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.     

Molecular evidence of cryptic species within the Liriomyza huidobrensis (Diptera: Agromyzidae)

Phylogenetic relationships among populations of the polyphagous pea leafminer, Liriomyza huidobrensis (Blanchard), were investigated using DNA sequence data. Maximum parsimony analysis of 941 bp of mitochondrial cytochrome oxidase I and II genes showed that L. huidobrensis contains two well-defined monophyletic groups, one composed of specimens from California and Hawaii and one composed of specimens from South and Central America together with populations that have been recently introduced into other parts of the world. The differentiation between the two clades within L. huidobrensis is equivalent to that seen between other agromyzid species, suggesting that L. huidobrensis as currently defined contains two cryptic species. This finding is consistent with field observations of differences in pest status and insecticide resistance between L. huidobrensis populations. Until additional studies are complete, no changes in L. huidobrensis taxonomy are proposed. However, researchers and quarantine officials may wish to consider the findings of the current study in designing research, pest management, and quarantine programs for L. huidobrensis.     

Influence of Liriomyza species (Diptera: Agromyzidae) and their host plants, on oviposition by Opius dissitus females (Hymenoptera: Braconidae)

Two factors, among others, influence the oviposition of Opius dissitus Muesebeck. The first was the host species, the second the plant upon which the pest host larvae develop. O. dissitus females prefer Liriomyza trifolii (Burgess) larvae over those of L. huidobrensis (Blanchard). O. dissitus females were more attracted to the larvae of L. trifolii or L. huidobrensis when these were present on courgette rather than tomato or lettuce.     

Foliar Application of the Entomopathogenic Nematode Steinernema feltiae Against Leafminers on Vegetables

Data from a comparative study of the efficacy of Steinernema feltiae for the control of three species of leafminer formed the basis of an application schedule which successfully suppressed an outbreak of the statutory leafminer pest, Liriomyza huidobrensis . All three instars of Liriomyza bryoniae and L. huidobrensis were similarly susceptible to S. feltiae at 20 o C and > 90% relative humidity (RH). Although all larval instars of Chromatomyia syngenesiae were susceptible to S. feltiae , mortality was lower than for L. bryoniae . Repeat applications of S. feltiae to L. bryoniae and C. syngenesiae indicated that a nematode treatment to the second/early third instar larvae was more effective than applying higher rates of nematodes when humidities were less than 90% RH. In a trial on lettuce at a commercial glasshouse a mean L. huidobrensis mortality of 82 +/- 5% was recorded after an S. feltiae application, significantly higher than the chemical treatment, heptenophos.     

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。     

The separation of Liriomyza huidobrensis (Diptera: Agromyzidae) from related indigenous and non-indigenous species encountered in the United Kingdom using cellulose acetate electrophoresis

The non-indigenous pest leaf miner Liriomyza huidobrensis and its close relatives L. bryoniae and L. strigata belong to a natural group. These species can be separated from one another by means of protein electrophoresis on a cellulose acetate membrane. The protocol for this diagnostic assay involves staining for two enzymes, glucose-6-phosphate dehydrogenase (G6PDH) and leucine-glycine peptidase (PEP). Other leaf miner species that might also be found under glass, Chromatomyia syngenesiae, C. horticola and the non-indigenous L. trifolii and L. sativae , are clearly distinguishable from the L. huidobrensis group of species by this assay. The effect of parasitism on L. huidobrensis by Dacnusa sibirica was investigated and shown unlikely to cause misidentification of the leaf miner host. Results obtained from practical use of the assay to identify unknown specimens from ports and nurseries are compared with those obtained from flies kept in laboratory culture and used during development of the assay. No new variation was found for G6PDH. New PEP variation was found for both L. huidobrensis and L. bryoniae but this did not affect the integrity of the assay. A biochemical key to these agromyzid leaf miners is presented.     

Toxicity of chemicals commonly used in Indonesian vegetable crops to Liriomyza huidobrensis populations and the Indonesian parasitoids Hemiptarsenus varicornis, Opius sp., and Gronotoma micromorpha, as well as the Australian parasitoids Hemiptarsenus varicornis and Diglyphus isaea

