Tomato chlorosis virus in pepper: prevalence in commercial crops in southeastern Spain and symptomatology under experimental conditions

Tomato chlorosis virus (ToCV), a member of the genus Crinivirus (family Closteroviridae), has been present in Spain since at least 1997, causing annual epidemics of yellowing in protected tomato crops. In 1999, sweet pepper plants exhibiting stunting and symptoms of interveinal yellowing and mild upward curling in the leaves, were found to be infected with ToCV in a greenhouse heavily infested with the whitefly Bemisia tabaci in the province of Almería, southeastern Spain. This study investigated the prevalence of ToCV in tomato and pepper crops in the major growing areas of southeastern Spain (Murcia, Almería and Málaga provinces) over a 3‐year period. In addition, an experimental system was developed for ToCV inoculation using B. tabaci as a vector, which allowed analysis of susceptibility of different pepper cultivars to the virus. The disease syndrome and yield losses induced by ToCV in pepper were also studied under experimental conditions, confirming severe yield reduction in infected plants.     

番茄褪绿病毒与番茄黄化曲叶病毒复合侵染对番茄褪绿病毒传播的影响

番茄褪绿病毒Tomato chlorosis virus(ToCV)是一种由烟粉虱Bemisia tabaci传播的正义单链RNA病毒,在田间常与番茄黄化曲叶病毒Tomato yellow leaf curl virus(TYLCV)复合侵染而造成番茄生产上重大的经济损失。为了明确ToCV与TYLCV的复合侵染对烟粉虱传播ToCV所造成的影响,本文采用RT-PCR以及qRT-PCR检测了复合侵染的番茄对烟粉虱获取和传播ToCV的影响。研究表明,烟粉虱取食复合侵染的番茄后对ToCV的传播效率显著提高,仅25头烟粉虱的传毒率即可达到100%,ToCV在烟粉虱以及番茄体内的累积量均显著提高。说明这种复合侵染促进了烟粉虱对ToCV的传播,在田间应当及时防控烟粉虱,警惕病毒与烟粉虱的蔓延。     

Effects of host plants on insecticide susceptibility and carboxylesterase activity in Bemisia tabaci biotype B and greenhouse whitefly, Trialeurodes vaporariorum

Bemisia tabaci (Gennadius) biotype B and the greenhouse whitefly, Trialeurodes vaporariorum (Westwood), have become serious pests of cotton and vegetable crops in China since the early 1990s. In recent years, however, B. tabaci have broken out more frequently and widely than have T. vaporariorum. The B. tabaci biotype B has also developed higher resistance to several insecticides. Here, the effects of four different host plants on the insecticide susceptibility of B. tabaci biotype B and T. vaporariorum have been compared. The LC(50) values of imidacloprid, abamectin, deltamethrin and omethoate in T. vaporariorum reared on cucumber were significantly higher than those in B. tabaci (the LC(50) values in T. vaporariorum were respectively 3.13, 2.63, 2.78 and 6.67 times higher than those in B. tabaci). On the other hand, the B. tabaci population reared on cotton was more tolerant to all four insecticides tested than the T. vaporariorum population from the same host, especially to abamectin (up to 8.4-fold). The effects of the four host plants on the activity of carboxylesterase (CarE) in B. tabaci biotype B and T. vaporariorum were also compared. The results showed that, although the CarE activity of B. tabaci and T. vaporariorum varied depending on the host plants, the B. tabaci population possessed significantly higher CarE activity than the T. vaporariorum population reared on the same host plant. This was especially so on cucumber and cotton, where the CarE activities of the B. tabaci population were over 1.6 times higher than those of T. varporariorum. The frequency profiles for this activity in B. tabaci and T. vaporariorum populations reared on same host plant were apparently different.     

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     

Transmission of Tomato Yellow Leaf Curl Virus by two invasive biotypes and a Chinese indigenous biotype of the whitefly Bemisia tabaci

The global invasion of certain Bemisia tabaci biotypes provides opportunities to compare the competency of virus transmission between invasive and indigenous biotypes. Here we report on the acquisition, retention and transmission of Tomato Yellow Leaf Curl Virus (TYLCV) by the invasive B, Q and indigenous ZHJ2 biotypes of B. tabaci from Zhejiang, China. For all whitefly biotypes, TYLCV DNA was detected within a 30-min acquisition access period (AAP) to infected leaves. The percentage of adults with viral DNA increased with the length of AAP and reached 100% after 10–12 h. Following acquisition, viruliferous B, Q and ZHJ2 adults retained TYLCV DNA for the rest of their lives. Transmission was achieved with one B/Q adult per plant at the frequency of 50–55%, which rose to 100% with 10 insects per plant. In contrast, transmission of the virus was not observed with one ZHJ2 adult per plant in the experiments, and the transmission frequency rose to only 30–45% when whitefly adults increased to 5–10 per plant. These new data will help in the determination of the pest status of the whitefly biotypes as virus vectors in the regions of invasion, and so help in the development of management strategies.     

