烟粉虱B型和Q型群体遗传结构的RAPD分析

近20年来,烟粉虱B型传入世界各地并暴发成灾,成为一种重要的农业入侵害虫; 烟粉虱Q型则是近几年引起人们高度重视的一种新的入侵生物型,目前已传入许多国家并造成一定危害。本文利用RAPD分子标记对烟粉虱B型和Q型不同地理种群的遗传结构进行了分析。结果表明：（1）引物H16对烟粉虱B型不同种群扩增的特异带,能有效区分烟粉虱B型和Q型、浙江非B/Q型种群;（2）烟粉虱Q型种群各项遗传多样性指数均比烟粉虱B型的要高;（3）我国烟粉虱Q型来自伊比利亚半岛的可能性比来自中东地区的可能性要大。另外,聚类分析结果提示,RAPD分子标记能有效地区分烟粉虱不同生物型,但可能不适用于生物型之间亲缘关系分析。     

Distribution patterns of the Q and B biotypes of Bemisia tabaci in the Mediterranean Basin based on microsatellite variation

At least five of the biotypes described in the Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) complex are known to be present in the Mediterranean Basin area. Only two of them, however, are economically relevant, that is, biotypes B and Q. Biological and genetic differences between the two biotypes have been well studied, but less is known about their patterns of genetic variation and population structure. To address these issues, a study was undertaken based on variation at six microsatellite loci among a subset of nine B. tabaci populations (five belonging to the Q and four to the B biotype). The data obtained show that (i) these loci showed considerable polymorphism in the Q and B biotypes populations although the presence of null alleles can obscure the picture; (ii) the Iberian‐Q, Canarian‐Q, and Egyptian‐B populations exhibit heterozygosity excess as a result of bottleneck events; (iii) the low genetic differentiation between the Israeli, Iberian Peninsula, and Italian populations suggest that these populations share a common gene pool; (iv) the genetic distances between the Canarian‐Q population and the geographically close population from Morocco indicates spatial isolation and a limited gene flow; and finally (v) the microsatellite data for the B populations indicate that the whiteflies from Egypt and Israel have a close phylogenetic relationship, but the source of these biotype B invasions into the Mediterranean area remains unknown.     

Rapid spread of tomato yellow leaf curl virus in China is aided differentially by two invasive whiteflies

Background

Tomato yellow leaf curl virus (TYLCV) was introduced into China in 2006, approximately 10 years after the introduction of an invasive whitefly, Bemisia tabaci (Genn.) B biotype. Even so the distribution and prevalence of TYLCV remained limited, and the economic damage was minimal. Following the introduction of Q biotype into China in 2003, the prevalence and spread of TYLCV started to accelerate. This has lead to the hypothesis that the two biotypes might not be equally competent vectors of TYLCV.

Methodology/Principal Findings

The infection frequency of TYLCV in the field-collected B. tabaci populations was investigated, the acquisition and transmission capability of TYLCV by B and Q biotypes were compared under the laboratory conditions. Analysis of B. tabaci populations from 55 field sites revealed the existence of 12 B and 43 Q biotypes across 18 provinces in China. The acquisition and transmission experiments showed that both B and Q biotypes can acquire and transmit the virus, however, Q biotype demonstrated superior acquisition and transmission capability than its B counterparts. Specifically, Q biotype acquired significantly more viral DNA than the B biotype, and reached the maximum viral load in a substantially shorter period of time. Although TYLCV was shown to be transmitted horizontally by both biotypes, Q biotype exhibited significantly higher viral transmission frequency than B biotype. Vertical transmission result, on the other hand, indicated that TYLCV DNA can be detected in eggs and nymphs, but not in pupae and adults of the first generation progeny.

Conclusions/Significance

These combined results suggested that the epidemiology of TYLCV was aided differentially by the two invasive whiteflies (B and Q biotypes) through horizontal but not vertical transmission of the virus. This is consistent with the concomitant eruption of TYLCV in tomato fields following the recent rapid invasion of Q biotype whitefly in China.     

