Coat protein-mediated transgenic resistance in tomato against a IB subgroup Cucumber mosaic virus strain

Transgenic tomato plants containing the coat protein (CP) gene of Cucumber mosaic virus (CMV) of subgroup IB were developed through Agrobacterium-mediated transformations. The progenies of transgenic plants showed the presence of transgene, its expression and translation of 26 KDa CP. The T₁ and T₂ generation plants were evaluated for resistance against challenge inoculations by a homologous strain of CMV. Visual observations of challenged transgenic plants categorized them into resistant, tolerant and susceptible as compared with untransformed control plants. Out of 33 plants of the T₁ generation, 36.3% showed resistance and remained symptomless throughout their life, 48.4% showed tolerance which developed delayed symptoms of mild mosaic, and 15.1% showed susceptibility to CMV which developed severe systemic mosaic and leaf distortion symptoms after 30?days of virus challenge. Out of 120 plants of the T₂ generation, 60% showed resistance, 26.6% were tolerant and only 13.3% were found susceptible to challenge inoculations of CMV. Resistant transgenic plants also showed less CP accumulation in systemic upper leaves as compared with challenged untransformed plants. In this study, CP of a CMV subgroup IB strain has demonstrated a significant level of resistance in transgenic tomato plants against the CMV strain. The strategy may be applied for better quality and productivity of tomato crops.     

Nucleocapsid Gene-Mediated Transgenic Resistance Provides Protection Against Tomato spotted wilt virus Epidemics in the Field

ABSTRACT Transformation of plants with the nucleocapsid (N) gene of Tomato spotted wilt tospovirus (TSWV) provides resistance to disease development; however, information is lacking on the response of plants to natural inoculum in the field. Three tobacco cultivars were transformed with the N gene of a dahlia isolate of TSWV (TSWV-D), and plants were evaluated over several generations in the greenhouse. The resistant phenotype was more frequently observed in 'Burley 21' than in 'KY-14' or 'K-326', but highly resistant 'Burley 21' transgenic lines were resistant to only 44% of the heterologous TSWV isolates tested. Advanced generation (R(3) and R(4)) transgenic resistant lines of 'Burley 21' and a 'K-326' F(1) hybrid containing the N genes of two TSWV isolates were evaluated in the field near Tifton, GA, where TSWV is endemic. Disease development was monitored by symptom expression and enzyme-linked immunosorbent assay (ELISA) analysis. Whereas incidence of TSWV infection in 'Burley 21' susceptible controls was 20% in 1996 and 62% in 1997, the mean incidence in transgenic lines was reduced to 4 and 31%, respectively. Three transgenic 'Burley 21' lines were identified that had significantly lower incidence of disease than susceptible controls over the two years of the study. In addition, the rate of disease increase at the onset of the 1997 epidemic was reduced for all the 'Burley 21' transgenic lines compared with the susceptible controls. The 'K-326' F(1) hybrid was as susceptible as the 'K-326' nontransformed control. ELISA analysis demonstrated that symptomless plants from the most resistant 'Burley 21' transgenic lines accumulated detectable nucleocapsid protein, whereas symptomless plants from more susceptible lines did not. We conclude that transgenic resistance to TSWV is effective in reducing incidence of the disease in the field, and that accumulation of transgene protein may be important in broad-spectrum resistance.     

Development of a Scale for Evaluation of Tomato yellow leaf curl virus Resistance Level in Tomato Plants

ABSTRACT We have developed a scale of differential hosts that enables the determination and comparison of level of resistance to Tomato yellow leaf curl virus (TYLCV) expressed by resistant tomato lines or by individual plants in a segregating population. The scale is composed of seven different homozygous tomato genotypes that exhibit different levels of TYLCV resistance, ranging from fully susceptible to highly resistant. The differential hosts composing the scale were inoculated with TYLCV under greenhouse conditions. Four weeks after inoculation the plants were evaluated for disease symptom severity, and virus DNA titer was determined. The different genotypes were arranged in the scale according to symptom severity score. The different genotypes were then tested under different environmental conditions, inoculated at different ages, and tested in a field experiment assaying TYLCV-induced yield reduction. While the symptom severity score of each individual resistant genotype changed under different environmental conditions, the relative position on the scale did not alter, except for one genotype. Thus, to evaluate disease resistance of a given tomato genotype, the genotype in question should be inoculated alongside the differential hosts composing the scale, and within 4 weeks one can determine the relative level of resistance of the tested genotype.     

