Criniviruses associated with cucurbit yellows disease in Greece and Cyprus: an ever-changing scene

Cucurbit yellow stunting disorder virus (CYSDV), Cucurbit chlorotic yellows virus (CCYV) and Beet pseudo-yellows virus (BPYV) are whitefly-transmitted criniviruses that cause foliar interveinal yellowing symptoms and result in high economic losses for cucurbit production. CYSDV and CCYV are transmitted by Bemisia tabaci, whereas BPYV is transmitted by Trialeurodes vaporariorum. During 2012–2017, an extensive survey was conducted to identify the viruses causing cucurbit yellows disease in Greece and Cyprus. The study sampled the main cucurbits and alternative hosts in these regions to determine crinivirus incidence, to identify the whitefly species present in the two countries and to characterize molecularly the virus populations. Results showed that CYSDV was the most widespread virus in Greece (49.9%), followed by CCYV (20.3%) and BPYV (18.4%). Bemisia tabaci and T. vaporariorum were identified in 54.5% and 45.6% of whitefly samples, respectively. In Cyprus, CYSDV was predominant (96.7%), followed by CCYV (19.2%), while no BPYV infection was detected. Approximately 15% of weed samples from 17 different species that belong to 12 botanical families were identified as hosts for one or more of these criniviruses. Finally, sequencing of the capsid protein gene of the crinivirus isolates revealed very low levels of genetic diversity, further supporting the genetic stability of crinivirus populations. The results of this long-lasting epidemiological study in two countries of the eastern Mediterranean revealed substantial changes in the relative incidence and distribution of cucurbit-infecting criniviruses and their whitefly vectors over the past 15 years, suggesting the need for adoption of novel management strategies.     

繁育寄主植物对浅黄恩蚜小蜂生防潜能的影响

为明确以雪莲果Smallanthus sonchifolius为寄主植物繁育的浅黄恩蚜小蜂Encarsia sophia的生防潜能,测定雪莲果繁育的浅黄恩蚜小蜂个体大小以及其对烟粉虱Bemisia tabaci MED隐种和温室白粉虱Trialeurodes vaporariorum的致死能力,并解析其寄生2种粉虱若虫后的子代发育情况。结果表明,雪莲果繁育的浅黄恩蚜小蜂雌雄蜂体长、头宽及后足胫节长度均显著高于番茄繁育的浅黄恩蚜小蜂。雪莲果繁育的浅黄恩蚜小蜂对烟粉虱和温室白粉虱的平均致死数量分别为24.7头和25.0头,显著高于番茄繁育的21.4头和21.0头。相对于番茄,雪莲果繁育的浅黄恩蚜小蜂寄生烟粉虱和温室白粉虱若虫后其子代发育时间更短,平均分别为13.2 d和12.5 d;而且子代羽化率也显著高于番茄繁育的子代羽化率,分别为84.1%和86.9%。表明与番茄相比,雪莲果为寄主植物繁育的浅黄恩蚜小蜂对烟粉虱和温室白粉虱具有更强的生防潜能。     

适度饥饿浅黄恩蚜小蜂对烟粉虱和温室白粉虱的寄生和取食选择

为评价释放前经历饥饿对浅黄恩蚜小蜂Encarsia sophia(GiraultDodd)寄生取食粉虱能力的影响,以3龄Q隐种烟粉虱Bemisia tabaci Q和温室白粉虱Trialeurodes vaporariorum若虫为寄主,在2种粉虱单独或同时存在的情况下,比较释放前经适度饥饿、初羽化未饥饿和初羽化喂饲蜂蜜水3种处理的浅黄恩蚜小蜂对2种粉虱寄主的寄生和取食选择情况。结果表明,在2种粉虱单独存在时,经适度饥饿6 h的浅黄恩蚜小蜂寄生的烟粉虱和温室白粉虱显著多于其它处理,而且能取食更多的温室白粉虱,经适度饥饿的寄生蜂在24 h内通过寄生和取食杀死烟粉虱和温室白粉虱的总量分别为12.5头和12.9头。在2种粉虱同时存在时,适度饥饿寄生蜂取食2种粉虱的总量明显高于其它处理,但不同处理间无显著差异,适度饥饿寄生蜂通过寄生和取食杀死2种粉虱的数量最多为11.5头,显著高于未饥饿处理的6.5头。表明释放前经历适度饥饿可以明显提高浅黄恩蚜小蜂寄生和取食粉虱若虫的能力。     

