蓟马的分子鉴定和种群遗传学研究进展

蓟马是一类个体微小的昆虫,形态学特征观察困难,种内遗传多样性高,且部分害虫类蓟马寄主广泛,繁殖能力强,为害严重,已成为重要的农林类害虫。采用分子生物学手段对蓟马进行物种鉴定,可以解决传统形态学鉴定困难、局限性较大等问题,能提高物种鉴定的效率与准确性。目前,关于蓟马分子生物学鉴定的研究较多,其中,基于线粒体细胞色素氧化酶亚基（ I cytochrome oxidase I,COI）基因片段的DNA条形码技术被广泛用于蓟马的快速准确鉴定、属内近缘种的区分及不同品系或生物型的鉴别。此外,开展蓟马类害虫种群遗传学的研究不仅有利于全面了解其遗传分化、适应性、入侵来源和扩散路径,还可为制订合理有效的监测预报和综合防治策略提供理论依据。目前,多种蓟马的种群遗传学研究表明地理隔离、寄主植物和微生物等因素影响其种内和种间的遗传多样性和遗传分化。本文综述了国内外有关蓟马的DNA条形码鉴定与种群遗传学研究进展,对目前蓟马物种鉴定中存在的问题进行讨论,并对未来的发展趋势进行展望。     

检疫性蛾类鉴定方法和标准的研究进展

由于检疫性蛾类多数种类个体小，形态难以观察，且与其他蛾类害虫在形态上具有一定的相似性，区别和鉴别难度大，造成该类检疫害虫传播扩散的风险加剧。一方面，该类害虫的幼虫啃食水果、木材等，使其品质下降，严重影响水果、木材等进出口贸易；另一方面，检疫害虫的扩散也对农林业生产构成了巨大威胁。因此，蛾类害虫的精准鉴定已成为国内外普遍关注的问题。该文对我国检疫性蛾类的种类进行系统性汇总，并对鉴定方法及鉴定标准等方面的研究进展进行综述，分析目前存在的问题和不足，并结合技术创新以及口岸实际检疫需求，提出今后的发展方向，以期为检疫性蛾类新型精准鉴定技术研发及其入侵防控策略制订提供参考。     

DNA条形码在昆虫学中的应用

DNA条形码技术是通过对一段标准化基因DNA序列的分析来实现对生物物种准确、快速鉴定的技术。自2003年提出以来,DNA条形码技术便备受关注,不仅被广泛地应用于生物分类学研究,也为生态学等学科的发展带来了机遇。昆虫是动物界的最大类群,目前条形码数据库中超过65%的序列都来自昆虫纲,DNA条形码的发展无疑为昆虫学带来了勃勃生机。本文综述了DNA条形码的产生、发展、优势及局限,总结并展望了DNA条形码技术在昆虫学基础研究与实践方面的应用。     

PM 7/129 (1) DNA barcoding as an identification tool for a number of regulated pests

Specific scope

This Standard describes the use of DNA barcoding protocols in support of the identification of a number of regulated pests and invasive plant species comparing DNA barcode regions with those deposited in publically available sequence databases. 1 It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

2016‐09     

Comparison of two short DNA barcoding loci (COI and COII) and two longer ribosomal DNA genes (SSU & LSU rRNA) for specimen identification among quarantine root-knot nematodes (Meloidogyne spp.) and their close relatives

