番茄黄化曲叶病毒对温室白粉虱适合度的影响

【目的】温室白粉虱Trialeurodes vaporariorum(Westwood)是为害北方地区花卉蔬菜的主要粉虱种类,烟粉虱Bemisia tabaci(Gennadius)则在近些年逐渐频繁的花卉贸易活动中扩散至黑龙江省部分地区并取代了白粉虱成为当地温室害虫的优势物种,番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)是烟粉虱传播的一种重要双生病毒,对作物的危害十分严重,然而该病毒对本地白粉虱的影响及对烟粉虱与白粉虱种间竞争关系的影响尚待研究。【方法】本研究观察记录感染番茄黄化曲叶病毒的番茄植株上温室白粉虱和烟粉虱的种群动态及番茄形态和部分生理指标的变化。【结果】结果表明:1)有烟粉虱滋生的番茄植株矮小,根系发达;2)有白粉虱滋生的番茄略微矮粗,影响较小;3)感染病毒的番茄矮粗或矮小,根部无明显变化;4)染毒带虫相对于带虫处理而言,在白粉虱试验中,番茄植株矮小,根系生物量也骤减;与此相反,在烟粉虱试验中,番茄的反应相对缓和;5)此外,不同酶类对植株染毒的响应不同:Ach E酶活高不利于植株,而GST酶活高则有利于植株。【结论】总体而言:烟粉虱单独作用很不利于苗期番茄,白粉虱对苗期番茄没有明显的直接影响;而感染病毒会缓解烟粉虱对番茄的强烈刺激,而加重白粉虱对番茄的作用,即染毒使带白粉虱的苗期番茄生长发育受到明显抑制。与番茄变化情况相对应的是,染毒番茄上烟粉虱产卵较少,但在发育前期(从卵到伪蛹)存活率较高;染毒番茄上白粉虱产卵较多,但在前期存活率较低。本研究可为高纬度地区粉虱综合防控提供参考。     

Efficiency of insect exclusion screens for preventing whitefly transmission of tomato yellow leaf curl virus of tomatoes in Israel

Tomato yellow leaf curl virus (TYLCV) is the most frequently occurring virus in tomatoes in the Middle East, and the most harmful one. It is transmitted solely by the whitefly Bemisia tabaci (Gennadius). Within 4-6h of inoculative feeding, a whitefly can transmit TYLCV to a healthy plant with 80% probability. The symptoms are apparent after two to three weeks whereupon fruit-set is effectively terminated. The only means of controlling TYLCV is by controlling the whitefly. Until 1990 this was exclusively by insecticides. Starting in 1990, growers of greenhouse tomatoes in Israel began adopting insect exclusion screens to prevent inoculation of TYLCV. This article reports on the methods used in the search for efficient screening materials and presents data on their relative efficiencies in excluding B. tabaci and several other greenhouse pests. Ten materials were tested, of which five were found to be effective in excluding B. tabaci under laboratory conditions. This number was reduced to three following field trials and trials in commercial tomato greenhouses. These materials are now in widespread use in Israel: by 2000 practically all table tomatoes in Israel were grown under exclusion screens. The use of exclusion screens has been shown to be an economically viable pest management method.     

番茄黄化曲叶病毒的快速分子检测

番茄黄化曲叶病毒是当前世界范围内危害番茄生产的毁灭性病害。文章针对番茄黄化曲叶病毒全基因组序列的特异区段自主设计了1对特异性PCR引物(上游引物TYLCV-F:5′-ACGCATGCCTCTAATCCAGTGTA-3′,下游引物TYLCV-R:5′-CCAATAAGGCGTAAGCGTGTAGAC-3′),依据PCR扩增特异片段543 bp的有无可以快速、准确、高效、特异地检测出是否感染了TYLCV病毒,这项技术可以方便地应用到工厂化育苗的带毒性检测、蔬菜大规模生产中植株发病情况的快速检测以及抗病毒育种,从而为蔬菜安全可持续生产提供科技支撑。     

番茄黄化曲叶病毒的快速分子检测

番茄黄化曲叶病毒是当前世界范围内危害番茄生产的毁灭性病害。文章针对番茄黄化曲叶病毒全基因组序列的特异区段自主设计了1对特异性PCR引物(上游引物TYLCV-F：5′-ACGCATGCCTCTAATCCAGTGTA-3′, 下游引物TYLCV-R：5′-CCAATAAGGCGTAAGCGTGTAGAC-3′), 依据PCR扩增特异片段543 bp的有无可以快速、准确、高效、特异地检测出是否感染了TYLCV病毒, 这项技术可以方便地应用到工厂化育苗的带毒性检测、蔬菜大规模生产中植株发病情况的快速检测以及抗病毒育种, 从而为蔬菜安全可持续生产提供科技支撑。     