微卫星位点BEM06与BEM23鉴别烟粉虱B型与Q型的有效性

烟粉虱Bemisia tabaci (Gennadius) B型与Q型是烟粉虱复合种中入侵性较强、分布较广的2种生物型, 当前在许多地区混合发生。这2种生物型的快速鉴别对其种群动态调查及入侵生态学研究具有重要价值。为了验证微卫星位点BEM06与BEM23鉴别B型与Q型烟粉虱的有效性, 本研究分析了这2个微卫星位点的等位基因在国内外17个B型、4个Q型、3个非B/Q型烟粉虱种群的分布特点。结果表明: 这2个微卫星位点的联合使用可鉴别B型与Q型烟粉虱, 但是无法有效地将B型、Q型与一些非B/Q型烟粉虱某些个体区分开来。结果提示: 利用微卫星位点BEM06与BEM23鉴别B型与Q型烟粉虱具有一定的局限性, 尤其是田间烟粉虱存在其他生物型时需要慎重使用这种鉴别方法。     

Biotype-dependent secondary symbiont communities in sympatric populations of Bemisia tabaci

The sweet potato whitefly, Bemisia tabaci, harbors Portiera aleyrodidarum, an obligatory symbiotic bacterium, as well as several secondary symbionts including Rickettsia, Hamiltonella, Wolbachia, Arsenophonus, Cardinium and Fritschea, the function of which is unknown. Bemisia tabaci is a species complex composed of numerous biotypes, which may differ from each other both genetically and biologically. Only the B and Q biotypes have been reported from Israel. Secondary symbiont infection frequencies of Israeli laboratory and field populations of B. tabaci from various host plants were determined by PCR, in order to test for correlation between bacterial composition to biotype and host plant. Hamiltonella was detected only in populations of the B biotype, while Wolbachia and Arsenophonus were found only in the Q biotype (33% and 87% infection, respectively). Rickettsia was abundant in both biotypes. Cardinium and Fritschea were not found in any of the populations. No differences in secondary symbionts were found among host plants within the B biotype; but within the Q biotype, all whiteflies collected from sage harboured both Rickettsia and Arsenophonus, an infection frequency which was significantly higher than those found in association with all other host plants. The association found between whitefly biotypes and secondary symbionts suggests a possible contribution of these bacteria to host characteristics such as insecticide resistance, host range, virus transmission and speciation.     

Evidence from molecular markers and population genetic analyses suggests recent invasions of the Western North Pacific region by biotypes B and Q of Bemisia tabaci (Gennadius)

Invasive events by Bemisia tabaci (Gennadius) biotypes in various parts of the world are of continuing interest. The most famous is biotype B that has caused great economic losses globally. In addition, biotype Q has also recently been reported to be a new invasive pest. These two biotypes have been monitored for some time in the Western North Pacific region, but the invasive events and population genetic structures of these two biotypes are still not clear in this region. In this study, the mitochondrial cytochrome oxidase I (COI) gene was used to reconstruct a phylogenetic tree for identifying biotypes B and Q and to study the relationships between invasive events and ornamental plants. Population genetic analyses of mtCOI sequences were also used to study the genetic relationships within and between populations. A combination of a phylogenetic tree and haplotype analysis suggested the recent invasion of biotype Q in this region is related to the international ornamental trade from the Mediterranean region. Low levels of haplotype diversity and nucleotide diversity indicate that the presence of biotypes B and Q in the Western North Pacific region are caused by multiple invasions. Hierarchical analysis of molecular variance supports the hypothesis of multiple invasions. In addition, high sequence identities and low genetic distances within and between populations of the two biotypes revealed that these invasive events occurred recently. The low levels of genetic differentiation revealed by pairwise F (ST) values between populations also suggests the invasions were recent. Therefore, results of this study suggested that biotypes B and Q entered this region through multiple recent invasions. A quarantine of agricultural crops may be necessary to prevent further invasions.     

Intra- and interspecific competition between biotypes B and Q of Bemisia tabaci (Hemiptera: Aleyrodidae) from Spain

Biotypes B and Q of Bemisia tabaci (Gennadius), collected from the islands of Tenerife and Majorca respectively, were exposed to competition conditions on tomato cv. Marmande in the laboratory. Both biotypes were established in single and mixed cultures at different densities. Increased mortality of biotype Q females and immature instars was observed together with a lower rate of fecundity and progeny size compared to biotype B, when reared in single or mixed cultures. The female:male sex ratio of F1 individuals of biotype Q was higher in single than in mixed cultures. However, the sex ratio of F1 individuals of biotype B was the same in single and mixed cultures, suggesting reproductive interference. Whitefly density did not affect interspecific interactions. It had a moderate effect on developmental rate of both biotypes, and on mortality of immature instars and progeny size of biotype B only. The results indicate that under laboratory conditions the studied biotype B should displace biotype Q.     