Root-knot nematodes (Meloidogyne spp.) are important pests of numerous crops worldwide. Some members of this genus have a quarantine status, and accurate species identification is required to prevent further spreading. DNA barcoding is a method for organism identification in non-complex DNA backgrounds based on informative motifs in short DNA stretches (≈600 bp). As part of the EU 7th Framework project QBOL, 15 Meloidogyne species were chosen to compare the resolutions offered by two typical DNA barcoding loci, COI and COII, with the distinguishing signals produced by two ribosomal DNA genes (small and large subunit rDNA; SSU?≈?1,700 and LSU?≈?3,400 bp). None of the four markers distinguished between the tropical species Meloidogyne incognita, M. javanica and M. arenaria. Taking P ID (Liberal) values ≥0.93 as a measure for species delimitation, the four mtDNA and rDNA markers performed well for the tropical Meloidogyne species complex, M. enterolobii, M. hapla, and M. maritima. Within cluster III A (Holterman et al. Phytopathology, 99, 227–235, 2009), SSU rDNA did not offer resolution at species level. Both mtDNA loci COI and COII did, whereas for LSU rDNA a longer fragment (≥700 bp) is required. The high level of mitochondrial heteroplasmy recently reported for M. chitwoodi (Humphreys-Pereira and Elling Nematology, 15, 315–327, 2013) was not found in the populations under investigation, suggesting this could be a regional phenomenon. For identification of RKNs, we suggest the combined use of SSU rDNA with one of three other markers presented here.     

QBOL: a new EU project focusing on DNA barcoding of Quarantine organisms

In 2009 a new three year EU funded project (QBOL) started on DNA barcoding of important plant pests. An international consortium of 20 partners (universities, research institutes, and phytosanitary organizations) from around the world, coordinated by Plant Research International (Wageningen, the Netherlands), will collect DNA barcodes from many plant pathogenic quarantine organisms, store these sequences in a database accessible over the internet, develop a DNA bank and train end-users. All these activities should help National Plant Protection Services in the correct identification and detection of plant pathogenic quarantine organisms.     

DNA条形码技术在入侵植物刺萼龙葵检验检疫中的应用

刺萼龙葵(Solanum rostratum)是危害严重的外来入侵植物,基于种子形态的传统检疫方法有一定的局限性,不依赖于形态学特征的DNA条形码技术是基于DNA序列差异对物种进行鉴定的新方法,是传统形态学鉴定方法的有力补充。以刺萼龙葵及其8种龙葵亚属近缘植物为材料,从Gen Bank数据库中下载rbc L、mat K和内转录间隔区(ITS)序列,进行DNA条形码分析,结果显示ITS鉴定效果优于其他片段,可把刺萼龙葵从其近缘植物中鉴别出来。     

利用条形码技术鉴定苹浅褐卷蛾

苹浅褐卷蛾原产澳大利亚,目前仅分布于少数地区,是澳大利亚、新西兰及英国等国家多种经济作物上的重要害虫,苹浅褐卷蛾形态具有多态性,对其形态鉴定一直非常困难,由此也增加了该虫在口岸上的入侵风险。本研究使用DNA条形码技术(barcoding)测定了采集自新西兰5个不同地区苹浅褐卷蛾种群26个样本的细胞色素酶I(COI)序列。研究表明,苹浅褐卷蛾种内COI序列差异远小于3%,而与BOLD数据库中卷蛾科其他种类的种间差异约为7%～16%,远大于3%。该结果肯定了条形码技术能够成为口岸快速、准确鉴定苹浅褐卷蛾的有效方法。     

植物DNA条形码技术在出入境检验检疫领域的应用

DNA条形码技术是一种通过标准的DNA片段鉴定物种的新技术,已成为近年来生物学研究热点之一。由于DNA条形码技术具有快速、准确、自动等优点,可以满足目前出入境检验检疫工作中提出的"检得出、检得快、检得准"的要求。本文对植物DNA条形码的研究进展进行了概述,分析了该技术在检验检疫应用领域中存在的不足,并针对如何更好地将该技术应用于检验检疫工作中提出了建议。     

贵州省黔东南地区发现草地贪夜蛾的5种寄生性天敌及其两种重寄生蜂

迁飞性重大农业入侵害虫草地贪夜蛾Spodoptera frugiperda(J. E. Smith)于2019年入侵贵州省黔东南地区。深入研究害虫的天敌发生情况有利于发掘和利用天敌资源开展生物防治工作。本文将贵州省黔东南地区采集的被寄生卵块和幼虫置于实验室条件下饲育,利用DNA条形码技术鉴定羽化出的天敌昆虫。共鉴定出5种寄生性天敌昆虫,其中卵寄生蜂为夜蛾黑卵蜂Telenomus remus,幼虫寄生蜂为菜粉蝶盘绒茧蜂Cotesia glomerata和半闭弯尾姬蜂Diadegma semiclausum,幼虫寄生蝇为厉寄蝇属寄生蝇Lydella sp.和日本追寄蝇Exorista japonica。另外,还发现了两种寄生于菜粉蝶盘绒茧蜂的重寄生蜂,分别为弯沟啮小蜂属寄生蜂Baryscapus sp.和吉氏金小蜂Nasonia giraulti。本文的发现为筛选防治草地贪夜蛾的优势天敌奠定了基础。     