Whitefly population dynamics and evaluation of whitefly-transmitted tomato yellow leaf curl virus (TYLCV)-resistant tomato genotypes as whitefly and TYLCV reservoirs

Sweetpotato whitefly, Bemisia tabaci (Gennadius), and whitefly-transmitted tomato yellow leaf curl virus (TYLCV) are major threats to tomato production in the southeastern United States. TYLCV was introduced to Florida from the Caribbean islands and has spread to other southern states of the United States. In Georgia, in recent years, the incidence of TYLCV has been steadily increasing. Studies were conducted to monitor population dynamics of whiteflies in the vegetable production belt of Georgia, to evaluate TYLCV-resistant genotypes against whiteflies and TYLCV, and to assess the potential role of resistant genotypes in TYLCV epidemiology. Monitoring studies indicated that the peak incidence of whiteflies varied seasonally from year to year. In general, whitefly populations were not uniformly distributed. Tomato genotypes exhibited minor differences in their ability to support whitefly populations. TYLCV symptoms were visually undetectable in all but one resistant genotype. The infection rates (visually) in susceptible genotypes ranged from 40 to 87%. Greenhouse inoculations with viruliferous whiteflies followed by polymerase chain reaction (PCR) indicated that up to 100% of plants of resistant genotypes were infected, although predominantly symptomless. TYLCV acquisition by whiteflies from TYLCV-infected genotypes was tested by PCR; TYLCV acquisition rates from resistant genotypes were less than from susceptible genotypes. Nevertheless, this difference did not influence TYLCV transmission rates from resistant to susceptible genotypes. Results emphasize that resistant genotypes can serve as TYLCV and whitefly reservoirs and potentially influence TYLCV epidemics.     

Genetic structure and population variability of tomato yellow leaf curl China virus

Geminiviruses have circular single-stranded DNA genomes and are important pathogens in tropical and subtropical regions, but their population diversity and variability are poorly understood. Here, we have investigated variations accumulating in Tomato yellow leaf curl China virus (TYLCCNV), a geminivirus in the genus Begomovirus of the family Geminiviridae. The population variation was analyzed in a naturally infected tomato (Solanum lycopersicom) plant and in Nicotiana benthamiana and tomato plants experimentally infected with a swarm of TYLCCNV DNA clones to provide an identical sequence for initiation of infection. Our results demonstrate that the population of TYLCCNV in a naturally infected tomato plant was genetically heterogeneous and that rapid mutation occurred in the populations amplified from N. benthamiana and tomato plants that had been infected with cloned DNA. This feature of the population of TYLCCNV in these plants consisted of the consensus sequence and a pool of mutants that are not identical but are closely related to the consensus sequence, and it coincides with the quasispecies concept described for many RNA viruses. The mutation frequency was circa 10(-4) in N. benthamiana and tomato at 60 days postinoculation, a value comparable to that reported for plant RNA viruses. The quasispecies-like nature of the TYLCCNV populations suggested that TYLCCNV is capable of rapid evolution and adaptation in response to changing agricultural practices.     

Identification of wild hosts of tomato yellow leaf curl virus in South-Eastern Iran

Abstract

In this research, to identify natural wild hosts of TYLCV, 44 symptomatic samples were collected in or around harvested tomato fields in south-eastern Iran and tested for the TYLCV infection by PCR. Among four PCR-positive plant species, full-length genome of TYLCV was amplified by rolling circle amplification (RCA) method only from a ground cherry (Physalis divaricata L., Solanaceae) sample. Cloning and full-length genome sequencing of a ground cherry isolate (G4) showed that it comprises 2756 nucleotides (nts) in length and shares 87.7%–95.2% nt sequence identities with TYLCV isolates reported from Iran, Oman and Pakistan. The G4 isolate of TYLCV was successfully transmitted to tomato plants and the original host (P. divaricata) via agroinoculation and showed typical symptoms. According to the results of this research, four symptomatic weed species appear to be alternative hosts of TYLCV and play the role of the primary inoculum for the viral infection.     

