A Variant of Cucumber mosaic virus Is Restricted to Local Lesions in Inoculated Tobacco Leaves with a Hypersensitive Response

A variant of Cucumber mosaic virus, CMV(Y/GM2), was isolated from a tobacco plant with mild green mosaic symptoms that was regenerated in vitro from a yellow strain of CMV [CMV(Y)]-infected tobacco leaves by tissue culture. CMV(Y/GM2) has two amino acid substitutions at 36 and 111 positions in the coat protein encoded on RNA3. CMV, assembled by mixing in vitro transcribed CMV(Y) RNA1 and RNA2 plus infectious RNA3 transcribed in vitro from cDNA to RNA3 of CMV(Y/GM2), was prepared and designated as CMV(Y/GM2)tr. When tobacco (Nicotiana tabacum cv. Xanthi nc) plants were inoculated with CMV(Y/GM2)tr, large necrotic local lesions in which the virus was localized, developed on the inoculated leaves. This host response unique to CMV(Y/GM2)tr was similar to the hypersensitive response (HR), which is a common resistance response to avirulent pathogens and was observed in five cultivars of Nicotiana tabacum and eight Nicotiana species. The revertant virus, however, accumulated to quite different levels in the various hosts. CMV(Y/GM2)tr induced pathogenesis-related 1 (PR-1) protein accumulation and systemic acquired resistance (SAR) which were generally observed in the HR. However, when tobaccos were inoculated with CMV(S36P)tr and CMV(V111I)tr, which have an amino acid substitution at either the 36 or 111 position in the coat protein of CMV(Y), respectively, CMV(S36P)tr was restricted to the primary infection site without necrotic local lesion formation and PR-1 protein and SAR induction. CMV(V111I)tr, however, systemically spread and induced mild green mosaic symptoms, while the host had the HR to CMV(Y/GM2)tr. The localization of CMV(Y/GM2)tr at the primary infection site may not only be caused by the HR, but also by the restriction of virus systemic movement resulting from the amino acid substitution at position 36 in the coat protein of CMV(Y). Received 15 December 1999/ Accepted in revised form 18 April 2000     

Occurrence and diversity of Tomato spotted wilt virus isolates breaking the Tsw resistance gene of Capsicum chinense in Yunnan,southwest China

Widely used resistant peppers (Capsicum spp.) bearing the Tsw locus triggered the rapid emergence of resistance‐breaking (RB) isolates of Tomato spotted wilt virus (TSWV) around the world. However, although TSWV‐induced diseases have rapidly increased in Yunnan, southwest China, in recent years, no information is available about the diversity of TSWV isolates in this region. In this study, the occurrence of natural TSWV RB variants among isolates collected in Yunnan is reported. Initially, a TSWV isolate from asparagus lettuce (TSWV‐LE) was collected in Yunnan in 2012. Surprisingly, this isolate of TSWV induced systemic necrosis on pepper carrying the Tsw resistance gene. Novel TSWV isolates, collected in 2015, included a tomato isolate (TSWV‐YN18) and a tobacco isolate (TSWV‐YN53) that also overcame Tsw‐mediated resistance. TSWV‐YN18 induced systemic ringspots, whereas TSWV‐YN53 caused systemic chlorotic mottling. Variations in the TSWV nonstructural (NSs) protein are the key determinants associated with Tsw resistance‐breaking isolates. It was found that TSWV‐LE NSs retained the hypersensitive response (HR) induction, whereas TSWV‐YN18 and TSWV‐YN53 NSs were unable to induce HR. However, the NSs of all three RB isolates suppressed RNA silencing. Sequence analysis of the NSs revealed that RB isolates of Yunnan have no amino acid mutation sites common to other previously reported RB isolates. However, two amino acids (F74 and K272) on TSWV‐LE NSs make it distinct from TSWV‐YN18 and TSWV‐YN53. The occurrence of different RB isolates and the failure of Tsw‐mediated resistance control pose serious threats to domestic pepper crops in southwest China.     