Resistance enhancement of transgenic tomato to bacterial pathogens by the heterologous expression of sweet pepper ferredoxin-I protein

ABSTRACT Expression of a foreign gene to enhance plant disease resistance to bacterial pathogens is a favorable strategy. It has been demonstrated that expressing sweet pepper ferredoxin-I protein (PFLP) in transgenic plants can enhance disease resistance to bacterial pathogens that infect leaf tissue. In this study, PFLP was applied to protect tomato (Lycopersicon esculentum cv. cherry Cln1558a) from the root-infecting pathogen, Ralstonia solanacearum. Independent R. solanacearum resistant T(1) lines were selected and bred to produce homozygous T(2) generations. Selected T(2) transgenic lines 24-18-7 and 26-2-1a, which showed high expression levels of PFLP in root tissue, were resistant to disease caused by R. solanacearum. In contrast, the transgenic line 23-17-1b and nontransgenic tomato, which showed low expression levels of PFLP in root tissue, were not resistant to R. solanacearum infection. The expansion of R. solanacearum populations in stem tissue of transgenic tomato line 24-18-7 was limited compared with the nontransgenic tomato Cln1558a. Using a detached leaf assay, transgenic line 24-18-7 was also resistant to maceration caused by E. carotovora subsp. carotovora; however, resistance to E. carotovora subsp. carotovora was less apparent in transgenic lines 26-2-1a and 23-17-1b. These results demonstrate that PFLP is able to enhance disease resistance at different levels to bacterial pathogens in individual tissue of transgenic tomato.     

西瓜抗小西葫芦黄花叶病毒基因的连锁分子标记研究

 小西葫芦黄花叶病毒中国株系(Zucchini yellow mosaic virus Chinese strain,ZYMV-CH)是危害我国西瓜的主要病毒。本实验以抗病毒病西瓜野生种质P.I.595203与感病的普通西瓜自交系98R为亲本,采用单粒传方式得到109个E代株系,分别对亲本、F₁及109个F₃代株系群体进行了苗期抗ZYMV-CH接种鉴定,通过F₃代群体的抗感分离情况,推测得到F₂代各单株的基因型,采用集团分离分析法(bulked segregant analysis,BSA)在F₂代建立抗感基因池,以亲本、F₁和抗感基因池为模板,对640条RAPD引物进行PCR扩增筛选,其中引物AK13在亲本、F₁和抗感基因池之间扩增出一条多态性片段(644bp),在F₂代群体上验证该多态性条带与ZYMV-CH的抗性基因呈现连锁关系,遗传连锁距离为8cM,定名为AK13_-644,该连锁标记在ZYMV-CH抗性转育后代自交系上得到了验证。最终将此RAPD标记成功转化成SCAR标记SCAK13_-644,该标记可以作为西瓜抗病毒病辅助选择的分子标记。     

Potato <Emphasis Type="Italic">R1</Emphasis> resistance gene confers resistance against <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in transgenic tomato plants

Tomato is challenged by several pathogens which cause loss of production. One such pathogen is the oomycete Phytophthora infestans which is able to attack all the aerial parts of the plant. Although a wide range of resistance sources are available, genetic control of this disease is not yet successful. Pyramiding R-genes through genetic transformation could be a straightforward way to produce tomato and potato lines carrying durable resistance to P. infestans. In this work the R1 potato gene was transferred into tomato lines. The tomato transgenic lines were analyzed by using q-RT-PCR and progeny segregation to determine the gene copy number. To test the hypothesis that R1 represents a specifically regulated R-gene, transgenic tomato plants were inoculated with P. infestans isolate 88133 and IPO. All the plants containing the R1 gene were resistant to the late blight isolate IPO-0 and susceptible to isolate 88133. These results provide evidence for specific activation of the R1 gene during pathogen challenge. Furthermore, evidence for enhancement of PR-1 gene expression during P. infestans resistance response was obtained.     