Pcr-amplification of tomato yellow leaf curl virus (TYLCV) DNA from squashes of plants and whitefly vectors: Application to the study of TYLCV acquisition and transmission

DNA of tomato yellow leaf curl virus (TYLCV), a geminivirus transmitted by the whitefly Bemisia tabaci, was amplified from squashes of infected tomato plants and of viruliferous vectors using the polymerase chain reaction (PCR). Samples of infected tissues as small as 1 mm² were squashed onto a nylon membrane. A 1 × 2 mm strip containing the squash was introduced into a 25 µl PCR reaction mix. The reaction products were subjected to gel electrophoresis, blotted and hybridized with a radiolabeled virus-specific DNA probe. TYLCV DNA was amplified from squashes of leaves, roots, and stem of infected tomato and from individual viruliferous whiteflies. The same squash could be used several times to amplify different virus DNA fragments with various sets of primers. Thus plant and insect squashes can be used as templates for the amplification of geminiviral DNA with no need to prepare tissue extracts or purify nucleic acids. The squash-PCR procedure was applied to study whitefly transmission of TYLCV. Tomato plants were inoculated by placing a single viruliferous insect in the center of a young leaflet. In some plants TYLCV DNA was detected at the site of inoculation as early as 5 min after the beginning of the access feeding and in all plants after 30 min. The squash-PCR procedure also was applied to the study of TYLCV acquisition by the insect vector. TYLCV DNA was detected in the head of whiteflies as early as 5 min after the beginning of the access feeding on infected tomato plants. Viral DNA was detected in the thorax after 10 min and in the abdomen after 25 min.     

番茄黄化曲叶病毒与番茄褪绿病毒复合侵染对番茄黄化曲叶病毒传播的影响

番茄黄化曲叶病毒(tomato yellow leaf curl virus, TYLCV)是一种由烟粉虱传播的单链环状DNA病毒, 在田间可与多种病毒发生复合侵染, 如番茄褪绿病毒(tomato chlorosis virus, ToCV)等?本文对比了TYLCV单独侵染和TYLCV与ToCV复合侵染对烟粉虱获取和传播TYLCV的影响?结果表明, 与取食TYLCV单独侵染的番茄相比, 取食复合侵染番茄的烟粉虱对TYLCV的传毒率显著提高, 且番茄植株和烟粉虱体内TYLCV的病毒积累量也显著提高?试验结果说明复合侵染会提高烟粉虱的传毒率, 促进TYLCV的发生与流行?     

B型烟粉虱携带传播烟草曲茎病毒的能力

B型烟粉虱近20年来入侵许多国家和地区,已成为重要的世界性作物害虫,其主要危害方式之一是传播双生病毒,造成作物病毒病大发生.应用PCR技术,研究了入侵我国的B型烟粉虱个体获取、存留及传播烟草曲茎病毒（Tobacco curly shoot virus,TbCSV）的能力.B型烟粉虱在感染TbCSV的普通烟毒株上饲毒30 min时,就可在40%的个体内检测到TbCSV DNA,个体带毒率随饲毒时间延长而增加,饲毒12 h后,带毒率达100%;TbCSV DNA在B型烟粉虱体内可存留10天左右,但不能终身存留;传毒处理植株的发病症状及PCR检测结果表明,B型烟粉虱是TbCSV的传播媒介.     