烟粉虱B型与Q型种群遗传多样性的AFLP分析

烟粉虱Bemisia tabaci(Gennadius)是由多种生物型或物种组成的复合种。Q型烟粉虱在我国部分地区正在取代B型烟粉虱成为优势生物型。烟粉虱Q型与B型遗传多样性比较研究为解析这2种生物型入侵的遗传学基础具有重要的意义。本文利用AFLP技术研究了Q型(包括Q1型、Q2型)、B型烟粉虱种群的遗传多样性。结果表明:Q型烟粉虱遗传多样性高于B型烟粉虱;Q1型烟粉虱各项遗传多样性指数均接近于Q2型。最后探讨了AFLP与SSR分子标记的各自特点。     

三种抗生素对B型和Q型烟粉虱内共生菌的去除效果比较研究

利用烟粉虱Bemisia tabaci(Gennadius)内共生菌特异性引物,研究了内共生菌在B、Q型烟粉虱种群中的分布和感染率,同时评价了3种不同的抗生素利福平、氨苄青霉素和硫酸卡那霉素分别在3种不同的浓度下(100.0、50.0及25.0μg/mL)对烟粉虱内共生菌的去除效果。结果表明:B、Q型烟粉虱原生内共生细菌Portiera的带菌率均为100.0%;B、Q型烟粉虱次生内共生菌Hamiltonella的带菌率分别为91.7%和100.0%;B型烟粉虱次生内共生菌Rickettsia的带菌率为87.5%,Q型为0;其它次生内共生菌在B、Q型烟粉虱中均未检测到。利福平、氨苄青霉素和硫酸卡那霉素在3种不同的浓度下均不能去除B、Q型烟粉虱Portiera;利福平、氨苄青霉素在3种不同的浓度下均能完全去除B型烟粉虱Rickettsia,硫酸卡那霉素在不同浓度下去除Rickettsia的效果不同;3种抗生素去除Hamiltonella的能力受抗生素种类以及浓度的影响。同一抗生素在不同浓度下去除Hamiltonella的效果均是100.0μg/mL>50.0μg/mL>25.0μg/mL;不同浓度的抗生素去除Hamiltonella的效果均是利福平>氨苄青霉素>硫酸卡那霉素,各浓度与各抗生素之间的去除Hamiltonella的效果均具有显著性差异。     

Further insights into the strange role of bacterial endosymbionts in whitefly, Bemisia tabaci: comparison of secondary symbionts from biotypes B and Q in China

The percentage infection of secondary symbionts (SS) (Wolbachia, Arsenophonus, Rickettsia, Hamiltonella, Fritschea and Cardinium) in the exotic Bemisia tabaci (Genn.) invaders, commonly known as biotypes B and Q from China, were determined by PCR. In total, 373 biotype B and 1830 biotype Q individuals were screened for the presence of SS. Biotype B was more abundant than biotype Q from 2005 to 2006, and biotype Q was more abundant from 2007 to 2009. Each of the SS, with the exception of Fritschea, was detected in both biotypes B and Q; Fritschea was found in none of the samples examined. For biotype B, the percentage infection of Hamiltonella was the highest (92.0%) followed by Rickettsia (70.2%). For biotype Q, the percentage infection of Hamiltonella was again the highest (73.3%). Arsenophonus was the least common of the SS observed in both biotypes B and Q. The percentage infection of Wolbachia, Rickettsia and Hamiltonella in biotype B was each significantly higher than in biotype Q, whereas the percentage infection of Cardinium in biotype B was significantly lower than in biotype Q. The percentage infection of SS in biotypes B and Q varied from year to year over the period 2005-2009. Furthermore, within biotype Q, two distinct subgroups were identified which differ from each other in terms of their SS complement. We discuss these results in the light of the potentially influential factors and roles of the SS.     