Virus-induced CRISPR-Cas9 system improved resistance against tomato yellow leaf curl virus

Molecular Biology Reports - Plant viruses are the most significant factors associated with massive economical losses in agricultural industries worldwide. Accordingly, many studies are dedicated to...     

Relationship between Tomato yellow leaf curl viruses and the whitefly vector

Tomato Yellow Leaf Curl Virus (TYLCV) and Tomato Leaf Curl Virus (ToLCV) are the currently known begomoviruses in Uganda. The relationship with their whiteflies (Bemisia tabaci) vector and its management were not known in Uganda. A direct relationship was expected between these begomoviruses and whiteflies at Buwama in Mpigi district of Uganda. Farmer practices were expected to have limited efficacy. To investigate this, a completely randomised block design was used for all trials with six treatments replicated three times. Treatments evaluated were farmer whitefly pest management practices. These included both chemical pesticide and non-pesticide applications. Data on whitefly population and tomato yellow leaf curl virus disease incidence was recorded weekly, and analysed using SAS and SPSS statistical programmes for ANOVA, and correlations. Ranked means, coefficients of variation and standard errors were noted. Virus-vector relationship field studies established that virus occurrence varied in space and time, and with management practices, crop development stage, and weather conditions. A negative relationship (R = -0.14, p 0.04) was established between number of plants infected with TYLCV (sensu lato) and percentage marketable tomato yield. Tomato maturity was inversely propotional to whitefly infestation (R = -0.5, p 0.0001). Uprooting and application of dimethoate was most effective of the six treatments. Tomato leaf curl virus diseases and whitefly management options were established in Uganda.     

Advances in diagnosing tomato yellow leaf curl geminivirus infection

As a result of the spread of TYLCV on tomato crops, reliable and rapid diagnostic tools to identify and isolate new sources of infection are necessary. We tested several methods, based both on antibodies and on nonradioactive DNA probes. Indirect plate-trapping ELISA was only effective in detecting the virus in purified preparations, but not in crude extracts. Dot-ELISA with chemiluminescence detection gave satisfactory results when young stems were directly squashed on membranes. A digoxigenin-labeled probe, detected with chemiluminescence, was used in leaf squashes and dot blots. Best results were obtained with dot blots of total nucleic acids prepared with a fast and safe procedure. TYLCV DNA was readily and reliably detected in spots corresponding to 15 μg fresh weight. When weak signals were observed, total extracts were analyzed by Southern blotting, to confirm the presence of viral DNA forms.     

Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes

The leaf disc agroinoculation system was applied to study tomato yellow leaf curl virus (TYLCV) replication in explants from susceptible and resistant tomato genotypes. This system was also evaluated as a potential selection tool in breeding programmes for TYLCV resistance. Leaf discs were incubated with a head-to-tail dimer of the TYLCV genome cloned into the Ti plasmid ofAgrobacterium tumefaciens. In leaf discs from susceptible cultivars (Lycopersicon esculentum) TYLCV single-stranded genomic DNA and its double-stranded DNA forms appeared within 2–5 days after inoculation. Whiteflies (Bemisia tabaci) efficiently transmitted the TYLCV disease to tomato test plants following acquisition feeding on agroinoculated tomato leaf discs. This indicates that infective viral particles have been produced and have reached the phloem cells of the explant where they can be acquired by the insects. Plants regenerated from agroinfected leaf discs of sensitive tomato cultivars exhibited disease symptoms and contained TYLCV DNA concentrations similar to those present in field-infected tomato plants, indicating that TYLCV can move out from the leaf disc into the regenerating plant. Leaf discs from accessions of the wild tomato species immune to whitefly-mediated inoculation,L. chilense LA1969 andL. hirsutum LA1777, did not support TYLCV DNA replication. Leaf discs from plants tolerant to TYLCV issued from breeding programmes behaved like leaf discs from susceptible cultivars.The Hebrew University of Jerusalem, Faculty of Agriculture, Department of Field and Vegetable Crops     

Severe outbreaks of tomato yellow leaf curl Sardinia virus in Calabria, Southern Italy

