基于小RNA深度测序和RT-PCR检测侵染广东省冬种马铃薯的病毒

为明确侵染广东省冬种马铃薯的病毒种类及优势病毒,结合小RNA深度测序技术及RTPCR检测方法,对采集于广东省冬种马铃薯7个主产区的189份疑似病样进行检测分析。结果表明,经小RNA深度测序技术检测马铃薯病毒病混合样,发现存在马铃薯Y病毒(Potato virus Y,PVY)、马铃薯S病毒(Potato virus S,PVS)和马铃薯卷叶病毒(Potato leaf-roll virus,PLRV)3种病毒。进一步设计3种病毒的特异性引物并利用国内已报道的其它5种马铃薯病毒的特异性引物进行RT-PCR检测,发现189份马铃薯病毒病疑似病样中仅检测到PVY、PVS和PLRV这3种病毒,检出率依次为75.13%、10.05%和4.76%,且3种病毒在马铃薯上还存在复合侵染,复合侵染率为14.19%,其中PVY在各马铃薯产区均可检测到。表明侵染广东省冬种马铃薯的病毒为PVY、PVS和PLRV,其中PVY是优势病毒。     

Ultrastructural aspects of tomato leaves infected by Tomato torrado virus (ToTV) and co‐infected by other viruses

Optical and electron microscopy studies were carried out to investigate the cytopathology induced in tomato leaves infected by Tomato torrado virus (ToTV), a new picorna‐like virus associated with the ‘Torrado’ disease. Infected leaves, showing typical Torrado disease symptoms were surveyed in commercial greenhouses in the main tomato production areas of Spain. The effect of the co‐infection of ToTV with other viruses which commonly infect tomato crops was also studied. Ultra‐thin sections of ToTV‐infected tomato leaves did not show a strong cellular alteration. However, crystalline arrays of isometric virus‐like particles (VLPs) of 20–30 nm in the inclusion bodies were observed in phloem parenchyma cells of the infected tissues. Tissues co‐infected by ToTV and either Tomato chlorosis virus (ToCV) or Pepino mosaic virus (PepMV) presented more severe cellular alterations. The most deleterious consequences for tomato cells were found in triple infections of ToTV, PepMV and Tomato spotted wilt virus (TSWV), where characteristic cell wall overgrowth was distinguishable, together with a large amount of necrotic cells.     

Virus transmission by aphids in potato crops

This paper reviews the contribution of vector activity and plant age to virus spread in potato crops. Determining which aphid species are vectors is particularly important for timing haulm destruction to minimize tuber infection by potato virus Y (PVY). Alate aphids of more than 30 species transmit PVY, and aphids such asRhopalosiphum padi, that migrate in large numbers before flights of the more efficient vector,Myzus persicae, appear to be important vectors. Differences in methodology, aphid biotypes and virus strains prevent direct comparisons between estimates of vector efficiencies obtained for aphids in different countries in north western Europe. M. persicae is also the most efficient vector of potato leafroll virus (PLRV), but some clones ofMacrosiphum euphorbiae transmit PLRV efficiently toNicotiana clevelandii and potato test plants. The removal of infected plants early in the season prevents the spread of PLRV in cool regions with limited vector activity. The proportion of aphids acquiring PLRV from infected potato plants decreases with plant age, and healthy potato plants are more resistant to infection later in the season. Severe symptoms of secondary leafroll developed on progeny plants of cv. Maris Piper derived from mother plants inoculated with PLRV in June or July of the previous year. Progeny plants derived from mother plants inoculated in August showed only mild symptoms, but the concentration of PLRV in these plants was as high as that in the plants with severe symptoms.     

Lack of synergistic effects in tomato plants co-infected with tomato severe rugose virus and tomato chlorosis virus