番茄褪绿病毒病在山东设施西葫芦上的发生及潜在危害

番茄褪绿病毒tomato chlorosis virus (ToCV)是我国蔬菜生产的重要新发病毒,其寄主范围逐年扩大。2019年10月项目组在山东寿光西葫芦温室调查时发现部分叶片呈现黄化、脉间褪绿,类似于ToCV侵染症状,并伴有烟粉虱发生。利用特异性引物检测发现,扩增的目的片段与GenBank中登录的侵染番茄的ToCV基因序列(登录号：KC887998.1)同源性高达99.58%,充分说明西葫芦植株已被ToCV侵染。通过对病毒病发生规律和烟粉虱虫口数量调查发现,西葫芦定植后1个月表现侵染症状的植株为2.0%,定植后2个月达到4.2%,定植后4个月后高达68.2%,病毒发生呈指数增长,而烟粉虱虫口数量却维持较低密度。从济南和德州采集的西葫芦疑似病叶和烟粉虱中也检出ToCV病毒,说明该病毒可能已经在山东省设施西葫芦主要种植区普遍发生,并经烟粉虱广泛传播,需引起高度重视。     

烟粉虱与温室白粉虱羧酸酯酶、谷胱甘肽转移酶和乙酰胆碱酯酶性质的比较研究

对温室白粉虱 Trialeurodes vaporariorum和烟粉虱 Bemisia tabaci羧酸酯酶(Car E)、乙酰胆碱酯酶 (ACh E)和谷胱甘肽转移酶 (GSTs)的生物化学性质进行了初步研究。比较 Car E活性 ,烟粉虱显著高于温室白粉虱 ,烟粉虱和温室白粉虱 Car E对底物乙酸 -α-萘酯的 Km 分别为 0 .073mmol/ L 和 3.458mmol/ L。烟粉虱 ACh E活性分布高峰在 2～3m OD/ (min·头 )之间 ,而温室白粉虱 ACh E活性分布高峰在 10～ 15 m OD/ (min·头 )之间 ,烟粉虱 ACh E的 Km 值是温室白粉虱 Km 值的 12 .6倍。烟粉虱的 GSTs比活力为2 .218OD/ (mg pro·min) ,显著高于温室白粉虱的 GSTs比活力 0 .663OD/ (mg pro· min) ,差异达极显著水平 ,烟粉虱 GSTs对底物的亲和力比温室白粉虱高。     

Tomato infectious chlorosis virus causes leaf yellowing and reddening of tomato in Italy

Since autumn 2000, severe and widespread chlorosis, sometimes associated with redness, has been observed in greenhouse tomatoes in different regions of Italy. A total of 104 samples were analyzed for tomato infectious chlorosis virus (TICV), by a one-step RT-PCR procedure. In some areas of central Italy and Sardinia, the symptom was consistently correlated with the presence of TICV. The RT-PCR procedure enabled rapid and reliable detection of TICV from field samples. Sequence analysis of the amplified 501-bp fragment, part of the HSP70 coding region, revealed an identity of 99% with the TICV sequence in the GenBank database. A digoxigenin-labeled DNA probe was also produced and successfully tested in dot blot assays. This is the first report of TICV causing epidemics in Europe.     

河北省番茄黄化曲叶病毒和番茄褪绿病毒复合侵染及分布

 番茄黄化曲叶病毒(Tomato yellow leaf curl virus, TYLCV) 及番茄褪绿病毒(Tomato chlorosis virus, ToCV)是危害番茄的主要病毒。2016~2017年,在河北省18个番茄主产区调查番茄病毒病危害情况,采集262份植株矮化、叶片黄化、褪绿、卷曲的番茄样品,利用分子生物学技术对病原进行鉴定。结果表明:2016~2017年河北省番茄病毒病发生普遍,所检样品中TYLCV的侵染率为68.66%,其中与ToCV复合侵染率为19.5%;除栾城、滦南、乐亭、永年4地样品暂未检测到ToCV外,其他14个采样点的样品均检测到ToCV,侵染率为19.5%,且全部表现为与TYLCV复合侵染,河北省番茄主产区暂未发现ToCV单独侵染的样品。     

Serological and molecular detection of Tomato chlorosis virus and Tomato infectious chlorosis virus in tomato

Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) are two criniviruses inducing similar yellowing symptoms in tomato. An approximately 4 kb central region of the genomic RNA2 of French ToCV and TICV isolates was sequenced. TICV, for which no other sequences were available, appeared as a distant species in the genus, being close only to LIYV ( Lettuce infectious yellows virus ) for some, but not all, proteins. ToCV has more than 98% nucleotide identity with isolates from the US and Spain, and sequencing the CP gene of several isolates collected in different regions in southern France during 2 years suggested a unique origin. Polyclonal antisera were produced using capsid proteins of both viruses expressed in Escherichia coli . DAS-ELISA assays were developed for routine diagnosis and conditions for preparing samples for an optimized detection were determined. No cross-reactions were observed. However, some false-negative results, corresponding to samples giving ELISA readings close to the detection limit were regularly detected, particularly for ToCV (approximately 5% of the samples). A triplex RT-PCR assay was thus developed, which allowed detection of both viruses in a one-step protocol. An internal PCR control was included, which in addition showed that it could be used as a control for the entire RT-PCR procedure. Finally, combining DAS-ELISA in a first round, and triplex RT-PCR for doubtful samples, appeared the best way to achieve a reliable diagnosis of these viruses.     

