Molecular identification key for pest species of Scirtothrips (Thysanoptera: Thripidae)

Effective plant quarantine and biological control initiatives require rapid and accurate identification of exotic and potentially invasive taxa that may cause high economic losses or environmental damage. The genus Scirtothrips Shull includes several species that are serious agricultural pests, and, because of their minute size and cryptic behavior, prone to undetected transport through international trade of plant material. Although assigning specimens to the genus Scirtothrips is straightforward using traditional taxonomic methods, identification of species is much more difficult and requires expert knowledge of the genus. Furthermore, the validity of some Scirtothrips species is questionable. Therefore, an easy, accurate, and highly reliable technique is desirable for Scirtothrips identification. Here, we provide a simple molecular key based on the internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) of nuclear ribosomal DNA. Individual specimens can be identified by amplification of their ITS1 and ITS2 regions with general primers and determining the size of the products by using standard agarose gel electrophoresis, followed in some instances by DNA digestion with the restriction enzymes SacII or PspOM I. The advantage of this identification system is that nonspecialists are able to quickly and cheaply identify individual specimens. Material analyzed for this work was collected in the United States (California), India, South Africa, Kenya, Mexico, Guatemala, Honduras, Nicaragua, Costa Rica, Panama, Australia, New Zealand and Raiatea (Society Islands French Polynesia). We have identified seven pest species with the molecular-based methods described here. It is hoped that this system can be extended to other members of the genus as their ITS1 and ITS2 sequences become available. We also provide molecular confirmation for two new Scirtothrips species, one species from Honduras and one species from New Zealand.     

安荔赤眼蜂在中国野外首次发现及其体内Wolbachia的检测

【目的】为丰富赤眼蜂Trichogramma的种类资源,明确野外新采集的一种赤眼蜂的种类,探明该赤眼蜂所感染Wolbachia的类型。【方法】采用挂米蛾Corcyra cephalonica卵卡法在华南农业大学树木园诱集到两批赤眼蜂,通过形态鉴定和PCR扩增ITS2序列并测序分析的分子鉴定手段对野外采集的赤眼蜂材料进行种类鉴定;通过PCR扩增Wolbachia的外膜蛋白基因(wsp)序列,检测赤眼蜂体内Wolbachia的感染情况;通过PCR扩增wsp序列和多位点序列分型(multilocus sequence typing, MLST)对检测到的赤眼蜂体内的Wolbachia进行同源性分析。【结果】所诱集到的两批赤眼蜂均被鉴定为安荔赤眼蜂Trichogramma oleae Voegelé & Pointel,体内Wolbachia的感染率达100%。该Wolbachia株系与安荔赤眼蜂(前南斯拉夫品系)、短管赤眼蜂T. pretiosum(乌拉圭品系)以及T. deion(荷兰品系)体内Wolbachia亲缘关系较近,属于B超组Sib亚组,对应MLST序列型为ST486。【结论】安荔赤眼蜂T. oleae为中国野外首次发现,是完全感染Wolbachia的产雌孤雌生殖品系。本研究为害虫生物防治提供了一种新的天敌种类资源,并为进一步探明Wolbachia与赤眼蜂的互作提供了研究材料。     

基于ITS条形码序列的刺桐姬小蜂分子鉴定

【目的】刺桐姬小蜂Quadrastichus erythrinae Kim体型小,传统的形态学鉴定方法难以快速准确识别。【方法】本研究测定了刺桐姬小蜂的rDNA ITS1和ITS2序列,根据18S rDNA部分序列,利用MEGA的最大相似法(Maximum Likehood)构建系统发育树。根据刺桐姬小蜂ITS1和ITS2序列设计了特异引物,应用特异引物对单只刺桐姬小蜂进行PCR扩增,可稳定地扩增出明显的目的DNA条带。【结果】研究表明,基于ITS基因的DNA条形码技术可以用于刺桐姬小蜂的快速准确鉴定。【结论】因此,采用ITS1和ITS2区的特异性引物可对刺桐姬小蜂进行快速分子鉴定。     