A selective barrier in the midgut epithelial cell membrane of the nonvector whitefly <Emphasis Type="Italic">Trialeurodes vaporariorum</Emphasis> to <Emphasis Type="Italic">Tomato yellow leaf curl virus</Emphasis> uptake

We studied the presence of a potential transmission barrier that blocks Tomato yellow leaf curl virus in the nonvector greenhouse whitefly, Trialeurodes vaporariorum. Because T. vaporariorum can ingest and retain the virus after acquisition feeding on an infected plant, comparable to the vector whitefly Bemisia tabaci, circumstance evidence suggested that a transmission barrier presents at location(s) where the virus moves from the digestive tract lumen to the hemolymph. To provide direct evidence for the site of a transmission barrier in the nonvector insect, we compared the accumulation levels and localization of the virus between the two species of whiteflies. Quantitative real-time and conventional PCR analysis showed that accumulation of the virus during acquisition feeding and retention after a short acquisition period were indistinguishable between the two species, but the circulation of the virus within the whiteflies differed significantly between the species. In an immunofluorescence analysis using an antibody specific to the coat protein of the virus, the virus was restricted to the luminal surface of the midgut epithelial cells and did not enter their cytoplasm or that of the salivary glands in T. vaporariorum. In contrast, the virus was localized within the cytoplasm of the midgut epithelial cells and in the paired salivary glands of B. tabaci adults. This direct evidence shows that a selective transmission barrier at the luminal membrane surface of midgut epithelial cells in the nonvector whitefly blocks entrance of the virus into the midgut epithelial cells, resulting in incompetence as a vector of the virus.     

The transmission of geminiviruses by Bemisia tabaci

The cosmopolitan whitefly species, Bemisia tabaci (Gennadius) and Trialeurodes taporariorum (Westwood) have always been regarded as pests to a large range of worldwide crops. Both species are capable of transmitting plant viruses, with T. vaporariorum being the vector of only a few ‘clostero’-like viruses and B. tabaci the vector of viruses in several groups. The largest group of viruses transmitted by B. tabaci are the geminiviruses and B. tabaci is known to transmit around 60 members. Until recently, B. tabaci had been associated with only a limited range of host plants within any one region, although its total potential host range was large. Virus transmission was confined within the plant host range of each regional population of B. tabaci. The emergence of the polyphagous ‘B’ biotype of B. tabaci with its increased host range of more than 600 plant species, has resulted in geminiviruses infecting previously unaffected crops. As the ‘B’ biotype spreads further into Europe, European field and glasshouse crops have been shown to be susceptible to whitefly-transmitted viruses already endemic to other parts of the world. More than 20 colonies of B. tabaci, including both ‘B’ and non-‘B’ biotypes from disparate global locations have been compared for their ability to transmit more than 20 geminiviruses. All but two highly host-specific colonies were capable of transmitting most geminiviruses tested. However, some viruses were transmitted more efficiently than others. The virus coat protein or capsid is essential for vector recognition and transmission. By comparing transmissible viruses at the molecular level to viruses that are no longer whitefly-transmissible, the active epitope on the virus coat protein could be identified for designing future virus control strategies.     

Situation of the whiteflies Bemisia tabaci and Trialeurodes vaporariorum in protected tomato crops in Algarve (Portugal)*

Protected tomato is the most important horticultural crop in the Algarve (south of Portugal). However, the growing area has decreased by almost 48% since 1995, mainly as a result of the pests Bemisia tabaci and Trialeurodes vaporariorum and the epidemics of Tomato yellow leaf curl virus (TYLCV), a Begomovirus transmitted by B. tabaci. Both whiteflies are vectors of economically important viruses. Recently, Tomato chlorosis virus (ToCV), a member of the genus Crinivirus, transmitted by both B. tabaci and T. vaporariorum, was reported infecting tomato crops in Algarve. A study was carried out to evaluate the dynamics of whitefly populations on tomato crops in Algarve. Population counts of B. tabaci were high in the first months of autumn, then decreased until January, when numbers of T. vaporariorum became higher. Counts of B. tabaci then increased again.     