From laboratory to point of entry: development and implementation of a loop‐mediated isothermal amplification (LAMP)‐based genetic identification system to prevent introduction of quarantine insect species

BACKGROUND

Rapid genetic on‐site identification methods at points of entry, such as seaports and airports, have the potential to become important tools to prevent the introduction and spread of economically harmful pest species that are unintentionally transported by the global trade of plant commodities. This paper reports the development and evaluation of a loop‐mediated isothermal amplification (LAMP)‐based identification system to prevent introduction of the three most frequently encountered regulated quarantine insect species groups at Swiss borders, Bemisia tabaci, Thrips palmi and several regulated fruit flies of the genera Bactrocera and Zeugodacus.

RESULTS

The LAMP primers were designed to target a fragment of the mitochondrial cytochrome c oxidase subunit I gene and were generated based on publicly available DNA sequences. Laboratory evaluations analysing 282 insect specimens suspected to be quarantine organisms revealed an overall test efficiency of 99%. Additional on‐site evaluation at a point of entry using 37 specimens performed by plant health inspectors with minimal laboratory training resulted in an overall test efficiency of 95%. During both evaluation rounds, there were no false‐positives and the observed false‐negatives were attributable to human‐induced manipulation errors. To overcome the possibility of accidental introduction of pests as a result of rare false‐negative results, samples yielding negative results in the LAMP method were also subjected to DNA barcoding.

CONCLUSION

Our LAMP assays reliably differentiated between the tested regulated and non‐regulated insect species within <1 h. Hence, LAMP assays represent suitable tools for rapid on‐site identification of harmful pests, which might facilitate an accelerated import control process for plant commodities. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

基于DNA条形码的广西苦瓜中实蝇幼虫分子鉴定研究*

实蝇是多种热带、亚热带水果和蔬菜的重要害虫,其卵、幼虫、蛹难以通过形态特征进行种类鉴定。DNA条形码是近年来出现的物种分子鉴定技术,能够实现对昆虫非成熟虫态的快速鉴定。本研究利用DNA条形码技术和BOLD系统,选取线粒体DNA细胞色素氧化酶I（COI）基因片段,针对采自于广西南宁地区苦瓜中的实蝇幼虫样品进行了序列测定、比对和分析。结果显示,在所检测的10头实蝇幼虫样品中,有4头为瓜实蝇［Bactrocera cucurbitae (Coquillett)］、6头为南亚果实蝇［Bactrocera tau (Walker)］。     

DNA barcoding as an identification tool for selected EU‐regulated plant pests: an international collaborative test performance study among 14 laboratories

DNA barcoding protocols for selected EU‐regulated arthropods, bacteria, fungi, nematodes and phytoplasmas were developed within the Quarantine organisms Barcoding of Life (QBOL) project financed by 7th framework program of the European Union. DNA barcodes generated with the developed protocols were stored in the Q‐bank database. An test performance study (TPS) was set up involving 14 participating laboratories to validate the use of the developed protocols as a diagnostic tool and to identify possible difficulties in the use of the protocols and Q‐bank. This paper describes the steps that were used to set up the TPS, to validate the protocols and to identify difficulties. TPS data shows that the developed tests are very robust and produce highly reproducible results. Participants managed to define good consensus sequences which allowed them to correctly identify their samples using Q‐bank in 78% of all cases. Q‐bank outperformed NCBI and BOLD in terms of diagnostic sensitivity and diagnostic specificity for all organism groups. Using general qualifiers, performance criteria and feedback from TPS participants, difficulties in the set‐up of the TPS, the use of the protocols and databases, and the proficiency of participants were identified/evaluated and recommendations for future work were made. The developed DNA barcoding protocols and Q‐bank have proven to be useful tools in support of the identification of selected EU‐regulated plant pests and pathogens on the desired taxonomical level.     