Further spread of and domination by Bemisia tabaci (Hemiptera: Aleyrodidae) biotype Q on field crops in China

The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), causes severe crop losses to many crops. The worst of these losses are often associated with the invasion and establishment of biotypes B and Q of this pest. Previous research in 2007 showed that biotype Q occurred with other biotypes in most field populations in China. To determine the current status of the biotype composition in the field, an extensive survey covering mainly eastern parts of China was conducted in 2009. Using polymerase chain reaction primers specific for the mitochondrial cytochrome oxidase I of biotypes B and Q and gene sequencing, we determined the biotypes composition in 61 whitefly populations and their distribution across 19 provinces in China. Our research revealed that only biotypes B and Q have been found in the field in 2009 in China. Among them, biotype Q was dominant in 44 locations (100.0%) and biotype B was dominant in 17 locations (100.0%). The current survey indicates that biotype Q has rapidly displaced biotype B in most locations in China.     

中国部分地区烟粉虱生物型种类及系统发育关系分析

烟粉虱是一种危害严重的世界性害虫,是一个快速进化的复合种.本文利用mtCOI分子标记方法,对2010和2011年采自我国9个省(市)的33个烟粉虱种群进行了生物型鉴定和系统发育分析.结果表明： 我国目前存在着B型、Q型、ZHJ-1型、ZHJ-3型、An型以及Nauru型等6种生物型,且不同生物型的分布是不均匀的.遗传距离及系统发育树的分析结果显示,海南省的An型和台湾省的An型聚为一支,为同一来源;中国B型与来自法国和乌干达的B型的亲缘关系较近,同源性达到99%以上;中国的Q型与来自摩洛哥和法国的Q型聚为一个分支,而来自以色列和土耳其的Q型烟粉虱单独聚为一支,说明中国的Q型烟粉虱与来自地中海西部的Q型烟粉虱亲缘关系更近,可以推断中国的Q型烟粉虱的起源地为地中海西部地区.
     

烟粉虱生物型对浅黄恩蚜小蜂寄主选择及个体发育的影响

为探讨寄生蜂在Q型烟粉虱Bemisia tabaci替代B型烟粉虱的过程中是否起作用, 我们在实验室条件(温度27±1℃, 光周期16L∶8D, 相对湿度RH 70%～80%)下, 观察了浅黄恩蚜小蜂Encarsia sophia寄生B型和Q型烟粉虱若虫的行为, 研究了浅黄恩蚜小蜂对B型和Q型烟粉虱若虫的选择性、 烟粉虱生物型对浅黄恩蚜小蜂取食数量及个体发育的影响。结果发现, 浅黄恩蚜小蜂体外检测时间在B型和Q型烟粉虱若虫间差异不显著, 而寄生Q型烟粉虱若虫时的体内检测和产卵时间(190.2±14.6 s）显著高于寄生B型时所用时间(140.0±7.5 s)。在非选择条件下, 浅黄恩蚜小蜂寄生B型烟粉虱若虫的数量(8.1±0.5头)及总产卵量（9.3±0.6粒）显著高于仅提供Q型烟粉虱的寄生数量(6.3±0.5头)及总产卵量(7.0±0.6粒); 而被寄生若虫单头着卵量在处理间差异不显著。在选择性条件下, 该蜂寄生B型烟粉虱若虫量(3.1±0.4头)、总产卵量(3.8±0.5粒)及被寄生若虫单头着卵量(1.2±0.1粒)都显著高于寄生Q型烟粉虱时的情况(1.8±0.3头、1.8±0.4粒、0.7±0.1粒)。被寄生蜂取食的B型与Q型烟粉虱数量间差异不显著, 但对于同一生物型而言, 交配过的雌蜂能够取食更多的烟粉虱若虫。以B型烟粉虱为寄主时, 浅黄恩蚜小蜂雌蜂卵-黑蛹(7.2±0.1 d)、黑蛹-羽化(5.2±0.1 d)的发育时间与以Q型烟粉虱若虫为寄主时的相应发育时间(7.3±0.1 d, 5.6±0.1 d)间无显著性差异。以B型烟粉虱为寄主时寄生蜂的羽化率（73.55%±1.42%）与以Q型烟粉虱为寄主时的羽化率（68.42%±13.01%）间差异不显著。这些结果表明, 虽然浅黄恩蚜小蜂发育时间、 羽化率在烟粉虱2种生物型间无显著差异, 但该小蜂倾向于B型烟粉虱若虫作为寄主, 而且, 以B型烟粉虱若虫为寄主时, 小蜂的产卵量和寄生若虫数量均增加。但田间浅黄恩蚜小蜂的存在是否有助于Q型烟粉虱成为B型和Q型混合种群的优势种群, 还需进一步研究。     