Liriomyza huidobrensis (Blanchard) and Liriomyza sativae (Blanchard) are important pests of vegetable crops in Indonesia and are likely to spread to neighboring countries. Three pesticides (dimehypo, abamectin, and cyromazine) are currently used to control these pests, but there is little information on their effectiveness against field populations and on their impact on parasitoids controlling Liriomyza species. The toxicity of these chemicals to L. huidobrensis and three common parasitoids (Hemiptarsenus varicornis Gerault, Opius sp., and Gronotoma micromorpha Perkins) was therefore evaluated in Indonesia with mortality laboratory assays. All three chemicals were effective against larvae of three populations of L. huidobrensis with different histories of chemical exposure. Dimehypo caused mortality in adult Opius sp., G. micromorpha, and H. varicornis, whereas abamectin was toxic only at concentrations substantially higher than the field rate. Cyromazine did not influence survival of the parasitoids. A commonly used fungicide, mancozeb, had no impact on parasitoid mortality. Trials were repeated with a strain of H. varicornis from Australia and a different parasitoid (Diglyphus isaea) recently found in Australia. Neither parasitoid was influenced by mancozeb or cyromazine. Abamectin applied at field rates caused some mortality among the adults of both species, but was less toxic than chlorpyrifos. Abamectin produced lower LC50s against Australian H. varicornis than against Indonesian H. varicornis. These results suggest that cyromazine can be incorporated into Liriomyza control programs in Indonesia that conserve parasitoids, whereas dimehypo and abamectin need to be used cautiously. Local Australian parasitoids should help control L. huidobrensis as long as only cyromazine and nontoxic fungicides are applied.     

Morphological and genetic characteristics of Liriomyza huidobrensis (Blanchard) (Diptera: Agromyzidae) infesting potato crops in Korea

The pea leafminer fly, Liriomyza huidobrensis (Blanchard), was found infesting and damaging Korean potato crops during a 2012 field survey. Localized outbreaks of L. huidobrensis were observed in Miryang and Goryeong counties in the south of the Korean Peninsula. This paper describes the morphological and genetic characteristics of the Korean population of L. huidobrensis. The dorsal color pattern on the abdomen, and an array of microsetae on the thorax, were the main diagnostic characteristics of this species. On the basis of an analysis of a partial mitochondrial cytochrome c oxidase subunit 1 (COI) DNA sequence, we found that intraspecific variation in L. huidobrensis was negligible, and that the distance between L. huidobrensis and a closely related species, L. trifolii, was estimated to be 7.4–7.6 K2P. This is the first report of the infestation of field crops by the invasive leafminer L. huidobrensis in Korea.     

Host plant-related parasitism and host feeding activities of Diglyphus isaea (Hymenoptera: Eulophidae) on Liriomyza huidobrensis, Liriomyza sativae, and Liriomyza trifolii (Diptera: Agromyzidae)

Host plant species can affect the behavior and attributes of parasitoids, such as host searching, oviposition, and offspring fitness. In this study, parasitism, host feeding, and sex ratios of Diglyphus isaea (Walker) (Hymenoptera: Eulophidae) on Liriomyza huidobrensis (Blanchard), Liriomyza sativae Blanchard, and Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) larvae reared on Phaseolus vulgaris L., Pisum sativum L., Solanum lycopersicum L., and Vicia faba L. were determined. In no-choice tests, L. huidobrensis had the highest rate of parasitism when reared on P. vulgaris (46%), L. sativae when reared on V. faba (59%) and P. vulgaris (59%), and L. trifolii when reared on S. lycopersicum (68%). Host feeding in no-choice tests ranged between 2% and 36% and was highest on L. trifolii reared on V. faba. Results of choice tests showed a significant interaction effect for host plant and Liriomyza species on parasitism and host feeding. Within plant mixtures, L. sativae reared on P. vulgaris had the highest rate of parasitism (31%), followed by L. trifolii on S. lycopersicum (29%) and L. huidobrensis on V. faba (28%). Host feeding was highest on L. trifolii reared on S. lycopersicum (14%) and lowest on L. huidobrensis reared on P. sativum and S. lycopersicum (1%). In some instances, plant mixtures resulted in a higher proportion of females of D. isaea than single plant species. The highest proportion of females was obtained in plant mixtures on L. huidobrensis and L. trifolii on V. faba (71 and 72%, respectively). This study suggests that planting crop mixtures can potentially lead to higher proportions of females, thus improving parasitism and host feeding, depending on Liriomyza and host plant species.     