Occurrence and Diagnosis of Tomato chlorosis virus in Portugal

Tomato chlorosis virus (ToCV), a new whitefly-transmitted and phloem-limited Crinivirus infecting tomatoes in Europe, is reported for the first time in Portugal. Tomato plants with symptoms of interveinal chlorosis, collected during autumn 1998 and summer and autumn 1999 in Algarve, southern Portugal, were positive in RT-PCR assays using ToCV-specific primers. The amplified 439bp fragment was sequenced and showed 99% homology with the ToCV sequence in the GenBank database. A digoxigenin–DNA probe was produced and tested in dot-blot with total RNAs extracted from tomato samples. Both the RT-PCR and dot-blot hybridisation procedures enabled rapid and reliable detection of ToCV from field samples.     

北京地区番茄黄化曲叶病毒病的鉴定及防治对策

番茄黄化曲叶病毒病是一种由烟粉虱传播的病毒病,给番茄生产造成严重威胁。2009年在北京郊区调查时发现部分保护地种植的番茄植株表现典型黄化曲叶症状。通过提取典型症状样品总DNA利用粉虱传双生病毒检测简并引物PA/PB,进行PCR扩增到541bp的特异条带。通过测序和核苷酸序列比对表明该序列与番茄黄化曲叶病毒序列相似性最高为99%。分子检测结果表明北京郊区部分保护地种植的番茄已被烟粉虱传播的番茄黄化曲叶病毒侵染危害。     

沼泽红假单胞菌PSB-06菌剂对番茄褪绿病的防效及其作用机制

为明确沼泽红假单胞菌Rhodopseudomonas palustris PSB-06菌剂对番茄褪绿病毒（tomato chlorosis virus,ToCV）的抑制作用,连续3年在田间进行沼泽红假单胞菌PSB-06菌剂对ToCV的药效试验,并在室内研究其抗病毒的作用机制。田间试验结果显示,PSB-06菌剂处理能降低ToCV的发生,植株发病率均低于50%,均显著低于清水和氨基寡糖素处理;室内试验结果显示,PSB-06菌剂处理能抑制病毒传播,喷施PSB-06菌剂后的感病番茄的SA含量达532.67 mg/g,PI II和NPR1基因表达量也相对上升;同时,健康番茄与感病番茄的叶绿素含量均明显升高;经PSB-06喷施后番茄植株上的烟粉虱Bemisia tabaci生存数量降低。蛋白组学分析结果表明,PSB-06处理促使植物代谢及防御的相关蛋白均上调。表明沼泽红假单胞菌PSB-06菌剂能增强植物免疫和防御烟粉虱的能力,从而有效地控制ToCV的发生及传播。     

A study of viral coat protein accumulation in lily chloroplasts from mixed virus infections of Lily mottle virus and Cucumber mosaic virus

Two viruses that frequently occur in many Lilium species are Lily mottle virus (LMoV) and Cucumber mosaic virus (CMV), which usually co-infect lilies causing severe disease symptoms. Recent reports have revealed that the viral coat protein (CP) affects chloroplast ultrastructure and symptom development. This study used western blot analysis to confirm that in leaves infected by mixed virus infections of LMoV and CMV, CPs of both viruses were accumulated in lily chloroplasts. Immunogold labelling further demonstrated that both the LMoV CP and CMV CP were localized in the stroma and the thylakoid membranes of the chloroplasts. In addition, it was found that CPs of both viruses were rapidly transported into isolated, intact chloroplasts (in vitro), and their transport efficiencies were positively related to CP concentrations. The lowest transmembrane concentration of CMV CP decreased from 38 μg mL⁻¹ recorded in the single CMV CP import system to 10 μg mL⁻¹ in the mixed import system of LMoV CP and CMV CP. CPs of both viruses exhibited species selection in their transmembrane transport into chloroplasts. This is the first report that the CPs from two viruses (LMoV and CMV) are simultaneously present in lily chloroplasts. Accumulation of high levels of LMoV CP and CMV CP inside the chloroplast appears to contribute to a synergistic interaction inducing the development of mosaic symptoms.