米尔顿姬小蜂rDNA ITS1和ITS2的序列分析及其分子鉴定

本研究测定了米尔顿姬小蜂Anselmella miltoni Girault的rDNA ITS1和ITS2序列,以探讨其分子鉴定方法。米尔顿姬小蜂的ITS1和ITS2侧翼区（18S和5.8S）序列相对稳定,ITS1和ITS2序列存在种间差异。根据18S rDNA部分序列,利用DNAMAN的Maximum Likelihood方法构建了与膜翅目其它科的系统发育树。根据米尔顿姬小蜂ITS1和ITS2序列设计了特异性引物,应用特异性引物对样品进行了PCR扩增,扩增效果理想,采用上述特异性引物可从单头米尔顿姬小蜂稳定地扩增出明显的目的DNA条带。因此,可以采用ITS1和ITS2区的特异性对米尔顿姬小蜂进行快速的分子鉴定。     

Host-foraging success of three species of Trichogramma (Hymenoptera: Trichogrammatidae) in a simulated retail environment

Three species of trichogrammatid egg parasitoids (Trichogramma deion Pinto & Oatman, Trichogramma ostriniae Pang & Chen, and Trichogramma pretiosum Riley) (Hymenoptera: Trichogrammatidae) were evaluated under laboratory conditions as potential biological control agents for the Indianmeal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae), on retail shelves. A single shelving unit was used in each trial and a grid of sentinel egg disks was used to evaluate foraging success. The shelving consisted of pallet units with five shelves that were either bare or stocked with empty cereal boxes. In each replicate, approximately 500 female Trichogramma were released at the center of the shelving unit and allowed to forage for 48 h. Percentage of egg parasitism and percentage of host egg mortality were recorded after 7 d. Foraging success as well as the spatial pattern of parasitism differed significantly among the three Trichogramma species. Percentage of egg parasitism was approximately 4 times greater for T. deion than for T. ostriniae or T. pretiosum. The vertical distribution of parasitism by T. deion was also more uniform than for the other two species. In addition, the presence of packaging affected the foraging efficiency of T. ostriniae and T. pretiosum but not T. deion. Based on these findings, Trichogramma deion may be the best-suited candidate for augmentative biological control of P. interpunctella in retail stores, and a central release point of T. deion will likely provide adequate coverage of products on pallet-type shelving.     

M-PCR： a powerful method for rapid molecular identification of Trichogramma wasps （Hymenoptera： Trichogrammatidae）

Two species-specific primers were designed depending on ITS2 sequence variation of 37 Trichogramma wasps, and these primers were applied to establish an assay,multiplex PCR (M-PCR), for molecular diagnosis of two important Trichogramma wasps,T. confusum and T. dendrolimi, in China. Multiplex-PCR results showed that only target species produced two PCR products, one product of ITS2 region species-specific amplification and one product of its ITS 1 region universal amplification, but other species produced only one ITS1 universal PCR product. Using this method, the target Trichogramma species can be distinguished from other Trichogramma species. Molecular identification based on M-PCR has particular value over morphological technology and other approaches, such as normal molecular and biochemical methods. Furthermore, because M-PCR assay can avoid false negative results, which frequently happen in PCR reaction, this method will be much more accurate and useful for Trichogramma identification, and can be developed as an easy and rapid diagnostic kit applied in the identification and quality monitoring of Trichogramma mass products both in the factory and in the field. Such an easy and rapid diagnostic kit will be valuable in the application of Trichogramma species as a biological control.     

Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences.

Laccate polypores of the Ganoderma lucidum species complex are widespread white rot fungi of economic importance, but isolates cannot be identified by traditional taxonomic methods. Parsimony analysis of nucleotide sequences from the internal transcribed spacers (ITS) of the ribosomal gene (rDNA) distinguished six lineages in this species complex. Each ITS lineage may represent one or more putative species. While some isolates have identical ITS sequences, all of them could be clearly differentiated by genetic fingerprinting using random amplified polymorphic DNA (RAPD). To investigate the suitability of RAPD markers for taxonomic identification and grouping of isolates of the G. lucidum complex, RAPD fragments (RAPDs) were used as phenotypic characters in numerical and parsimony analyses. Results show that data from RAPDS do not distinguish the same clades as ITS data do. Groupings based on analysis of RAPD data were very sensitive to the choice of the grouping method used, and no consistent grouping of isolates could be proposed. However, analysis with RAPDs did resolve several robust terminal clades containing putatively conspecific isolates, suggesting that RAPDs might be helpful for systematics at the lower taxonomic levels that are unresolved by ITS sequence data. The limitations of RAPDs for systematics are briefly discussed. The conclusion of this study is that ITS sequences can be used to identify isolates of the G. lucidum complex, whereas RAPDs can be used to differentiate between isolates having identical ITS sequences. The practical implications of these results are briefly illustrated.     