Involvement of a vascular hypersensitive response in quantitative resistance to Ralstonia solanacearum on tomato rootstock cultivar LS‐89

Ralstonia solanacearum causes bacterial wilt disease in Solanaceae spp. Expression of the Phytophthora inhibitor protease 1 (PIP1) gene, which encodes a papain‐like extracellular cysteine protease, is induced in R. solanacearum‐inoculated stem tissues of quantitatively resistant tomato cultivar LS‐89, but not in susceptible cultivar Ponderosa. Phytophthora inhibitor protease 1 is closely related to Rcr3, which is required for the Cf‐2‐mediated hypersensitive response (HR) to the leaf mould fungus Cladosporium fulvum and manifestation of HR cell death. However, up‐regulation of PIP1 in R. solanacearum‐inoculated LS‐89 stems was not accompanied by visible HR cell death. Nevertheless, upon electron microscopic examination of inoculated stem tissues of resistant cultivar LS‐89, several aggregated materials associated with HR cell death were observed in xylem parenchyma and pith cells surrounding xylem vessels. In addition, the accumulation of electron‐dense substances was observed within the xylem vessel lumen of inoculated stems. Moreover, when the leaves of LS‐89 or Ponderosa were infiltrated with 10⁶ cells mL^?1 R. solanacearum, cell death appeared in LS‐89 at 18 and 24 h after infiltration. The proliferation of bacteria in the infiltrated leaf tissues of LS‐89 was suppressed to approximately 10–30% of that in Ponderosa, and expression of the defence‐related gene PR‐2 and HR marker gene hsr203J was induced in the infiltrated tissues. These results indicated that the response of LS‐89 is a true HR, and induction of vascular HR in xylem parenchyma and pith cells surrounding xylem vessels seems to be associated with quantitative resistance of LS‐89 to R. solanacearum.     

转CP基因线辣椒对CMV和CMV-RNA的抗病性比较

 本试验以转化CMV-CP和TMV-CP基因线辣椒纯合系作试材,比较了接种CMV粒体和CMV-RNA后的发病特点和叶片中的病毒含量。结果表明:转化线辣椒不仅能抵抗CMV粒体的侵染,而且还能抵抗CMV-RNA的侵染。不论接种CMV粒体或CMV-RNA,CP(+)线辣椒的系统症状都延迟出现,显症株率和病害严重度级别大幅度降低,病毒增殖和运转受到抑制,接种叶片与新生叶片中的病毒含量明显减低。这一结果证实CMV-RNA不能克服线辣椒由CP基因介导的抗病性。     

Production of discernible disease phenotypes in Canna by five plant viruses belonging to the genera Potyvirus,Cucumovirus and Badnavirus

Cannas are tropical and subtropical flowering perennial plants. The genus contains many species but most commercially grown cultivars are interspecific hybrids selected for their attractive foliage and flowers. Canna production is so lucrative that there are farmers and nurseries dedicated solely to its production. The specific issue that the canna industry faces is virus diseases. In this study, rhizomes of 24 canna cultivars were gathered and diagnostics conducted to detect Bean yellow mosaic virus (BYMV, Potyvirus), Canna yellow mottle virus (CaYMV, Badnavirus), Canna yellow streak virus (CaYSV, Potyvirus), Cucumber mosaic virus (CMV, Cucumovirus) and Tomato aspermy virus (TAV, Cucumovirus). Visual assessment of disease symptoms and diagnostic tests were carried out to identify the prevalent diseases and describe the symptoms that are associated with virus infection. BYMV, CaYMV and CaYSV caused severe mosaic and necrosis either in the leaf lamina or veins of infected leaves. Potyvirus infection suppressed red colouration in the foliage of some varieties. CaYMV and CaYSV often appeared in the same plant, suggesting they might represent a viral complex. CMV and TAV were rarely seen in these populations. Interestingly, CaYMV but not CaYSV could be mechanically inoculated to Phaseolus vulgaris plants.     