Comparison of Resistance to Tomato Leaf Curl Virus (India) and Tomato Yellow Leaf Curl Virus (Israel) among Lycopersicon Wild Species, Breeding Lines and Hybrids

The objective of this study was to screen wild and domesticated tomatoes for resistance to Tomato yellow leaf curl virus, Israel (TYLCV-Is) and Tomato leaf curl virus from Bangalore isolate 4, India (ToLCV-[Ban4]) to find sources of resistance to both viruses. A total of 34 tomato genotypes resistant/tolerant to TYLCV-Is were screened for resistance to ToLCV-[Ban4] under glasshouse and field conditions at the University of Agricultural Sciences, Bangalore, India. Resistance was assessed by criteria like disease incidence, symptom severity and squash-blot hybridization. All the tomato genotypes inoculated with ToLCV-[Ban4] by the whitefly vector Bemisia tabaci (Gennadius) produced disease symptoms. In some plants of the lines 902 and 910, however, the virus was not detected by hybridization. The tomato genotypes susceptible to ToLCV-[Ban4] by whitefly-mediated inoculation were also found susceptible to the virus under field conditions. However, there were substantial differences between genotypes in disease incidence, spread, symptom severity and crop yield. Despite early disease incidence, many genotypes produced substantially higher yields than the local hybrid, Avinash-2. Sixteen tomato genotypes from India resistant/tolerant to ToLCV-[Ban4] were also tested for TYLCV-Is resistance at the Hebrew University of Jerusalem, Rehovot, Israel. Accessions of wild species, Lycopersicon hirsutum LA 1777 and PI 390659 were the best sources of resistance to both viruses. Lines 902 and 910, which were, resistant to TYLCV-Is were only tolerant to ToLCV-[Ban4] and accession Lycopersicon peruvianum CMV Sel. INRA, resistant to ToLCV-[Ban4], was only tolerant to TYLCV-Is. Implications of using the resistant lines in breeding programme is discussed.     

烟草品种对烟草花叶病毒和黄瓜花叶病毒的抗性鉴定

 2010-2011年采用大田人工接种鉴定的方法,对生产中大面积推广使用的24份烟草品种进行了烟草花叶病毒（TMV）和黄瓜花叶病毒（CMV）的抗性鉴定。结果表明,供试品种对TMV和CMV的抗病性存在较大差异,对TMV表现抗病的有Coker86、吉烟5号、Coker176、CV87、辽烟8号、CV91、中烟90等7份材料;表现中抗的有秦烟98、双抗70、C151等3份材料;表现中感的有秦烟96、G80、金星6007、龙江981、K326、秦烟201、NC89等7份材料;表现感病的有G28、云烟97、净叶黄、红花大金元、RG11、云烟85、云烟87等7份材料。对CMV表现抗病的有Coker86、龙江981、C151、秦烟201、云烟87等5份材料;表现中抗的材料是金星6007;表现中感的有CV91、RG11、Coker176、中烟90、K326、红花大金元、净叶黄、G80、G28等9份材料;表现感病的有秦烟98、云烟85、秦烟96、NC89、双抗70、云烟97、CV87、辽烟8号、吉烟5号等9份材料。其中兼抗TMV和CMV两种病毒病的材料有2份,分别是Coker86和C151。同时研究还发现,抗病性不同的烟草品种在受到病毒危害以后,对烟叶的产量和品质的影响也不同。明确了中国24个烟草品种的抗病性水平,为抗病品种的利用与品种合理布局提供科学依据,同时为烟草抗病毒病育种的亲本选择提供抗源信息。     

Broad-Spectrum Resistance to Different Geographic Strains of Papaya ringspot virus in Coat Protein Gene Transgenic Papaya