果园螟蛾总科部分种类DNA条形码鉴定

为检验DNA条形码在鳞翅目螟蛾总科蛾类鉴定中的可行性,对采自山西省太原市晋源区螟蛾总科26种78头蛾类标本分别提取了DNA,扩增了全部78头标本的线粒体cox1基因和其中75头标本的核糖体28S基因,并通过构建系统发育树、计算遗传距离及种间差异阈值等方法,对所有标本进行了鉴定和比较分析,检验了国际DNA条形码数据库BOLD(the barcode of life data)系统的鉴定成功率。结果表明,基于cox1基因和28S基因的系统发育树鉴定成功率分别为100.00%和97.14%,BOLD系统的鉴定成功率达到了67.94%。基于最大简约法、邻接法和最大似然法构建的系统发育树,鉴定结果均相同。基于cox1基因的种内遗传距离全部小于1.00%,种内种间的遗传距离形成明显的3.00%阈值现象。研究表明,cox1及28S基因均适用于供试螟蛾总科种类的鉴定,核糖体28S基因可以作为DNA条形码鉴定的辅助基因;BOLD系统数据库仍有待充实,且标本鉴定工作相对滞后;不同聚类分析方法对结果影响很小,其中邻接法计算速度快,更适合DNA条形码大数据的分析。     

Molecular taxonomy and its application to ecological studies of Pythium species

More than 140 Pythium species have been described; 41 of these species have been described since 2000. Many Pythium species cause seed rot and damping off of seedlings. Recent changes in crop culture systems, such as the introduction of hydroponic cultures, have resulted in Pythium diseases of mature plants as well, with symptoms such as root rot, crown rot, and blight. In Japan, newly introduced ornamental and floral plants are vulnerable to high temperatures and humidity, resulting in severe Pythium infections. Once a pathogen invades a greenhouse, it quickly propagates and disperses, resulting in serious yield loss. It is very difficult to identify an isolate at the species level based on morphological characteristics because of overlaps in the size and shape of reproductive structures, heterothallism, and the continuing discovery of new species. New procedures based on DNA sequences have thus been introduced to identify species; in particular, the rDNA ITS region and the coxI gene have been recommended for DNA barcoding. BLAST searches and phylogenic analyses allow for easy and precise identification. PCR techniques are also being used to diagnose plant diseases, and real time PCR can be used for quantifying the pathogen to assess the risk of disease loss. Molecular techniques to analyze population structure help clarify transmission routes of pathogens and the ecology of Pythium flora in natural ecosystems. In this review we discuss recent advances in molecular methods for identifying, detecting, and classifying Pythium species.     

DNA条形码技术在小花蝽属昆虫分类鉴定中的应用

本研究利用DNA条形码技术对13种小花蝽属Orius Wolff昆虫进行了鉴定,进行了59条COI基因序列碱基组成及种内、种间遗传距离的分析,采用邻接法、最大简约法、贝叶斯推论法构建了系统发育树。结果表明,小花蝽属昆虫COI基因序列碱基组成与典型的昆虫线粒体DNA一致,A+T平均含量(66.4%)明显高于G+C含量,密码子的第3位A+T含量高达90.7%,碱基替换多为同义替换;13种小花蝽种内平均遗传距离为0.008,种间平均遗传距离为0.128,种内、种间遗传距离没有重叠区域。3种方法构建的系统发育树的聚类分析与形态学鉴定结果基本一致,除微小花蝽Orius minutus(Linnaeus)可能存在隐存种现象外,其他同一种群的不同个体单独聚为一支。利用DNA条形码技术对小花蝽属昆虫进行物种快速分子鉴定具有可行性。     