The begomovirus Tomato severe rugose virus (ToSRV) and the crinivirus Tomato chlorosis virus (ToCV), in single and co-infections, are very common in tomato crops in Brazil. Both viruses are transmitted by the whitefly Bemisia tabaciMEAM1 (biotype B). The objective of this study was to analyse the interaction between ToSRV and ToCV in tomato plants of cultivars Santa Clara and Kada. Plants at 15, 30 and 45 days after emergence were inoculated with 30 viruliferous B. tabaci per plant. The following treatments were compared: plants inoculated with ToSRV, ToCV, ToSRV + ToCV, and healthy (control). The interaction between these viruses was analysed by measuring the virus titre by qPCR and the fresh and dry weights of the aerial parts of the tomato plants. Based on two independent assays, no significant effects for co-infection of ToSRV and ToCV on virus titres and plant development were observed compared to single infections. The dry weight of tomato plants of both cultivars infected with ToSRV, ToCV, or co-infected did not differ significantly. However, the dry weight of Santa Clara tomato plants infected with ToSRV, ToCV and ToSRV + ToCV showed mean reductions of 21.5%, 25.5% and 32%, respectively, compared to healthy plants, and mean reductions for Kada were 31.7%, 37.5% and 38%, respectively.     

Differential reaction of sweet pepper to infection with the crinivirus tomato chlorosis virus probably depends on the viral variant

The tomato chlorosis virus (ToCV), transmitted by whitefly species of the genera Bemisia and Trialeurodes in a semipersistent manner, causes significant losses in solanaceous crops including tomato (Solanum lycopersicum) and sweet pepper (Capsicum annuum). Worldwide reports of natural and experimental infection of sweet pepper plants with ToCV are contradictory, raising the question of whether the critical factor determining infection is related to the susceptibility of sweet pepper cultivars or the genetics of virus isolates. In this work, ToCV isolates obtained from different hosts and geographical origins were biologically and molecularly analysed, transmitted by B. tabaci MEAM1 and MED, and the reaction of different sweet pepper cultivars was evaluated under different environmental conditions. Brazilian ToCV isolates from tomato, potato (S. tuberosum), S. americanum, and Physalis angulata did not infect plants of five sweet pepper cultivars when transmitted by B. tabaci MEAM1. Temperatures did not affect the sweet pepper susceptibility to tomato-ToCV isolates from São Paulo, Brazil, and Florida, USA. However, sweet pepper-ToCV isolates from Spain and São Paulo, Brazil, were transmitted efficiently to sweet pepper plants by B. tabaci MEAM1 and MED. Although the results indicated that ToCV isolates from naturally infected sweet pepper plants seem to be better adapted to plants of C. annuum, phylogenetic analyses based on the complete nucleotide sequences of RNA1 and RNA2 as well as the p22 gene did not reveal significant nucleotide differences among them. Additional studies are needed to identify intrinsic characteristics of ToCV isolates that favour infection of sweet pepper plants.     

Virus transmission by aphids in potato crops

This paper reviews the contribution of vector activity and plant age to virus spread in potato crops. Determining which aphid species are vectors is particularly important for timing haulm destruction to minimize tuber infection by potato virus Y (PVY). Alate aphids of more than 30 species transmit PVY, and aphids such asRhopalosiphum padi, that migrate in large numbers before flights of the more efficient vector,Myzus persicae, appear to be important vectors. Differences in methodology, aphid biotypes and virus strains prevent direct comparisons between estimates of vector efficiencies obtained for aphids in different countries in north western Europe.M. persicae is also the most efficient vector of potato leafroll virus (PLRV), but some clones ofMacrosiphum euphorbiae transmit PLRV efficiently toNicotiana clevelandii and potato test plants. The removal of infected plants early in the season prevents the spread of PLRV in cool regions with limited vector activity. The proportion of aphids acquiring PLRV from infected potato plants decreases with plant age, and healthy potato plants are more resistant to infection later in the season. Severe symptoms of secondary leafroll developed on progeny plants of cv. Maris Piper derived from mother plants inoculated with PLRV in June or July of the previous year. Progeny plants derived from mother plants inoculated in August showed only mild symptoms, but the concentration of PLRV in these plants was as high as that in the plants with severe symptoms.     

二重RT-PCR快速检测马铃薯病毒的方法

本研究采用传统的蛋白酶K法和病毒RNA简易浸提法,从马铃薯块茎、茎干、叶梗、叶片中提取马铃薯X病毒,马铃薯Y病毒,马铃薯A病毒及马铃薯卷叶病毒RNA,并设计了4种马铃薯病毒引物,优化了二重RT-PCR反应条件,可以同步扩增出上述4种病毒,扩增产生的靶带分别为562bp(PVX)、480bp(PVY)、336bp(PLRV)、255bp(PVA).应用病毒RNA简易制样技术和优化的二重RT-PCR反应条件,可以同步快速检测田间自然感染的马铃薯病毒,此研究还可适合于检测马铃薯脱毒种薯及试管苗,对马铃薯病毒病早期监测有一定的作用.     