Identification of biotypes and secondary endosymbionts of Bemisia tabaci in Korea and relationships with the occurrence of TYLCV disease

Bemisia tabaci is a species complex that consists of at least 24 genetically diverse biotypes. Here, we determined the biotypes of 27 populations collected in 17 different regions of Korea. Nucleotide sequence comparisons of cytochrome oxidase showed that 26 populations were Q biotype and that one population, the Goyang population, was B biotype. Further subgroup analysis of the Q biotype showed that all populations belonged to the Q1 subgroup, which originates from Western Mediterranean countries. Five endosymbiotic bacteria from various B. tabaci populations were analyzed by comparing rDNA sequences. Hamiltonella was detected in all the populations tested regardless of biotype. Cardinium was detected in all Q biotype populations but not in the B biotype population, while Rickettsia was detected in the B biotype population but not in Q biotype populations. Arsenophonus and Wolbachia were detected in 35% and 58% of Q biotype populations, respectively, but not in the B biotype population. Our results show that the endosymbiont profile is strongly associated with each biotype and with subgroups of the Q biotype. Survey of TYLCV disease from 2008 to 2010 indicated that this disease is widely spread in Korea. This study suggests that the rapid spread of TYLCV may be associated with endosymbiont infection, particularly Hamiltonella infection of B. tabaci.     

烟粉虱生物型的监测及其遗传结构研究

烟粉虱Bemisia tabaci(Gennadius)是一种重要的农业害虫并包括许多生物型,其中B型和Q型是入侵性较强的2种生物型。文章着重介绍近年来在烟粉虱生物型的监测及其遗传结构方面的研究进展。B型烟粉虱和Q型烟粉虱这2个生物型均已入侵我国,其中多数地区烟粉虱是B型烟粉虱,局部地区有Q型烟粉虱并呈现不断蔓延趋势。微卫星(SSR)分子标记分析结果表明我国B型烟粉虱的入侵来源具有多元化,而云南地区Q型烟粉虱来源比较单一。化学农药的使用能够影响室内种群的遗传结构,降低种群的遗传多样性。基于RAPD、ISSR分子标记的分析结果表明,Q型烟粉虱种群各项遗传多样性指数均比B型烟粉虱的高。今后加强烟粉虱入侵生物型的遗传结构及其种群动态关系等方面的研究,对于揭示烟粉虱的灾变机制及其控制具有重要的意义。     

Biotype status and genetic polymorphism of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) in Greece: mitochondrial DNA and microsatellites

The genetic polymorphism and the biotype identity of the tobacco whitefly Bemisia tabaci (Gennadius) have been studied in population samples taken from different localities within Greece from cultivated plants growing in greenhouses or in open environments and from non-cultivated plants. Two different approaches were used: sequencing of the mitochondrial cytochrome oxidase I (mtCOI) gene and genotyping using microsatellite markers. Analyses of the mtCOI sequences revealed a high homogeneity between the Greek samples which clustered together with Q biotype samples that had been collected from other countries. When genetic polymorphism was examined using six microsatellite markers, the Greek samples, which were all characterized as Q biotype were significantly differentiated from each other and clustered into at least two distinct genetic populations. Moreover, based on the fixed differences revealed by the mtCOI comparison of known B. tabaci biotype sequences, two diagnostic tests for discriminating between Q and B and non-Q/non-B biotypes were developed. Implementation of these diagnostic tools allowed an absence of the B biotype and presence of the Q biotype in the Greek samples to be determined.     