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。     

福州郊区美洲斑潜蝇、南美斑潜蝇寄主植物初步名录

本记录了福州郊区美洲斑潜蝇、南美斑潜蝇寄主植物的初步名录。发现该地区美洲斑潜蝇可取食20科85种植物,南美斑潜蝇可取食20科73种。最后讨论了这两种斑潜蝇的主要寄主作物和杂草,并提出套种和清除田间杂草的农防措施;斑潜蝇的适应性很强,寄主范围正逐步扩大,应密切关注其动态。     

Application of molecular techniques to distinguish Liriomyza trifolii from L. sativae (Diptera: Agromyzidae) on tomato cultivation in Japan

A molecular method is applied for differentiating the morphologically related leafminers Liriomyza trifolli and L. sativae on tomato cultivation. The method requires multiplex polymerase chain reaction (PCR) amplification of a region of mitochondrial cytochrome oxidase DNA using multiprimer sets. The method divides the mitochondrial fragment of L. trifolli into two fragments and L. sativae into three fragments. It is faster, less costly, and easier than random amplified polymorphic DNA-PCR, PCR-restriction fragment-length polymorphism, and DNA sequencing and more sensitive than the enzyme electrophoresis method, which are other ways to differentiate these two species. We applied the method to samples from populations of another place, sex, and stage and obtained the same results.     

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。     

Primer design for identifying economically important Liriomyza species (Diptera: Agromyzidae) by multiplex PCR

Leafminer flies, especially, Liriomyza huidobrensis, Liriomyza sativae and Liriomyza trifolii, are quarantine species in many countries. Their morphological similarity makes identification difficult. To develop a rapid, reliable, sensitive and simple molecular identification method using multiplex PCR, we newly sequenced the mitochondrial cytochrome oxidase I (COI) genes of Liriomyza bryoniae, Liriomyza chinensis, L. huidobrensis, L. sativae, L. trifolii, Chromatomyia horticola and four parasitoid species. We aligned them with all the COI sequences of the leafminer flies found in the international DNA nucleotide sequence databases (DDBJ/EMBL/GenBank). We then designed species‐specific primers to allow us to differentiate between L. bryoniae, L. chinensis, L. huidobrensis, L. sativae, and L. trifolii.     

入侵害虫南美斑潜蝇在西藏首次发现及其寄生蜂调查

【目的】南美斑潜蝇Liriomyza huidobrensis为我国重要的入侵害虫,我们于2017年7月首次发现其已扩散至西藏。本研究旨在了解西藏地区南美斑潜蝇的发生现状及其寄生蜂种类。【方法】2017和2018年6月上旬-9月中旬对西藏拉萨、林芝、山南和日喀则4个地区的南美斑潜蝇的发生危害及其寄生蜂组成进行了调查采样。【结果】调查的西藏这4个地区均发现南美斑潜蝇分布和危害;温室发生时间为6月上旬-9月中旬,而露地的发生高峰在7月下旬-8月上旬。其寄主植物有9科21种,主要有豆科(Fabaceae)、茄科(Solanaceae)和伞形科(Umbelliferae)等,主要嗜好经济作物有豇豆Vigna unguiculata, 扁豆Lablab purpureus , 莴笋Lactuca sativa var. angustana和白菜Brassica rapa ssp. pekinensis。其寄生蜂包括姬小蜂科(Eulophidae)、茧蜂科(Braconidae)和瘿蜂科(Cynipidae)共计3科9属16种,分别为双斑潜蝇姬小蜂Diglyphus bimaculatus, 豌豆潜蝇姬小蜂Diglyphus isaea, 小斑潜蝇姬小蜂Diglyphus minoeus, 万氏潜蝇姬小蜂Diglyphus wani, Diglyphus sp., 纵纹瑟姬小蜂Cirrospilus vittatus, Hemiptarsenus sp., 芙新姬小蜂Neochrysocharis formosa, 底比斯姬小蜂Chrysocharis pentheus, 普金姬小蜂Chrysocharis pubicornis, Chrysocharis sp., 潜蝇柄腹姬小蜂Pediobius metallicus, Pnigalio sp., 西伯利亚离颚茧蜂Dacnusa sibirica, Dacnusa sp.和Gronotoma sp.;其中优势种为西伯利亚离颚茧蜂、双斑潜蝇姬小蜂和豌豆潜蝇姬小蜂,其个体数量占比分别为总采集个体数的48.7%, 22.1%和12.9%。【结论】西藏南美斑潜蝇的寄生蜂种类丰富,发挥着重要的自然控制作用。建议进一步加强优势寄生蜂生物学、控害潜力评价以及利用研究,为南美斑潜蝇的绿色防控提供指导。     

温、湿度对南美斑潜蝇和美洲斑潜蝇羽化的影响

在实验条件下 ,研究了温湿度对南美斑潜蝇Liriomgzahuidobrensis和美洲斑潜蝇Liriomgzasativae羽化的影响。结果表明 ,温度对 2种斑潜蝇日羽化节律有显著的影响。随温度的升高 ,成虫的日羽化时段相应缩短 ,日羽化高峰期相应提前。且 2种斑潜蝇的羽化均集中在 1 2 :0 0之前。 2种斑潜蝇羽化所需要的最适温度 :南美斑潜蝇为 2 0℃ ,美洲斑潜蝇为 2 5℃ ;羽化所需要的最适相对湿度 :南美斑潜蝇为90 % ,美洲斑潜蝇为 1 0 0 %。     