临床常见镰刀菌的鉴别

目的从分子生物学角度寻找一种快速准确鉴定临床常见镰刀菌的方法。方法将受试镰刀菌接种于PDA培养基,观察其菌落及镜下形态,在此基础上PCR扩增受试镰刀菌的rDNA ITS并测其序列,在GenBank核酸序列数据库进行同源序列搜索及分析。选择限制性内切酶Dra Ⅱ和Cfr13 Ⅰ进行RFLP。设计了茄病镰刀菌的种特异性引物Sol1、Sol2,初步验证其特异性。结果形态学鉴定结果显示,茄病镰刀菌所占比例最高,除2株串珠镰刀菌外,其余镰刀菌ITS序列分析的结果与形态学鉴定结果一致。茄病、层生和串珠镰刀菌的Dra Ⅱ、Cfr13 I酶切带形互不相同。用Sol1、Sol2扩增受试菌的rDNA ITS,只有茄病镰刀菌为阳性。结论rDNA ITS序列测定及其PCR-RFLP可用于初步鉴别几种临床常见镰刀菌,合适的种特异性引物可以初步快速鉴定茄病镰刀菌。     

PCR-based strategy to detect and identify species of Phaeoacremonium causing grapevine diseases

Species of Phaeoacremonium (especially Phaeoacremonium aleophilum) are associated with two severe diseases in grapevines, Petri disease in young plants and Esca disease in adult plants. Phaeoacremonium species grow slowly on culture medium, and it is difficult to identify these species on the basis of morphological characteristics. Primers Pm1 and Pm2 were designed in the ribosomal DNA internal transcribed spacer (ITS) regions ITS1 and ITS2, respectively. They yielded a single amplicon of 415 bp for nine species of Phaeoacremonium that may occur in grapevines. A nested PCR (using general fungal primers ITS1F/ITS4 in the primary reaction) was developed to detect Phaeoacremonium directly in grapevine wood. Molecular detection was more sensitive than the traditional method of culturing in growth medium was. Identification of Phaeoacremonium species was achieved by digesting the PCR-amplified fragment with the restriction enzymes BssKI, EcoO109I, and HhaI. It was possible to distinguish these species by their restriction fragment length polymorphism patterns, except for Phaeoacremonium viticola and Phaeoacremonium angustius, which had 100% similarity in their ITS region sequences. A species-specific PCR amplification of the partial beta-tubulin gene using the primer pair Pbr4_1/T1 and Pbr8/T1 was necessary to differentiate P. angustius from P. viticola, respectively. An easy and fast protocol was developed to detect and identify species of Phaeoacremonium in a few hours. Primers defined here can be used in a plant nursery sanitation program to produce plants free of Phaeoacremonium spp. Use of healthy grapevine plants in new plantations is the most effective measure to manage Petri disease.     

A rapid single-step multiplex method for discriminating between Trichogramma (Hymenoptera: Trichogrammatidae) species in Australia

Inaccurate species identification confounds insect ecological studies. Examining aspects of Trichogramma ecology pertinent to the novel insect resistance management strategy for future transgenic cotton, Gossypium hirsutum L., production in the Ord River Irrigation Area (ORIA) of Western Australia required accurate differentiation between morphologically similar Trichogramma species. Established molecular diagnostic methods for Trichogramma identification use species-specific sequence difference in the internal transcribed spacer (ITS)-2 chromosomal region; yet, difficulties arise discerning polymerase chain reaction (PCR) fragments of similar base pair length by gel electrophoresis. This necessitates the restriction enzyme digestion of PCR-amplified ITS-2 fragments to readily differentiate Trichogramma australicum Girault and Trichogramma pretiosum Riley. To overcome the time and expense associated with a two-step diagnostic procedure, we developed a "one-step" multiplex PCR technique using species-specific primers designed to the ITS-2 region. This approach allowed for a high-throughput analysis of samples as part of ongoing ecological studies examining Trichogramma biological control potential in the ORIA where these two species occur in sympatry.     