<Emphasis Type="Italic">Bemisia tabaci</Emphasis> Biotype Q is present in Costa Rica

Whiteflies are an insect group that comprises multiple species and biotypes, capable of affecting crops by phloem feeding, virus transmission and promotion of fungal colonization. The distribution of these pests is worldwide. In Costa Rica, a country located in the tropics, the most problematic whiteflies are Bemisia tabaci biotype B and Trialeurodes vaporariorum. In September 2009, two greenhouses in the Alfaro Ruiz region, northwest of the country’s capital, San Jose, were surveyed as part of a larger effort to determine the occurrence of species and races of whiteflies in this agronomically important region. In addition, the insect samples were analyzed to determine the presence of Tomato chlorosis virus (ToCV), a yield-affecting crinivirus transmitted by whiteflies. The results revealed the presence of the Q biotype of B. tabaci, and important invasive species, as well as the expected T. vaporariorum. Viral detection assays identified potentially viruliferous individuals for Tomato chlorosis virus. These results identified a new pest capable of harbouring plant viruses has been identified, as well as a viral agent (ToCV) in a region where it was not reported, and which might cause significant yield losses.     

新疆两种粉虱的酯酶和过氧化物酶同工酶的比较研究

应用电泳分析方法 ,对乌鲁木齐地区所发生的温室白粉虱和烟粉虱进行了酯酶同工酶、过氧化物同工酶的比较研究。结果表明 :两种粉虱的酶谱有明显的种间差异。温室白粉虱的酯酶酶谱可分 2组区带 ;过氧化物酶酶谱可分 5组区带。烟粉虱的酯酶酶谱可分 5组区带 ;过氧化物酶酶谱可分 4组区带     

Host Range Studies for Tomato chlorosis virus, and Cucumber vein yellowing virus Transmitted by Bemisia tabaci (Gennadius)

The Bemisia tabaci (Gennadius) biotype B transmitted host range of Tomato chlorosis virus (ToCV), genus Crinivirus, Family Closteroviridae, and Cucumber vein yellowing virus (CVYV), genus Ipomovirus, Family Potyviridae, was studied. New experimental hosts were identified for each of these viruses. Seventeen species in eight plant families were assessed as potential hosts for ToCV. Infection in asymptomatic Anthriscus cereifolium (chervil) test plants by ToCV was confirmed by using a Real-Time PCR assay designed for ToCV. The presence of readily transmissible, infectious ToCV virions in A. cereifolium was confirmed by re-isolation of the virus via whitefly-transmission from A. cereifolium to Lycopersicon esculentum and A. cereifolium. This is the first report of the experimental transmission of ToCV by B. tabaci to a species within the Umbelliferae. All other hosts assessed for the presence of ToCV were found to be uninfected. Ten species in five families were assessed as potential hosts for CVYV. The CVYV host range identified included some important crops and common weeds, such as L. esculentum, Nicotiana tabacum, A. cereifolium, Datura stramonium, Nicotiana benthamiana, Nicotiana clevlandii and Cucumis sativus. Symptoms were present on D. stramonium, N. benthamiana and C. sativus control plants. The presence of infectious whitefly transmitted CVYV virions was confirmed solely for D. stramonium and N. tabacum, following re-isolation of the virus via B. tabaci transmission from all infected species to C. sativus. This is the␣first report of experimental CVYV transmission by B. tabaci to non-cucurbitaceous crop and weed hosts belonging to the Solanaceae or Umbelliferae.     