Genetic relationships among biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae) based on AFLP analysis

Genetic similarities between 13 samples belonging to nine reference biotypes and two field populations of Bemisia tabaci (Gennadius), one field population of B. medinae Gómez-Menor and another of B. afer Priesner & Hosny, were evaluated using amplified fragment length polymorphism (AFLP) markers. The results indicate that B. tabaci biotypes can be grouped together with a minimum similarity coefficient of 0.32 and separated from the two other species with a similarity coefficient of 0.07. Bemisia tabaci biotypes were grouped in four clusters which comprised: (i) Near East and Indian subcontinent biotypes; (ii) B and Q biotypes plus a Nigerian population from cowpea; (iii) New World A biotype; and (iv) S biotype and a Nigerian population from cassava. These results were consistent with a previous grouping of biotypes based on RAPD-PCR analysis. The AFLP assay allowed the scoring of a total of 354 polymorphic bands in two reaction events with the use of two primer combinations.     

DNA markers for identifying biotypes B and Q of Bemisia tabaci (Hemiptera: Aleyrodidae) and studying population dynamics

The two most widespread biotypes of Bemisia tabaci (Gennadius) in southern Europe and the Middle East are referred to as the B and Q-type, which are morphologically indistinguishable. In this study various DNA markers have been developed, applied and compared for studying genetic diversity and distribution of the two biotypes. For developing sequence characterized amplified regions (SCAR) and cleaved amplified polymorphic sequences (CAPS) techniques, single random amplified polymorphic DNA (RAPD) fragments of B and Q biotypes, respectively, were used. The CAPS were investigated on the basis of nuclear sodium channel and the mitochondrial cytochrome oxidase I genes (mtCOI) sequences. In general, complete agreement was found between the different markers used. Analysis of field samples collected in Israel for several years, using these markers, indicated that the percentage of the Q biotype tends to increase in field populations as time progresses. This may be attributed to the resistance of the Q biotype to neonicotinoids and pyriproxyfen and the susceptibility of the B biotype to these insecticides.     

Analysis of genetic diversity among different geographical populations and determination of biotypes of Bemisia tabaci in China

Abstract:  Analysis of the genetic diversity among 27 different geographical populations of Bemisia tabaci and determination of biotypes of B. tabaci in China based on amplified fragment-length polymorphism (AFLP) and the mitochondrial cytochrome oxidase I (mtDNA COI) gene sequences were conducted. In AFLP assay, the use of five primer combinations selected from 64 primer combinations allowed the identification of 229 polymorphic bands (97.03%) from 60 to 500 bp, suggesting abundant genetic diversity among different geographical populations of B. tabaci. To further identify biotypes of B. tabaci in China, the mtDNA COI gene sequences of nine representative populations from China, Israel and Spain were obtained. Molecular phylogenetic tree based on AFLP and mtDNA COI gene analyses revealed the presence, in China, of at least four different genetic groups of B. tabaci. B biotype, Q biotype and two non-B/Q biotype. B biotype was distributed nationwide. Q biotype was present only in the local region of China including the YunNan province and BeiJing city. This was also the first report about the invasion of Q biotype into China. Of the other two non-B/Q biotype groups, one was found in ShanDong and HeBei provinces, and another in ZheJiang province. The non-B/Q biotype ZheJiang population showed very high similarity with another Asian population India-IW ( AF110704 ) in mtDNA COI sequences and was possibly a Chinese indigenous population. The close monitoring of the Q biotype in locales of China where commercial plants were exported or imported, is now essential to avoid the further accidental distribution of the Q biotype.     

Influence of plant combinations on population characteristics of Bemisia tabaci biotypes B and Q

In recent years, the dominant biotype of Bemisia tabaci (Gennadius) has shifted from biotype B to Q in many locations of China. Our field monitoring showed that B. tabaci biotype Q could be found on pepper Capsicum frutescens L. while biotype B could not be found on the plant. To study the role of the host plant in the displacement of biotype B by Q, the adult mortality, female fecundity, and adult emergence of both biotypes B and Q on different host combinations were studied using a model system involving pepper and eggplant. The results showed that pepper is not a preferred host for either biotype B or Q adults in comparison with eggplant. The negative impact of pepper to biotype B is, however, much greater than that to biotype Q. Because both the survival rates of the adult whitefly and the average number of emerged adults per female of biotype Q on pepper are higher than that of biotype B, biotype Q showed higher fitness in comparison with biotype B. Our results suggest that the existence and differential impact of a nonpreferred host might affect the population fitness and therefore could play a role in the displacement of biotype B in some regions.