Potential phylogenetic utility of the low-copy nuclear gene pistillata in dicotyledonous plants: comparison to nrDNA ITS and trnL intron in Sphaerocardamum and other Brassicaceae.

We report the potential phylogenetic utility of DNA sequence data from the last 700 bp of a ca. 1-kb intron of the MADS-box gene pistillata from a sampling of Sphaerocardamum species and other Brassicaceae. These results are compared with nrDNA ITS and the chloroplast trnL intron for the same taxa to demonstrate the potential phylogenetic utility of this pistillata intron and to identify potential historically independent sequences for an ongoing study of relationships within Sphaerocardamum. Analyses of the DNA sequence data for Brassicaceae indicated that pairwise divergences and potentially informative characters were higher in the pistillata intron (0.6-30.8%, 284 characters) and ITS (0-24%, 94 characters) than in the chloroplast trnL intron (0-4.2%, 17 characters). A comparison of Sphaerocardamum sequences identified low divergences and numbers of informative characters for trnL intron (0-2.4%, 1 character) and nrDNA ITS (0-2.5%, 2 characters) and substantially more variation among the pistillata sequences (0.15-3.7%, 19 characters). Phylogenetic analyses of these pistillata sequences fully resolve ingroup relationships without character conflict. Results of pistillata PCR amplifications from a broader dicot sample showed that some primers may be useful in amplifying orthologous pistillata sequences. Ultimately this pistillata intron may be a valuable source of phylogenetic characters at lower taxonomic levels.     

Molecular approaches to identify cryptic species and polymorphic species within a complex community of fig wasps

Cryptic and polymorphic species can complicate traditional taxonomic research and both of these concerns are common in fig wasp communities. Species identification is very difficult, despite great effort and the ecological importance of fig wasps. Herein, we try to identify all chalcidoid wasp species hosted by one species of fig, using both morphological and molecular methods. We compare the efficiency of four different DNA regions and find that ITS2 is highly effective for species identification, while mitochondrial COI and Cytb regions appear less reliable, possibly due to the interference signals from either nuclear copies of mtDNA, i.e. NUMTs, or the effects of Wolbachia infections. The analyses suggest that combining multiple markers is the best choice for inferring species identifications as any one marker may be unsuitable in a given case.     

基于诊断引物的赤眼蜂鉴定和检测技术

通过对松毛虫赤眼蜂Tichogramma dendrolimi Matsumura和玉米螟赤眼蜂T.ostriniae Pang et Chen（膜翅目：赤眼蜂科）核内可转录第二间隔区（简称：ITS2）的克隆、测序,并获取和分析了GenBank中已登录的同源序列,然后设计了松毛虫赤蜂的特异引物以用于该蜂的分子鉴定和检测,经过反复筛选发现：采用鉴定引物通过PCR扩增不仅可以区分鑫头样品,单头样品（雌蜂或雄峰）,而且可鉴定幼期虫和卵,这用传统方法是无法办到的。该鉴定技术比基于形态学鉴定检测技术用来鉴定了从中国大陆不同地域和寄主上采集到的12个样品,结果表明：该方法可用于赤眼蜂田间分子监测和实验室拟寄生行为研究。     

A new cultivation independent approach to detect and monitor common Trichoderma species in soils

A set of primers was developed for the detection, identification and quantification of common Trichoderma species in soil samples. Based on a broad range master alignment primers were derived to amplify an approximate 540 bp fragment comprising the internal transcribed spacer region 1 (ITS 1), 5.8S rDNA and internal transcribed spacer region 2 (ITS 2) from all taxonomic Clades of the genus Trichoderma. The primer set was applied to test strains as well as community DNA isolated from arable and forest soil. For all tested isolates the corresponding internal transcribed spacer regions of Trichoderma spp. strains were amplified, but none of non-Trichoderma origin. PCR with community DNA from soil yielded products of the expected size. Analysis of a clone library established for an arable site showed that all amplified sequences originated exclusively from Trichoderma species mainly being representatives of the Clades Hamatum, Harzianum and Pachybasioides and comprising most of the species known for biocontrol ability. In a realtime PCR approach the primer set uTf/uTr also proved to be a suitable system to quantify DNA of Trichoderma spp. in soils.     