During the winter 2003--2004 a serious disease was observed in protected tomato crops in Castrovillari, Reggio Calabria province, Southern Italy. Symptoms consisted in marginal leaf yellowing, leaf curling, plant stunting, flower abortion. The disease was detected in a group of greenhouses (about 10ha) where several tomato cultivars were grown hydroponically. The highest incidence of infection (60-100%) was observed in tomatoes grafted on Beaufort DRS tomato rootstock. Since the symptoms were similar to those described for Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), detection assays for these viruses were used. In DAS-ELISA positive results were obtained with a abroad-spectrums reagent combination (distributed by Bioreba AG) detecting TYLCV, TYLCSV, and other begomoviruses. When DNA probes were used in tissue print assays, positive reactions were obtained for TYLCSV, but not for TYLCV. The two probes consisted of digoxigenin-labelled DNAs representing the coat protein gene of either TYLCSV or TYLCV. Attempts to isolate the viral agent by mechanical inoculation failed, except in few cases where Potato virus Y and Tobacco mosaic virus were identified following transmission from symptomatic plants to herbaceous indicatorpplants. By contrast, grafting onto tomato seedlings always successfully transmitted the disease. In the Castrovillari area TYLCSV was not reported before. The rootstocks that nurseries used for grafting were obtained from Sicily, where the disease is endemic and both TYLCSV and TYLCV are widespread. Probably the grafted plantlets represented the primary source of infection from which subsequent diffusion by way of the vector Bemisia tabaci followed. In fact the vector had previously been detected in both the glasshouse-grown and open field tomato crops in Calabria region. TYLCV was previously reported in a different area of Calabria in 1991, but apparently it was an occasional outbreak, and B. tabaci was not detected. Since in the Castrovillari area surveyed in the present study tomato is grown throughtout the year in protected crops, the whitefly vector of the virus is present, and some natural hosts of the virus are found, it is feared that TYLCSV may become endemic, as already happened in Sicily, Sardinia, and Spain several years ago. In Spain and Sicily TYLCV, together with TYLCSV, was reported as the causal agent of very severe tomato crop losses. Therefore the danger exists that also TYLCV will reach this area, furthermore complicating the management of tomato crops.     

Screening additional wild tomatoes for resistance to the whitefly-borne tomato yellow leaf curl virus

Plants of 25 wild Lycopersicon accessions were screened in the greenhouse for resistance to the whitefly-borne tomato yellow leaf curl virus (TYLCV). High levels of resistance were detected in 7 of 9 accessions of L. peruvianum and in all 5 accessions of L. chilense tested. In contrast, plants of 7 accessions of L. hirsutum and 3 of 4 accessions of L. pimpinellifolium were highly susceptible. Plants of accession CIAS 27 (L. pimpinellifolium) showed moderate resistance to TYLCV.     

The spread of tomato yellow leaf curl virus from the Middle East to the world

The ongoing global spread of Tomato yellow leaf curl virus (TYLCV; Genus Begomovirus, Family Geminiviridae) represents a serious looming threat to tomato production in all temperate parts of the world. Whereas determining where and when TYLCV movements have occurred could help curtail its spread and prevent future movements of related viruses, determining the consequences of past TYLCV movements could reveal the ecological and economic risks associated with similar viral invasions. Towards this end we applied Bayesian phylogeographic inference and recombination analyses to available TYLCV sequences (including those of 15 new Iranian full TYLCV genomes) and reconstructed a plausible history of TYLCV's diversification and movements throughout the world. In agreement with historical accounts, our results suggest that the first TYLCVs most probably arose somewhere in the Middle East between the 1930s and 1950s (with 95% highest probability density intervals 1905-1972) and that the global spread of TYLCV only began in the 1980s after the evolution of the TYLCV-Mld and -IL strains. Despite the global distribution of TYLCV we found no convincing evidence anywhere other than the Middle East and the Western Mediterranean of epidemiologically relevant TYLCV variants arising through recombination. Although the region around Iran is both the center of present day TYLCV diversity and the site of the most intensive ongoing TYLCV evolution, the evidence indicates that the region is epidemiologically isolated, which suggests that novel TYLCV variants found there are probably not direct global threats. We instead identify the Mediterranean basin as the main launch-pad of global TYLCV movements.     