Incidence of potato viruses and characterisation of <Emphasis Type="Italic">Potato virus Y</Emphasis> variability in late season planted potato crops in Northern Tunisia

Surveys were conducted of symptomatic potato plants in late season crops, from the major potato production regions in Northern Tunisia, for infection with six common potato viruses. The presence of Potato leafroll virus (PLRV), Potato virus Y (PVY), Potato virus X (PVX), Potato virus A (PVA), Potato virus S (PVS) and Potato virus M (PVM) was confirmed serologically with virus infection levels up to 5.4, 90.2, 4.3, 3.8, 7.1 and 4.8%, respectively. As PVY was prevalent in all seven surveyed regions, further biological, serological and molecular typing of 32 PVY isolates was undertaken. Only one isolate was shown to induce PVY^O-type symptoms following transmission to tobacco and to react only against anti-PVY^O-C antibodies. Typical vein necrosis symptoms were obtained from 31 samples, six of which reacted against both anti-PVY^N and anti-PVY^O-C antibodies showing they contained mixed isolates, while 25 of them reacted only with anti-PVY^N antibodies. An immunocapture RT-PCR molecular test using a PVY^NTN specific primer pair set in the 5’NTR/P1 genomic region and examination of recombinant points in three genomic regions (HC-Pro/P3, CI/NIa and CP/3’NTR) showed that all 25 serotype-N PVY isolates were PVY^NTN variants with similar recombinations to the standard PVYNTN-H isolate. This is the first report of the occurrence of the PVY^NTN variant and its high incidence in late season potatoes in Tunisia.     

<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.     

Tomato infectious chlorosis virus — a new clostero-like virus transmitted byTrialeurodes vaporariorum

A previously undescribed virus disease of tomato, other crops and weed hosts was found in California. Affected tomato plants exhibited interveinal yellowing, necrosis and severe yield losses. Leaf dips and purified preparations contained closterovirus-like long flexuous, filamentous particles approximately 12×850–900 nm. The virus, designated as tomato infectious chlorosis virus (TICV), is transmitted in a semipersistent manner by the greenhouse whitefly,Trialeurodes vaporariorum. The host range of the virus is moderate (26 species in 8 plant families) but includes some important crops and ornamental species including tomato, (Lycopersicon esculentum), tomatillo (Physalis ixocarpa), potato (Solanum tuberosum), artichoke (Cynara scolymus), lettuce (Lactuca sativa) and petunia (Petunia hybrida). The virus has been found in a number of different locations in California and has a number of potential vehicles of movement including greenhouse grown ornamentals, tomato transplants, artichoke cuttings and potato seed. The virus has the potential to spread to other growing regions with resident populations of the greenhouse whitefly. The host range, particle size, insect transmission, and serology clearly distinguish TICV from previously described viruses.     

马铃薯主产区病毒病发生情况调查分析

在我国马铃薯主产区黑龙江、内蒙古、甘肃、贵州采集了752份具有典型病毒病症状的样品和疑似样品,应用DAS ELISA方法进行检测。主要筛查6种病毒：PVX、PVY、PVS、PLRV、PVM、PVA。结果显示: 270份样品检测到病毒,总体上,PVY的检出率最高,PVS次之,33份样品是由多种病毒混合侵染造成的,田间PVY+PLRV混合侵染率最高,PVS+PVY次之。试管苗PVS病毒发生较多,原原种和大田种薯PVY病毒发生比例最高。     

