胸小蠹的分子鉴定,该方法不仅可以提高物种鉴定的准确率,还可以减少PCR过程中的时间和DNA消耗。     

基于mtDNA Cytb 的六种果实蝇的分子鉴定

本研究首次对果实蝇属的桔小实蝇Bactrocera dorsalis、瓜实蝇B. cucurbitae、南瓜实蝇B. tau、番石榴实蝇B. correcta、具条实蝇B. scutellata、黑漆实蝇B. scutellaris等6种实蝇mtDNA Cytb基因进行了测序。对这6种实蝇72个个体mtDNA Cytb基因中段420 bp的碱基序列进行分析,得到38种单倍型,发现了116个变异位点,其中30个位点较为稳定。对这6种实蝇与其各自鉴别位点的对应关系研究表明,mtDNA Cytb基因可以作为这6种实蝇种类鉴别的分子标记。     

Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory

Aims:  To facilitate efficient identification of commonly encountered mycobacteria species ( Mycobacterium tuberculosis , Mycobacterium avium , Mycobacterium intracellulare , Mycobacterium fortuitum complex , Mycobacterium chelonae/abscessus , Mycobacterium kansasii , Mycobacterium gordonae ) in high throughput laboratories, a 16s rDNA sequence based real-time PCR assay was developed and evaluated.
Methods and Results:  Oligonucleotide primers and hybridization probes were designed based on sequence differences of the mycobacterial 16S rDNA gene. This assay was evaluated with 1649 suspected non-tuberculosis mycobacterial isolates. Apart from 3 out of 40  M. avium isolates that showed false signal with M. intracellulare specific probe, 100% specificity was obtained for all tested probes. Assay sensitivity varied from 88·9 to 100% depending on species. Average cost for obtaining a definite identification was only USD 1·1 with an average turn around time of less than 3 days.
Conclusions:  A rapid, simple and inexpensive real-time PCR assay was developed for the identification of common encountered mycobacteria in a high throughput laboratory setting.
Significance and Impact of the Study:  With this assay, more than 80% of the clinically isolated nontuberculous mycobacteria could be identified in a highly cost effective manner. This helped to save resources for other laboratory activities especially in high throughput mycobacterial laboratories.     

Polyandry and Reproduction in the Serpentine Leaf Miner Liriomyza trifolii (Diptera: Agromyzidae)

We investigated the hypotheses that Liriomyza trifolii is a polyandrous species and that multiple mating is required to maximize female fecundity. In addition, the hypothesis that male copulating frequency reduces female fecundity was examined. Using sterile and fertile males, we found that L. trifolii females mate more than once during their life span, and that multiple mating is required to maximize female fecundity. A similar amount of sperm was transferred during five successive male copulations with an equal insemination probability. However, the probability of reproduction failure was strongly correlated with the male copulating sequence. Moreover, the failure rate of sperm transfer was found to be 6.8%. In light of these results, possible selection pressures driving for polyandry in L. trifolii are discussed.     

Entomopathogenic nematodes for biological control of the leafminer,Liriomyza trifolii (Dipt.: Agromyzidae)

In the laboratory, mortality rates of the agromyzid leafminer larvae,Liriomyza trifolii (Burgess), ranged from 48 to 98% by 20 strains and/or species of steinernematid and heterorhabditid nematodes. In the greenhouse, abamectin provided superior control of larval leafminers, killing 100% of them as compared withSteinernema carpocapsae (Weiser) All strain (24 to 43% leafminer mortality) orS. carpocapsae Liriomyza-selected strain (8 to 44% leafminer mortality); the maximum relative humidity (r.h.) ranged between 81 and 91% and the minimum r.h. between 50 and 70%. In the foghouse under high r.h., the commercially available All strain and the Hawaiian isolate ofS. feltiae (Filipjev) MG-14 strain caused 69 and 67% mean mortality, respectively. There was a significant correlation (P<0.01) between nematode mortality of leafminers and r.h., including the mean, standard deviation, and minimum r.h. during the 48 h after treatment. Average r.h. >92% with a standard deviation of <9% r.h. and a minimum of 72% r.h. providedS. carpocapsae All strain mortality rates of leafminers >65%. The major constraint against the use of nematodes against leafminers in the foliar environment is low r.h. The use of nematodes againstL. trifolii can be successful if the r.h. remains high and if nematodes enter leafmines before desiccation, and the nematodes should be integrated with chemical insecticides such as abamectin to manage pesticide resistance inL. trifolii.     