ABSTRACT Papaya ringspot virus (PRSV) is a major limiting factor for cultivation of papaya (Carica papaya) in tropical and subtropical areas throughout the world. Although the coat protein (CP) gene of PRSV has been transferred into papaya by particle bombardment and transgenic lines with high resistance to Hawaii strains have been obtained, they are susceptible to PRSV isolates outside of Hawaii. This strain-specific resistance limits the application of the transgenic lines in other areas of the world. In this investigation, the CP gene of a local strain isolated from Taiwan, designated PRSV YK, was transferred into papaya via Agrobacterium-mediated transformation. A total of 45 putative transgenic lines were obtained and the presence of the transgene in papaya was confirmed by polymerase chain reaction amplification. When the plants of transgenic lines were challenged with PRSV YK by mechanical inoculation, they showed different levels of resistance ranging from delay of symptom development to complete immunity. Molecular analysis of nine selected lines that exhibited different levels of resistance revealed that the expression level of the transgene is negatively correlated with the degree of resistance, suggesting that the resistance is manifested by a RNA-mediated mechanism. The segregation analysis showed that the transgene in the immune line 18-0-9 has an inheritance of two dominant loci and the other four highly resistant lines have a single dominant locus. Seven selected lines were tested further for resistance to three PRSV heterologous strains that originated in Hawaii, Thailand, and Mexico. Six of the seven lines showed varying degrees of resistance to the heterologous strains, and one line, 19-0-1, was immune not only to the homologous YK strain but also to the three heterologous strains. Thus, these CP-transgenic papaya lines with broad-spectrum resistance have great potential for use in Taiwan and other geographic areas to control PRSV.     

Occurrence of Tomato spotted wilt virus (TSWV) in Jordan

Tomato spotted wilt virus was recorded for the first time in Jordan on tomato plants. Severe disease symptoms were observed in different tomato farms in the Jordan Valley. Using a specific primer pair a fragment of the capsid protein gene of the virus has been amplified by RT-PCR and IC-RT-PCR. The amplified PCR product was cloned and sequenced. Sequence analysis revealed that the Jordanian isolate of TSWV shared high nucleotide similarities with other isolates from different countries. The sequence of the capsid protein gene was deposited in GenBank under the accession number AY646682 . The response of different tomato breeding lines and hybrids, previously developed for resistance against Tomato yellow leaf curl virus (TYLCV) were tested for their reaction to TSWV infection. All tested lines and hybrids were susceptible to TSWV infection. This has been confirmed at the molecular level by using the SCAR 421 marker linked to the TSWV resistance gene Sw-5 .     

Effect of Host Plant Resistance to Tomato yellow leaf curl virus (TYLCV) on Virus Acquisition and Transmission by Its Whitefly Vector

ABSTRACT The effect that Tomato yellow leaf curl virus (TYLCV)-infected resistant tomato plants may have on virus epidemiology was studied. Four tomato genotypes that exhibit different levels of viral resistance, ranging from fully susceptible to highly resistant, served as TYLCV-infected source plants. Viral acquisition and transmission rates by white-flies following feeding on the different source plants were evaluated. TYLCV transmission rate by whiteflies that had fed on infected source plants 21 days postinoculation (DPI), shortly after the appearance of TYLCV symptoms, was negatively correlated with the level of resistance displayed by the source plant. Therefore, the higher the resistance, the lower the transmission rate. In addition, TYLCV DNA accumulation was shown to be lower in the resistant source plants compared with the susceptible plants. Whitefly survival rate, following feeding on source plants 21 DPI, was similar for all the cultivars tested. Significant differences in whitefly survival were found, however, following feeding on the infected source plants at 35 DPI; here, whitefly survival rate increased with higher levels of resistance displayed by the source plant. At 35 DPI, the susceptible plants had developed severe TYLCV disease symptoms, and transmission rates from these plants were the lowest, presumably due to the poor condition of these plants. Transmission rates from source plants displaying a medium level of resistance level were highest, with rates declining following feeding on source plants displaying higher levels of TYLCV resistance. TYLCV DNA accumulation in whiteflies following feeding on infected source plants at both 21 and 35 DPI was directly correlated with viral DNA accumulation in source plants. Results show that, in essence, the higher the resistance expressed, the less suitable the plant was as a viral source. Consequently, following acquisition from a highly resistant plant, TYLCV transmission by whiteflies will be less efficient.     