烟粉虱及海氏桨角蚜小蜂对中国番茄黄化曲叶病毒感染烟草的嗅觉反应

双生病毒可通过调控寄主植物促进媒介昆虫烟粉虱种群增长,然而病毒侵染植物后是否通过调控植物挥发物来影响烟粉虱及其天敌的嗅觉反应还未见报道。【目的】本文旨在研究烟草植株感染中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus,TYLCCNV)后对烟粉虱Bemisia tabaci(Gennadius)及其重要寄生性天敌海氏桨角蚜小蜂Eretmocerus hayati(Zolnerowich and Rose)嗅觉反应行为的影响。【方法】利用Y形嗅觉仪方法,我们测试了烟粉虱及海氏桨角蚜小蜂对带毒植株、健康植株及烟粉虱危害植株的选择偏好性。【结果】烟粉虱及海氏桨角蚜小蜂选择携带TYLCCNV病毒的烟草显著多于健康烟草植株,但烟草被病毒与烟粉虱共同侵染时,烟粉虱对带毒烟草的选择仍显著多于无毒植株,而寄生蜂虽然仍较多选择带毒植株,但无显著差异。【结论】这些结果表明烟粉虱及海氏桨角蚜小蜂偏好选择携带TYLCCNV病毒的烟草,但这种偏好作用在烟粉虱取食共同危害时有一定程度的减弱。本研究首次报道了双生病毒侵染植物可增加烟粉虱及其天敌对植物的选择作用,并就其功能及机制进行了讨论。     

Tomato yellow leaf curl virus DNA in callus cultures derived from infected tomato leaves

Callus cultures were induced from leaves of a tomato plant infected with tomato yellow leaf curl virus (TYLCV) and analyzed for viral DNA presence during successive subcultures. No TYLCV DNA was detected in calli sampled after eight months of culture. Considerable differences in the presence of TYLCV DNA were found within sectors of a callus culture and between different callus cultures, throughout the entire eight months period. Infected calli which were cultured at sub-optimal temperature (15°C) retained the viral DNA longer than at 25 °C. The results suggested that TYLCV disappearance during callus culture was due to a disruption of some of the cell-to-cell connections, resulting in islands of infected cells in the midst of uninfected tissue and/or to the competition between the rate of cell division and that of viral DNA replication.Abbreviations BA benzyladenine - CMV cucumber mosaic virus - NAA naphthaleneacetic acid - TMV tobacco mosaic virus - TYLCV tomato yellow leaf curl virus     

Fine mapping of the tomato yellow leaf curl virus resistance gene Ty-2 on chromosome 11 of tomato

Resistances to begomoviruses, including bipartite tomato mottle virus and monopartite tomato yellow leaf curl virus (TYLCV), have been introgressed to cultivated tomato (Solanum lycopersicum) from wild tomato accessions. A major gene, Ty-2 from S. habrochaites f. glabratum accession “B6013,” that confers resistance to TYLCV was previously mapped to a 19-cM region on the long arm of chromosome 11. In the present study, approximately 11,000 plants were screened and nearly 157 recombination events were identified between the flanking markers C2_At1g07960 (82.5 cM, physical distance 51.387 Mb) and T0302 (89 cM, 51.878 Mb). Molecular marker analysis of recombinants and TYLCV evaluation of progeny from these recombinants localized Ty-2 to an approximately 300,000-bp interval between markers UP8 (51.344 Mb) and M1 (51.645 Mb). No recombinants were identified between TG36 and C2_At3g52090, a region of at least 115 kb, indicating severe recombination suppression in this region. Due to the small interval, fluorescence in situ hybridization analysis failed to clarify whether recombination suppression is caused by chromosomal rearrangements. Candidate genes predicted based on tomato genome annotation were analyzed by RT-PCR and virus-induced gene silencing. Results indicate that the NBS gene family present in the Ty-2 region is likely not responsible for the Ty-2-conferred resistance and that two candidate genes might play a role in the Ty-2-conferred resistance. Several markers very tightly linked to the Ty-2 locus are presented and useful for marker-assisted selection in breeding programs to introgress Ty-2 for begomovirus resistance.     

Chinese tomato yellow leaf curl virus--a new species of geminivirus

Chine tomato yellow leaf curl virus (TYLCV-CHI) and other geminiviruses were analysed with 20 monoclonaI antibodies. It was shown that TYLCV-CHI is serclogicaIly close to Chinese tabacco Ieaf curl virus (TbLCV-CHI). The fragment of TYLCV-CHI DNA including the common region (CR), N-terminal of coat protein gene and AV1 gene was amplified by PCR and cloned, and its DNA sequence was determined. These raults showed that TYLCV-CHI is different from other known geminiviruses in the world, and is a new whitefly-transmitted gerninivirus. Project supported by tile National Natural Science Foundation of China (Grant No. 39470034) and Chins-Israel Fund for Scientific and Strategic Reserch and Development. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and Genebank nucleotide sequence databases with the accession number D88773.