甘肃白银地区潜叶蝇及其寄生蜂的组成和发生调查

【目的】明确严重危害甘肃省白银地区作物的潜叶蝇及天敌寄生蜂的多样性和种群组成。【方法】采用五点取样法田间调查潜叶蝇危害的蔬菜、花卉、杂草并采样,室内对潜叶蝇及其寄生蜂进行形态鉴定和分子鉴定。【结果】在2016-2020年在甘肃白银地区采集的样本中发现6种常见潜叶蝇,包括本地种豌豆彩潜蝇Chromatomyia horticola和葱斑潜蝇Liriomyza chinensis,入侵种南美斑潜蝇L. huidobrensis、三叶草斑潜蝇L. trifolii、番茄斑潜蝇L. bryoniae和美洲斑潜蝇L. sativae。其中,豌豆彩潜蝇为优势种,可危害多科寄主植物;葱斑潜蝇仅在大葱上发现危害;南美斑潜蝇在温室内的蔬菜上发生严重;三叶草斑潜蝇首次在白银市发现;番茄斑潜蝇与美洲斑潜蝇的危害相对较轻。此外,调查的潜叶蝇寄生蜂包括3科12属24种,优势种包括豌豆潜蝇姬小蜂Diglyphus isaea、芙新姬小蜂Neochrysocharis formosa、万氏潜蝇姬小蜂D. wani和西伯利亚离颚茧蜂Dacnusa sibirica。【结论】一方面白银市应加强监测和预警入侵性潜叶蝇的发生,另一方面白银市潜叶蝇的天敌寄生蜂种类丰富,对田间主要危害的潜叶蝇可能发挥着重要的自然防控作用,建议保护利用本地寄生蜂天敌。     

南美斑潜蝇在温室蔬菜上种群动态的初步观察

系统观察结果表明,南美斑潜蝇Liriomyza huidobrensis(Blanchard)在平凉市日光温室蔬菜全生产期内的种群消长明显呈“两头高中间低”的态势,成虫发生量以春夏生产期最大,可达2.1头/叶;深冬生产期最小,仅为0.1~0.2头/叶;秋冬生产期较大,保持在1.0~1.2头/叶之间。温室内温、湿度条件的变化是引起该虫种群数量波动的主要原因。根据有效积温计算结果,该虫在平凉市日光温室蔬菜全生产期发生9.1代,世代平均发育速率0.030,不同生产期的发生代数和发育速率差异较大。生产上宜采取“前控后治中间松”的防治策略。     

Application of molecular techniques to identification of three plusiine species, Autographa nigrisigna, Macdunnoughia confusa, and Thysanoplusia intermixta (Lepidoptera: Noctuidae), found in integrated pest management lettuce fields in Japan

Three plusiine species, Autographa nigrisigna, Macdunnoughia confusa, and Thysanoplusia intermixta (Lepidoptera: Noctuidae), are commonly found together in lettuce, Lactuca sativa L., fields in Japan. Given the marked morphological similarities between these species and the difficulty associated with discriminating between them using only visual cues, we used multiplex polymerase chain reaction (PCR) assay to distinguish between the three target species. Multiplex PCR uses four primers to simultaneously amplify a specific region of the mitochondrial DNA and produce species-specific banding patterns. The stringency of the method was tested using specimens of different sex, location, and developmental stage, and consistent results were obtained for all samples. Indeed, our method has the potential to clarify the species structure of plusiine species in lettuce fields.     

Is host selection in leafminer adults influenced by pre‐imaginal or early adult experience?

Abstract:  Olfactometer and feeding/oviposition preference tests were carried out to study if host plant preference in two agromyzid leafminers, Liriomyza trifolii and Liriomyza huidobrensis , is influenced by pre-imaginal or imaginal experience, and at what level (host plant species, host plant variety or host age). It was found that the choice of host plant species (potato or tomato) and host plant variety had a pre-imaginal component, which could be reinforced (or modified) by an imaginal component. On the other hand, preference for potato leaves of different ages did not appear to be based on either pre-imaginal or imaginal factors, but resulted from differential performance and oviposition preference. The Hopkins' host selection principle appears to operate in these leafminers at the level of host species, and to some extent at the level of host variety, but not with respect to host age.