根据核DNA的ITS序列的RFLP分析鉴定稻属CD染色体组物种

稻属物种的染色体组类型有10种,其中具CD染色体组的物种有3种(Oryza alta Swallen,O.grandiglumis(Doell) Prod．和O.latifolia Desv．),仅分布在美洲中部和南部。这3个种在形态上和稻属其他染色体组的种比较容易区别,但它们彼此之间鉴别比较困难。近年来的研究表明,O.alta和O.grandiglumis应归并为1个种(O．gandiglumis),而O．latifolia应保持不变。本文基于代表不同分布区11个样品的核糖体转录间隔区(ITs)的77个克隆序列数据,利用DNA striderl.2软件进行了限制性酶切位点分析,提出了一个鉴别CD染色体组物种的方法。方法的具体步骤是：(1)用通用引物扩增ITS片段;(2)用特异性的限制性内切酶Fok I 和／或Dra III消化PCR扩增产物;(3)用1％的琼脂糖电泳并根据消化产物的多态性特征来鉴别不同物种。基于本文提出的核糖体ITS限制性片段多态性(RLFP)分析,可以快速和可靠地将稻属CD染色体组物种区别开来。     

根据核DNA的ITS序列的RFLP分析鉴定稻属CD染色体组物种

稻属物种的染色体组类型有10种,其中具CD染色体组的物种有3种(Oryza alta Swallen,O.grandiglumis(Doell)Prod.和O.latifolia Desv.),仅分布在美洲中部和南部.这3个种在形态上和稻属其他染色体组的种比较容易区别,但它们彼此之间鉴别比较困难.近年来的研究表明,O.alta和O.grandiglumis应归并为1个种(O.grandiglumis),而O.latifolia应保持不变.本文基于代表不同分布区11个样品的核糖体转录间隔区(ITS)的77个克隆序列数据,利用DNA Striderl.2软件进行了限制性酶切位点分析,提出了一个鉴别CD染色体组物种的方法.方法的具体步骤是:(1)用通用引物扩增ITS片段;(2)用特异性的限制性内切酶Fok Ⅰ和/或Dra Ⅲ消化PCR扩增产物;(3)用1%的琼脂糖电泳并根据消化产物的多态性特征来鉴别不同物种.基于本文提出的核糖体ITS限制性片段多态性(RLFP)分析,可以快速和可靠地将稻属CD染色体组物种区别开来.     

Molecular phylogeny in Indian Tinospora species by DNA based molecular markers

The phylogenetic relationships among the three species of Tinospora found in India are poorly understood. Morphology does not fully help to resolve the phylogeny and therefore a fast approach using molecular analysis was explored. Two molecular approaches viz Random Amplified Polymorphic DNA (RAPD) assay and restriction digestion of ITS1-5.8S-ITS2 rDNA (PCR-RFLP) were used to evaluate the genetic similarities between 40 different accessions belonging to three species. Of the 38 random primers used only six generated the polymorphism, while as three out of 11 restriction enzymes used gave polymorphic restriction patterns. The average proportion of polymorphic markers across primers was 95%, however restriction endonucleases showed 92% polymorphism. RAPD alone was found suitable for the species diversions. In contrast PCR- RFLP showed bias in detecting exact species variation. The correlation between the two markers was performed by Jaccard's coefficient of similarity. A significant (r= 0.574) but not very high correlation was obtained. Further to authenticate the results obtained by two markers, sequence analysis of ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) was performed. Three independent clones of each species T. cordifolia, T. malabarica and T. crispa were sequenced. Phylogenetic relationship inferred from ITS sequences is in agreement with RAPD data.     