利用RNA干扰介导抗病性获得兼抗四种病毒的转基因马铃薯

为获得兼抗马铃薯X病毒(Potato virus X,PVX)、马铃薯Y病毒(Potato virus Y,PVY)、马铃薯卷叶病毒(Potato leaf roll virus,PLRV)和马铃薯潜隐花叶病毒(Potato virus S,PVS)4种病毒的转基因马铃薯新材料,分别以这4种病毒全长CP基因为模板,通过设计PCR引物和亚克隆获得4种病毒CP基因相对保守区段的基因片段,并将其拼接成融合基因,以载体pHANNIBAL和pBI121为基础,构建RNA干扰(RNA interference,RNAi)载体,利用农杆菌介导的转基因体系进行马铃薯遗传转化,并对获得的转基因马铃薯进行病毒抗性检测。结果表明,所获得的融合基因片段RH1和RH2,酶切鉴定分别得到长度为1 200 bp的条带,与预期片段相符;构建了含pdk内含子和RH1、RH2融合基因的RNAi植物表达载体,经Bam H I/Sac I双酶切,获得长度约3 200 bp的片段,表明RNAi植物表达载体pBI121-pRH构建成功;转化易感病毒马铃薯品种陇薯11号,PCR检测和PCRSouthern杂交分析表明融合基因已整合到陇薯11号马铃薯基因组中;抗病性检测显示4株转基因马铃薯植株对4种病毒均免疫。表明利用RNAi可筛选出抗多种病毒的转基因马铃薯新种质。     

Molecular phylogenetic,morphological, and pathogenic analyses reveal a single clonal population of Septoria lycopersici with a narrower host range in Brazil

Septoria leaf spot, caused by Septoria lycopersici, is considered one of the most important diseases of tomato in Brazil. Despite its importance, the disease agent is still poorly studied. Septoria isolates collected from different production regions of Brazil were characterized by molecular, morphological, and pathogenic methods. A set of 104 isolates was sequenced for the DNA Tub, Cal, and EF1-α loci. Ten isolates were selected, according to geographical region of origin and type of leaf lesion (typical or atypical), for morphological characterization and for evaluation of aggressiveness on tomato cultivar Santa Clara. To evaluate the pathogen host range, cultivated and wild Solanaceae plants were inoculated with four selected isolates. The results showed that all isolates grouped with the type isolate of S. lycopersici in maximum likelihood and Bayesian inference trees. The isolates were morphologically similar. All isolates selected for pathogenicity testing on tomato were able to induce typical symptoms of the disease, but differed in their aggressiveness. A total of eight species of Solanaceae were also identified as potential alternative hosts for S. lycopersici. This information will provide a more accurate assessment of the risks involved with the introduction of new crops, especially of the genus Solanum, in areas where the species is already present. In addition, it will provide the basis for the establishment of more efficient methods in the management of Septoria leaf spot of tomatoes in natural conditions and in the different production systems.     

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.     

甘肃省马铃薯主要病毒病发生情况调查

2015年-2016年,在甘肃省10个地市24个马铃薯主栽县(区)146个生态区域(乡镇)采集了757份具有典型症状的马铃薯样品,应用DAS-ELISA法进行检测,筛查6种主要病毒(PVX、PVY、PLRV、PVA、PVS和PVM)。结果表明:631份样品检测到病毒,PVS的检出率最高,达47.03%,PVY次之,为33.82%,PVA最低,只有0.63%;发生复合侵染的病毒主要为PVY+PVS,复合侵染率达到10.13%,三种病毒复合侵染主要是PVY+PVS+PVM;病毒种类和感病程度与品种、地域有关。     

湖南省马铃薯病毒病发生情况调查

为了解湖南省马铃薯种薯质量和主要病毒病发生情况,2019年-2020年马铃薯秋作和冬作期间,对长沙、益阳、湘潭、澧临等马铃薯生产区的155个马铃薯样品,运用反转录-聚合酶链式反应(RT-PCR)和双抗体夹心酶联免疫吸附检测(DAS-ELISA)技术,筛查6种主要马铃薯病毒,包括马铃薯X病毒Potato virus X(PVX)、马铃薯Y病毒Potato virus Y(PVY)、马铃薯M病毒Potato virus M(PVM)、马铃薯S病毒Potato virus S(PVS)、马铃薯A病毒Potato virus A(PVA)、马铃薯卷叶病毒Potato leaf roll virus(PLRV)。检测结果表明:6种马铃薯病毒病在湖南均有不同程度的发生,单一和两种病毒复合感染植株占比最高,其次是3种病毒复合感染,存在极少数植株复合感染4～5种病毒病情况。在秋作马铃薯中,PVY检出率达到29.41%;PVS和PVA检出率均为27.94%;PVM、PVX、PLRV的检出率分别为20.59%、19.12%、17.65%。在冬作马铃薯中,PVX检出率最高,达到31.03%;其次是PLRV,...