Differentiation of three whitefly-transmitted geminiviruses from the Republic of Yemen

Three viruses collected in southern Yemen in 1990, infecting watermelon, tobacco and tomato were shown to be transmitted by the whiteflyBemisia tabaci and to have particle morphologies typical of geminiviruses. Colonies ofB. tabaci collected from different locations and from different hosts were used in virus transmission tests with the same host range of plants. Colonies established from both watermelon and cotton in the Yemen were identified as the squash silverleaf-inducing B biotype. The culture host of the colony did not influence virus acquisition and transmission efficiencies to and from other hosts. The tobacco and tomato geminiviruses had a similar host range, but differed in their severity in some hosts. Both these viruses differed from the watermelon geminivirus in host range and symptoms.Datura stramonium, an alternative host for all three viruses, could be co-infected by the watermelon and tobacco viruses.B. tabaci was able to acquire both viruses from the co-infectedD. stramonium and infect seedlings of either original host plant species with their respective viruses orD. stramonium with both. The viruses were identified as watermelon chlorotic stunt virus, tobacco leaf curl virus and tomato yellow leaf curl virus and were distinguished by cross hybridisation.     

两种蚜小蜂对烟粉虱MED隐种的田间笼罩控效评价

为明确烟粉虱Bemisia tabaci MED隐种优势寄生蜂海氏桨角蚜小蜂Eretmocerus hayati ZolnerowichRose与浅黄恩蚜小蜂Encarsia sophia GiraultDodd对其控制效果的影响,在棉田尼龙纱网笼罩中释放烟粉虱之后,再分别单独释放海氏桨角蚜小蜂、浅黄恩蚜小蜂以及二者以不同比例组合(1∶1、1∶3、3∶1)释放,定期调查统计2种蚜小蜂对烟粉虱的寄生量和烟粉虱的种群动态。结果表明,相对于不放蜂对照,自首次放蜂后40 d开始,所有放蜂处理均能显著降低烟粉虱若虫种群密度,每100 cm~2叶片上均少于1.00头,但各处理间的烟粉虱种群密度无显著差异;海氏桨角蚜小蜂和浅黄恩蚜小蜂以3∶1比例组合释放的处理中对烟粉虱的寄生量最高,每100 cm~2棉花叶片上能达到4.25头。表明在棉田中对烟粉虱进行生物防治时,以初级寄生蜂海氏桨角蚜小蜂与复寄生蜂浅黄恩蚜小蜂为3∶1的比例释放,可以到达较好的控制效果。     

PCR-RFLP identification ofBemisia tabaci biotypes in the Mediterranean Basin

At least 20 genetic biotypes, with varying degrees of biological characterization, are currently recognized within theBemisia tabaci (Hemiptera: Aleyrodidae) species complex. Their identification relies on a set of different molecular techniques. However, none of the available markers is completely adequate, due to technical difficulties or lack of reproducibility. We therefore developed a method for rapid biotyping ofB. tabaci populations. The five biotypes (B, Q, M, S and T) reported until now in the Mediterranean Basin have been tested by PCR amplification of the cytochrome oxidase I mitochondrial gene followed by restriction with the enzymeTru9I. The digestion patterns produced by this enzyme were able to identify the five biotypes clearly. Digestion with another enzyme,TaqI, discriminated only between biotypes B and Q. The newly developed method enables rapid biotyping and can be applied in studies aimed at assessing biotype distribution and competition at least in the Mediterranean area. http://www.phytoparasitica.org May 14, 2006.     

Tomato yellow leaf curl virus accumulates in vesicle-like structures in descending and ascending midgut epithelial cells of the vector whitefly, Bemisia tabaci, but not in those of nonvector whitefly Trialeurodes vaporariorum

Begomoviruses are transmitted by a single species of vector insect, the whitefly Bemisia tabaci, in a circulative manner. However, the mechanisms of this strict vector specificity have not been clarified. By immunoelectron microscopy, we showed that a begomovirus, Tomato yellow leaf curl virus (TYLCV), can enter midgut epithelial cells of the vector whitefly B. tabaci but not those of a nonvector whitefly, Trialeurodes vaporariorum, belonging to the same family. In midgut epithelial cells of viruliferous B. tabaci, the virus was localized in vesicle-like structures, suggesting endocytosis as an entry mechanism. These structures were also observed in midgut cells of nonviruliferous B. tabaci that had fed on healthy plants and in those of the nonvector T. vaporariorum that had fed on virus-infected plants. Vesicles containing TYLCV particles were observed most frequently in cells in the anterior part of the descending midgut, suggesting that this is the major entry site. These results clearly demonstrated that the virus-containing vector and nonvector whiteflies differ in the cellular localization of the virus and strongly suggest that a critical step in determining the vector insect specificity of begomoviruses is the entry of the viruses into midgut epithelial cells.     