百合黄瓜花叶病毒及其检测

应用酶联免疫吸附法(ELISA)检测了浙江丽水和杭州的东方百合、亚洲百合等栽培品种上黄瓜花叶病毒(CMV),64个田间样品中有26个带毒,检出率为40.6%;dsRNA检测结果与已知CMV-FQS株系的电泳条带相同,但百合组织中CMVdsRNA含量较低;电镜观察,受CMV侵染的样品中大多含有线状病毒,复合侵染率为35.9%;寄主反应测定显示从百合植株上获得的CMV株系均不能通过汁液摩擦接种侵染昆诺藜、苋色藜、普通烟、心叶烟等6科10种指示植物。     

Identification and Subgrouping of Cucumber mosaic virus with Mouse Monoclonal Antibodies

ABSTRACT Using a mixture of isolates of Cucumber mosaic virus (CMV) from subgroups I and II as immunogens, 20 mouse hybridoma cell lines secreting monoclonal antibodies were produced. A reliable method for efficient detection and accurate subgrouping of CMV isolates has been developed. Tests with 12 well-characterized strains of CMV and other cucumoviruses demonstrated the presence of epitopes that were virus and subgroup specific. Analyses of 109 accessions of CMV isolates collected from various parts of the world revealed 70% were subgroup I, with 20% identified as subgroup II. Seven isolates (6%) did not react with group-specific antibodies but did react with antibodies that recognized all CMV isolates. Differential reactions among isolates suggested a total of 10 epi-topes were recognized. The antigenic diversity among subgroup II CMVs was greater than for the subgroup I isolates, even though fewer subgroup II isolates were tested.     

Occurrence of Cucumber mosaic virus Subgroup IA in tomato in Pakistan

Journal of Plant Diseases and Protection - Infection of tomato in Faisalabad, Pakistan with subgroup IA strain of Cucumber mosaic virus (CMV) is reported for the first time. The virus was detected...     

A transgenic RNAi approach for developing tomato plants immune to Pepino mosaic virus

RNA interference (RNAi) or gene silencing is a natural defence response of plants to invading viruses. Here, we applied this approach against pepino mosaic virus (PepMV) isolates in their natural host, tomato. PepMV isolates differ in their genetic sequences, the severity of the disease they induce, and their worldwide distribution. PepMV causes heavy crop losses, mainly due to impaired tomato fruit quality. Resistant varieties are not yet available, despite many years of resistance breeding efforts within the tomato seed industry. To generate broad resistance to PepMV strains, conserved sequences from three different strains of PepMV (US1, LP, and CH2) were synthesized as a single insert and cloned in a hairpin configuration into a binary vector, which was used to transform tomato plants. Transgenic tomato lines that expressed a high level of transgene-siRNA exhibited immunity to PepMV strains, including a new Israeli isolate. This immunity was maintained even after graft inoculation, in which a transgenic scion was grafted onto nontransgenic infected rootstocks. However, an immune transgenic rootstock was unable to induce resistance in a nontransformed scion. These results provide the first example of engineered immunity to diverse PepMV strains in transgenic tomato based on gene silencing.     