Pest management programmes in vineyards using male mating disruption

Israeli vine growers have been reluctant to adopt the mating disruption technique for control of the European vine moth, Lobesia botrana Den. & Schiff. Since the chemically controlled honeydew moth, Cryptoblabes gnidiella Mill., coexists with the European vine moth, growers have maintained that the use of mating disruption would fail to bring about a significant reduction in pesticide use. In this study, the efficacy of mating disruption techniques against C. gnidiella was tested, as well as the effect of these methods on pesticide use and damage to clusters when the method was employed against both of the pests in wine grapes. Comparisons were made between plots treated with (1) L. botrana mating disruption pheromone, (2) L. botrana and C. gnidiella mating disruption pheromones and (3) control plots. A significant difference in the number of clusters infested with the developmental stages of the moths was seen between pheromone-treated plots and controls, while no such difference was observed between plots treated with one versus two pheromones. A similar pattern was observed in the number of insecticide applications; the greatest number of applications was used in control plots, followed by plots treated with L. botrana mating disruption pheromone and by plots treated with pheromones against both pests, in which no pesticides were applied.     

The mass rearing and utilization of Trichogramma to control lepidopterous pests: Achievements and outlook

The release of mass-produced egg parasites of the genus Trichogramma to control Lepidopterous pests has gained increasing attention in the last few years. About 18 different species of this egg parasite are being used to control pests on corn, sugar-cane, rice, soybean, cotton, sugar-beet, vegetables and pine. Some information on the global use of Trichogramma is given. Trichogramma evanescens(Westw.) has been used commercially to control the European corn borer Ostrinia nubilalis (Hb.) in Germany since 1980. The treated area reached about 6200 ha in 1992. The use of Trichogramma dendrolimi Matsumura to control the codling moth Cydia pomonella (L.) and the summer fruit tortrix moth Adoxophyes orana (F.R.) was started in 1990 and covered an area of c. 15 ha in 1992. Research is being carried out to select effective Trichogramma species to control other pests such as grape berry moth Eupoecilia ambiguella (Hb.), plum fruit moth Cydia funebrana Tr. and diamondback moth Plutella xylostella (L.).     

吉林省中西部地区灯下蛾类群落结构及多样性

为探明吉林省中西部地区蛾类昆虫的种类及组成,提供蛾类群落的多样性资料,本研究于2017年－2020年利用高空探照灯逐日诱集蛾类昆虫并分类,明确不同昆虫季节性种群动态,在此基础上对蛾类群落多样性进行分析。结果共采集、鉴定蛾类昆虫18科227种,优势科为夜蛾科,种数占比达到49.34%。按为害植物类别,可分为农业害虫、林业害虫、果树害虫、蔬菜害虫等。蛾类昆虫4月始见,7、8月群落丰富度最高,7月群落多样性最高,群落均匀性随时间变化不明显。明确了不同月份优势度指数较高的种类。7、8月的蛾类群落相似性高,4、10月与各月的相似性低。本研究结果可为此区域蛾类害虫的监测预警与综合治理提供基础理论依据。     

世界主要储粮书虱环介导等温扩增试剂盒研发与应用

为利用分子生物学技术快速准确鉴定国内外主要储粮书虱,基于环介导等温扩增 （loop-mediated isothermal amplification, LAMP）技术拟研发一款世界主要储粮书虱LAMP试剂盒,利用该LAMP试剂盒对10种储粮书虱的DNA粗提液进行测试,并结合DNA条形码技术对采自中国山东省和内蒙古自治区粮库的未知种类储粮书虱成虫样品进行鉴定。结果显示,本试验研发的LAMP试剂盒可准确鉴定10种储粮书虱;使用LAMP试剂盒和DNA条形码技术鉴定未知种储粮书虱样品的结果一致,即山东省储粮书虱样品为嗜虫书虱 Liposcelis entomophila,内蒙古自治区储粮书虱样品为啮书虱 L. corrodens,表明本试验研发的世界主要储粮书虱LAMP试剂盒可以快速、精准完成主要储粮书虱的鉴定,且操作便捷,可为海关口岸和基层仓储保护部门书虱鉴定工作提供技术支持。