PCR-Based Strategy To Detect and Identify Species of Phaeoacremonium Causing Grapevine Diseases

Species of Phaeoacremonium (especially Phaeoacremonium aleophilum) are associated with two severe diseases in grapevines, Petri disease in young plants and Esca disease in adult plants. Phaeoacremonium species grow slowly on culture medium, and it is difficult to identify these species on the basis of morphological characteristics. Primers Pm1 and Pm2 were designed in the ribosomal DNA internal transcribed spacer (ITS) regions ITS1 and ITS2, respectively. They yielded a single amplicon of 415 bp for nine species of Phaeoacremonium that may occur in grapevines. A nested PCR (using general fungal primers ITS1F/ITS4 in the primary reaction) was developed to detect Phaeoacremonium directly in grapevine wood. Molecular detection was more sensitive than the traditional method of culturing in growth medium was. Identification of Phaeoacremonium species was achieved by digesting the PCR-amplified fragment with the restriction enzymes BssKI, EcoO109I, and HhaI. It was possible to distinguish these species by their restriction fragment length polymorphism patterns, except for Phaeoacremonium viticola and Phaeoacremonium angustius, which had 100% similarity in their ITS region sequences. A species-specific PCR amplification of the partial β-tubulin gene using the primer pair Pbr4_1/T1 and Pbr8/T1 was necessary to differentiate P. angustius from P. viticola, respectively. An easy and fast protocol was developed to detect and identify species of Phaeoacremonium in a few hours. Primers defined here can be used in a plant nursery sanitation program to produce plants free of Phaeoacremonium spp. Use of healthy grapevine plants in new plantations is the most effective measure to manage Petri disease.     

High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples

The kingdom Fungi is estimated to include 1.5 million or more species, playing key roles as decomposers, mutualists, and parasites in every biome on the earth. To comprehensively understand the diversity and ecology of this huge kingdom, DNA barcoding targeting the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat has been regarded as a prerequisite procedure. By extensively surveying ITS sequences in public databases, we designed new ITS primers with improved coverage across diverse taxonomic groups of fungi compared to existing primers. An in silico analysis based on public sequence databases indicated that the newly designed primers matched 99% of ascomycete and basidiomycete ITS taxa (species, subspecies or varieties), causing little taxonomic bias toward either fungal group. Two of the newly designed primers could inhibit the amplification of plant sequences and would enable the selective investigation of fungal communities in mycorrhizal associations, soil, and other types of environmental samples. Optimal PCR conditions for the primers were explored in an in vitro investigation. The new primers developed in this study will provide a basis for ecological studies on the diversity and community structures of fungi in the era of massive DNA sequencing.     

Individual and population variation in cercariae of bird schistosomes of the Trichobilharzia ocellata species group as revealed with the polymerase chain reaction

The polymerase chain reaction with arbitrary (RAPD-PCR) or specific primers was used to study the population variation and to identify the species in cercariae of schistosomes of the Trichobilharzia ocellata species group (Trematoda, Schistosomatidae). In total, 28 cercariae were obtained from two spontaneously invaded mollusks Lymnaea stagnalis (LS) and L. ovata (LO), which were collected in different water bodies of Moscow. RAPD-PCR was carried out with two arbitrary primers, OPA9 and OPB11, which each detected different levels of individual and among-group variation and revealed considerable genetic differentiation of cercariae from different host mollusks. To check whether the cercariae of the two samples belong to one species, sequencing was performed with a region corresponding to intergenic transcribed spacer 2 (ITS2), which was earlier proposed for cercaria identification in three European species of bird schistosomes of the genus Trichobilharzia (T. franki, T. regenti, and T. szidati). The ITS2 sequences of two LO cercariae were identical, each consisted of 319 bp, and showed 100% homology to the T. franki ITS2 sequence. The ITS2 sequences of two LS cercariae were identical, each consisted of 323 bp, and showed 99.4% homology to the T. szidati counterpart. The causes of genetic variation in cercariae and prospects of using RAPD markers to study different stages of the life cycle in trematodes are discussed.