江苏地区Q型烟粉虱的单倍型分析

为明确江苏地区Q型烟粉虱的遗传多样性及其入侵来源,基于mt DNA COI基因序列,对2010、2011年采自江苏13个市的Q型烟粉虱种群进行了单倍型分析。结果显示,江苏地区Q型烟粉虱有4个单倍型,分别为单倍型Q1、Q2、Q3、Q4,不同单倍型的分布和发生频率不同,其中单倍型Q2是13个地理种群的共享单倍型,2010、2011年的发生频率均超过50%;单倍型Q1和Q3分别是部分地理种群的共享单倍型,发生频率较低;单倍型Q4仅在扬州种群中出现,发生频率最低;单倍型Q1和Q3亲缘关系较近,单倍型Q2和Q4亲缘关系较近,表明各地理种群间既有一定的基因交流,也存在一定程度的遗传分化;系统发育分析表明,江苏地区的Q型烟粉虱可能来源于西部地中海地区,与日本的Q型烟粉虱具有相同的入侵来源。     

Aubian wheat mosaic virus, a new soil-borne wheat virus emerging in France

The properties of Aubian wheat mosaic virus (AWMV), a new soil-borne wheat virus in France, were investigated. Symptoms include foliar mosaic and severe stunting of winter wheat. The vector of the disease is unknown but the plants infected carry Polymyxa graminis in the roots. AWMV was transmitted mechanically to wheat and to two dicotyledoneous species: Lactuca sativa and Vicia faba. This virus was transmitted by seed to three winter wheat cultivars tested. Purified preparations contained rod-shaped particles with a variable length of 150–700 nm. Certain particles are very long and appear flexible. Antiserum raised against AWMV reacted specifically with AWMV in both indirect and direct enzyme-linked immunosorbent assays (ELISA). The incidence of AWMV in 26 winter wheat cultivars was investigated in the field during the growing season of 1999–2000. AWMV was detected in roots and shoots of all cultivars regardless of the symptoms. Twelve virus species belonging to the genera Benyvirus, Bymovirus, Furovirus, Pecluvirus and Pomovirus did not react with the AWMV antisera. A new tubular virus described in winter wheat in Bedfordshire in England reacted strongly with AWMV in ELISA. It is concluded that AWMV and probably the Bedford-virus constitute a previously undescribed tubular virus biologically and serologically distinct from other soil-borne viruses of wheat.     

烟粉虱传播的番茄褪绿病毒和番茄黄化曲叶病毒对不同番茄品种的复合侵染

为明确烟粉虱传播的番茄褪绿病毒(Tomato chlorosis virus,ToCV)与番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)对不同番茄品种的复合侵染情况,于2015年11月在山东省寿光市温室内采集13个番茄品种共390份疑似发病植株叶片,对不同番茄品种的TYLCV抗性和2种病毒的复合侵染以及温室内发病番茄植株上烟粉虱成虫的带毒率进行检测。结果表明,采集的13个番茄品种经分子标记检测鉴定均为TYLCV杂合抗性;不同番茄品种ToCV与TYLCV的复合侵染率存在明显差异,大果番茄粉宴和贝瑞上复合侵染率最高可达73.3%,而樱桃番茄八喜上未检测到这2种病毒的复合侵染。此外,在发病番茄植株上采集的烟粉虱成虫体内可检测到2种病毒,其中烟粉虱ToCV带毒率为90.7%,TYLCV带毒率为80.0%,同时检测到ToCV与TYLCV的概率为71.3%。表明ToCV和TYLCV的复合侵染在山东省番茄生产中普遍发生,烟粉虱可同时携带这2种病毒并广泛传播。     