Influence of temperature onOpius dissitus (Hym.: Braconidae), a parasitoid ofLiriomyza trifolii (Dipt.: Agromyzidae)

Rearing experiments at different temperatures (10, 15, 20, 25 and 30°C) were made onOpius dissitus Muesebeck, a parasitoid ofLiriomyza trifolii (Burgess). Temperatures between 20 and 30°C were optimal for the development ofO. dissitus, which was able to adapt to relatively high temperatures despite its holarctic origin.     

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 .     

Biological control of the leafminer,Liriomyza trifolii by introduction,releasing, evaluation of the parasitoids Diglyphus isaea and Dacnusa sibirica on vegetables crops in greenhouses in Egypt

Abstract

The serpentine leafminer, Liriomyza trifolii (Burgess) (Diptera: Agromyzidae), is one of the most serious pests of various floricultural and vegetable crops. The European strains of the parasitoids Diglyphus isaea (Walker) (Hymenoptera: Eulophidae) and Dacnusa sibirica Telenga (Hymenoptera: Braconidae) were imported from The Netherlands. A total of 90,000 of these parasitoids were reared and released on cucumber and tomato in greenhouses. The parasitism rates of D. sibirica reached maximum 11.6% and 7.2% in the 11th week from the releasing date, on cucumber and tomato, respectively. Also the parasitism rates of the European strain of D. isaea increased until it reached a maximum 2.1% and 1.4%, in the tenth week from the releasing date on cucumber and tomato, respectively. It is concluded that the parasitoids D. sibirica and the European strain of D. isaea can be established in Egypt.     

Utilisation of morphological features in life table studies of Liriomyza huidobrensis (Dipt., Agromyzidae) developing in lettuce

Abstract: A method of distinguishing different larval instars of Liriomyza huidobrensis morphologically, using measurements of the cephalopharyngeal skeleton was developed. The growth ratios of cephalopharyngeal skeletons between first and second and second and third instar larvae were 1.80 and 1.47, respectively, enabling clear separation to be achieved for experimental work. Using this method the development rates of the immature stages of L. huidobrensis feeding on Lactuca sativa were determined under constant temperatures of 11, 16, 19, 26 and 28 ± 1°C and were shown to increase linearly with temperature over the range investigated. The theoretical lower threshold temperatures for development from oviposition to the end of each larval instar or pupal stage were 5.35, 6.30, 6.20 and 5.70°C, respectively. The overall threshold temperature for development from oviposition to 50% adult emergence (5.70°C) was used to calculate degree‐day (DD) requirements for development from oviposition to each larval instar or pupal eclosion, which were 84.3, 30.1, 58.9, 143.7 DD, respectively. The use of these data for optimizing the timing of application of control agents which are effective against specific developmental stages is discussed.     

Avermectin类农药对美洲斑潜蝇的生物活性

Avermectin类农药对美洲斑潜蝇Liriomyzasattvae Blanchard生物活性的室内研究结果表明： 揭阳霉素对美洲斑潜蝇1、2、3龄幼虫的LC₅₀分别是1.54×10^-4 g/L、3.73×10^-4 g/L、1.99×10^-3g/L;害极灭对上述幼虫的LC₅₀分别是1.48×10^-4g/L、3.68x10^-4 g/L和1.97×10^-3 g/L。美洲斑潜蝇幼虫对Avermectln类农药以1龄最敏感,其中揭阳霉素对3龄幼虫的LC₅₀是1龄的12.9倍。揭阳霉素对雌成虫24 h、48 h的LC₅₀分别是3.12×10^-3 g/L和2.08×10^-3 g/L,其对美 洲斑潜蝇取食、产卵拒避持效期分别是4-8天和10天。使用浓度0.005 g/L揭阳霉素处理6天、8天和10天后接虫,幼虫的存活率分别是0、16.13%和28.07%。田间使用浓度0.005 g/L的揭 阳霉素和0.0045 g/L几的害极灭分别处理,6天后的校正虫口减退率分别为91.0%和90.9%,两者差异不显著,而使用浓度0.0067 g/L揭阳霉素处理,6天后校正虫口减退率为93.6%。