Resistance of <Emphasis Type="Italic">Solanum</Emphasis> species to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> subgroup IA and its vector <Emphasis Type="Italic">Myzus persicae</Emphasis>

Sixty-nine tomato genotypes representing nine Solanum species were evaluated for resistance to Cucumber mosaic virus (CMV) subgroup IA and its aphid vector Myzus persicae. Resistance was assessed by visual scoring of symptoms in the field under natural conditions, and in the greenhouse by artificial inoculations through aphid M. persicae and mechanical transmissions in the year 2007 and 2009. Considerable variation in responses was observed among the evaluation methods used. Field evaluations were found liable to errors as different levels were observed for the same genotypes in the different years, however mechanical inoculation was found to be the most useful in identifying CMV subgroup IA resistance, in contrast aphid transmission was most useful in identifying insect transmission resistance. All genotypes observed as highly resistant to CMV subgroup IA in the field or through vector transmission became systemically infected through mechanical inoculations. Using mechanical inoculation, six genotypes (TMS-1 of S. lycopersicum, LA1963 and L06049 of S. chilense, LA1353, L06145 and L06223 of S. habrochaites) were found resistant and another six (L06188 and L06238 of S. neorickii, L06219 of S. habrochaites, L05763, L05776 and L06240 of S. pennellii) were found tolerant showing mild symptoms with severity index (SI) ranging 1-2 and with delayed disease development after a latent period (LP) of 18–30 days. However, these genotypes were found to be resistant to highly resistant in the field and through inoculation by M. persicae; and they also supported low population levels of M. persicae except TMS-1. Another nine genotypes (LA2184 of S. pimpinellifolium L., LA2727 of S. neorickii, LA0111, L06221, L06127 and L06231 of S. peruvianum L., LA1306, L06057 and L06208 of S. chmielewskii) showing a susceptible response after mechanical inoculation were highly resistant, resistant and tolerant after M. persicae transmission. The resistant genotypes, identified in the present study can be exploited in the breeding programmes aimed at developing tomato varieties resistant to CMV subgroup IA and broadening the genetic base of CMV-resistant germplasm. The differences observed between mechanical and aphid transmission suggests that one should consider both evaluation methods for tomato germplasm screening against CMV subgroup IA.     

马铃薯Y病毒复制酶基因不同位置cDNA区段介导对PVY的不同抗性

为探究靶序列位置对RNA介导的病毒抗性产生的影响,利用聚合酶链式反应(polymerase chain reaction,PCR)技术扩增马铃薯Y病毒(Potato virus Y,PVY)复制酶基因(nuclear inclusion b,NIb)不同位置的cDNA区段,反向插入双元载体pROKII中,构建了发夹RNA(hairpin RNA,hpR-NA)结构的植物表达载体。将构建的植物表达载体采用冻融法转入农杆菌LBA4404,叶盘法转化烟草NC89,获得转基因植株。攻毒试验表明:PVYNIb基因不同位置cDNA区段介导的对PVY的抗性存在显著差异;3′端1/2处和中间位置的序列可介导高水平的病毒抗性,抗性植株的比例在50%以上,而5′端、5′端1/2处和3′端的序列介导的抗性效率较低,抗性植株的比例仅为10%~30%。Northern杂交显示:抗病植株中RNA的积累量明显低于同类型的感病植株,抗性与RNA积累量呈负相关;抗病转基因植株中有siRNA存在,表明病毒抗性是由RNA介导的。     

Synergistic Interaction Between Tomato chlorosis virus and Tomato spotted wilt virus Results in Breakdown of Resistance in Tomato

ABSTRACT Multiple viral infections frequently are found in single plants of cultivated and wild hosts in nature, with unpredictable pathological consequences. Synergistic reactions were observed in mixed infections in tomato plants doubly infected with the positive-sense and phloem-limited single-stranded RNA (ssRNA) crinivirus Tomato chlorosis virus (ToCV) and the negative-sense ssRNA tospovirus Tomato spotted wilt virus (TSWV). Synergism in a tomato cultivar susceptible to both viruses resulted in a rapid death of plants. A pronounced enhancement of ToCV accumulation mediated by TSWV co-infection was observed with no evident egress of ToCV from phloem tissues. No consistent alteration of TSWV accumulation was detected. More remarkable was the synergism observed in tomato cultivars which carry the Sw-5 resistance gene, which are resistant to TSWV. Pre-infection with ToCV resulted in susceptibility to TSWV, whereas co-inoculations did not. This suggested that a threshold level or a time lapse is needed for ToCV to interfere or downregulate the defense response in the TSWV-resistant plants.