天津市烟粉虱隐种鉴定及其携带TYLCV、内共生菌情况和抗药性监测

为明确天津市烟粉虱Bemisia tabaci隐种的类别及其寄主适应性、传毒能力、携带内共生菌情况和抗药性,采用mt COI酶切法对从武清、西青、蓟州和宁河4个区的番茄、黄瓜及辣椒3种寄主上采集的12个烟粉虱种群进行隐种鉴定,采用PCR检测其携带番茄黄化曲叶病毒（tomato yellow leaf curl virus,TYLCV）和内共生菌情况,并采用浸叶法测定其对4种常用药剂的抗性。结果表明,采集的烟粉虱种群以MED隐种为主,占所有检测个体的93.33%,有3个种群为MED和MEAM1隐种混合发生。所有检测个体中有36.25%的个体携带TYLCV,在6个种群中检测到TYLCV,其中5个种群有超过50%的个体携带TYLCV。在12个种群中共检测到Hamiltonella、立克次氏体Rickettsia、Cardinium和杀雄菌属Arsenophnus共4种内共生菌,携带个体比例分别为90.63%、48.96%、43.75%和8.33%,进一步对内共生菌协同感染情况进行分析,发现有HARC、HRC、HAC、HR、HC和AC共6个协同感染型,感染率分别为4.17%、28.13%、3....     

烟粉虱基因组和转录组研究及展望

烟粉虱Bemisia tabaci(Gennadius)是世界性的重要害虫,为物种复合体,其中少数隐种近30年来入侵世界各国,并暴发成灾。在我国,最近几年MED隐种已经逐渐替代了之前入侵的MEAM1隐种,成为我国农田生态系统中的主要致害隐种。基因组和转录组研究的不断深入极大地推动了烟粉虱入侵机制、隐种替代机制、种系发生、不同隐种或地理种群鉴定、抗药性机制、烟粉虱与共生菌互作等领域的研究。本文简要综述了烟粉虱基因组和转录组测序的研究进展,介绍了转录组数据在种的分化和鉴定、共生菌的互作及抗药性研究领域的应用。     

Biomolecular relationships among isolates of<Emphasis Type="Italic">Tomato Yellow Leaf Curl Tanzania Virus</Emphasis>

An investigation of the biological properties of the virus causing tomato yellow leaf curl disease in Tanzania was initiated to compare it with other known tomato yellow leaf curl viruses. Properties relating to acquisition and inoculation feeding time, persistence, mechanical inoculation, seed transmission and host range were studied. Results obtained indicate that the virus was transmitted persistently byBemisia tabaci Genn., but it was not mechanically, sap- or seed-transmissible. Minimum acquisition and inoculation feeding time was 30 min.Capsicum annuum, Datura stramonium, Nicotiana glutinosa, N. tabacum andLycopersicon esculentum were found to be hosts of the virus among the plant species tested, whereasPhaseolus vulgaris was not. It is concluded that the properties of the agent causing yellow leaf curl symptoms in tomato plants from different regions in Tanzania are similar to those ofTomato yellow leaf curl Sardinia virus species studied elsewhere. http://www.phytoparasitica.org posting Feb. 20, 2003.     

Analysis of genetic relations between <Emphasis Type="Italic">Broad bean wilt virus 1</Emphasis> and <Emphasis Type="Italic">Broad bean wilt virus 2</Emphasis>

We determined the complete nucleotide sequence of RNA-1 and the 5-terminal region of RNA-2 from Broad bean wilt virus 1 (BBWV-1) isolate PV132. This report is the first analysis of the genome organization of BBWV-1. We also determined the complete nucleotide sequence of RNA-1 from Broad bean wilt virus 2 (BBWV-2) isolate IP and analyzed the genetic relations between BBWV-1 and BBWV-2. Similar to the BBWV-2 isolates, both RNAs of PV132 encoded a single large polyprotein, which was predicted to contain some functional proteins in a manner similar to those of comovirus. With respect to the deduced amino acid sequences of the mature proteins, PV132 and IP had only 20%–40% homology to comovirus. On the other hand, IP was 73%–98% homologous to BBWV-2 isolates, but PV132 was 39%–67% homologous to the isolates. Although the extent of the homologies differed, the homologies were limited between BBWV-1 and BBWV-2 not only for the coat protein but also for the other proteins. These results clearly support the placement of BBWV-1 and BBWV-2 in the genus Fabavirus as distinct species, proposed on the basis of double immunodiffusion tests.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB084450 (RNA-1 of isolate PV132), AB084451 (RNA-2 of isolate PV132), and AB023484 (RNA-1 of isolate IP)