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。     

Overexpression of barley oxalate oxidase gene induces partial leaf resistance to Sclerotinia sclerotiorum in transgenic oilseed rape

Sclerotinia stem rot (SSR) is a severe disease of oilseed rape, which severely impacts the crop productivity worldwide. Sclerotinia sclerotiorum causes SSR, resulting in the secretion of oxalic acid (OA), which can be further degraded to carbon dioxide (CO₂) and hydrogen peroxide (H₂O₂) by oxalate oxidase (OXO). In the present investigation, the barley oxalate oxidase (BOXO, Y14203) gene was introduced into oilseed rape by Agrobacterium‐mediated transformation to investigate the mechanism by which OXO promotes resistance to S. sclerotiorum. Compared to the control 72 h post‐inoculation, there were c. 15–61% fewer lesions on leaves of the transgenic oilseed rape, which thus exhibited a detectable level of partial resistance in leaf tissue to S. sclerotiorum. Transgenic oilseed rape also showed decreased oxalate and increased hydrogen peroxide levels compared to the control, and the expression of defence response genes involved in the hydrogen peroxide signalling pathway was also induced. Therefore, the improved resistance of oilseed rape could be attributed to the enhanced OA metabolism, production of hydrogen peroxide and the hydrogen peroxide‐mediated defence levels during infection.     

Detection of Tomato yellow leaf curl virus in imported tomato fruit in northern Europe

Imported tomato fruits infected with Tomato yellow leaf curl virus (TYLCV) were identified on the market in northern Europe using paper‐based FTA Classic Cards (Whatman), polymerase chain reaction (PCR) and partial DNA sequence analysis. Trade tomatoes originating from southern Europe, Africa and the Middle East were sampled in Estonia and Sweden, and tested for infection with begomoviruses. Out of 100 batches analysed with five fruits sampled in each batch (58 batches from Estonia and 42 from Sweden), 20 batches were positive (16 from Estonia and four from Sweden). Rolling circle amplification (RCA) and full‐length genome sequence analysis of one isolate collected in Estonia and one isolate in Sweden, revealed highest nucleotide sequence identity at 99% to TYLCV‐IL for the Estonian isolate and at 97% to TYLCV‐Mld for the Swedish isolate. In this study, TYLCV was identified for the first time in imported tomato fruits on the market in northern Europe. FTA cards proved to be an effective means to collect, extract and store begomovirus DNA from tomato fruits and the subsequent molecular analysis.     

Cucumber mosaic virus populations in Tunisian pepper crops are mainly composed of virus reassortants with resistance‐breaking properties

Cucumber mosaic virus is one of the most prevalent viruses in Tunisian pepper crops, where it has been detected in 68% of plants developing mosaic symptoms, making it essential to characterize the molecular and biological properties of local CMV populations. Two hundred and seventy‐eight isolates collected in the late 1990s, 2006 and 2008–2010 were characterized genetically. Isolates belonging to the three phylogenetic subgroups of CMV (IA, IB and II) were detected, but surprisingly, 90% of the isolates were reassortants between subgroups IA and IB, with two predominant haplotypes, IB‐IA‐IA and IB‐IA‐IB (nomenclature according to the subgrouping of the three genomic RNAs). The IB‐IA‐IA haplotype was present in all regions surveyed, while IB‐IA‐IB was observed only in northern Tunisia. This situation was unexpected, because CMV reassortants were previously thought to be counterselected in nature, and this raises the questions of the origin of IB strains in Tunisia and of the widespread distribution of these two reassortant types. Phylogenetic studies revealed low diversity within haplotypes, whatever the locality or the year of sampling. However, analysis of haplotype frequencies revealed a high genetic differentiation between CMV populations, which was better explained by the localities of sampling than by years. Geographic distances affected the differentiation of CMV populations, mainly between north and central Tunisia. When tested against a polygenic resistance to CMV movement in pepper, 55 of 57 isolates tested were able to break the resistance, indicating that this resistance would not be useful for controlling CMV in Tunisian pepper fields.     

Survey for CMV in field samples of Musa spp. in southern Nigeria

Symptomatic and asymptomatic plantain and banana (Musa spp.) leaf samples were collected in 1998, 1999, 2000 and 2004 to determine the frequency of infection by Cucumber mosaic virus (CMV) in 11 major plantain- and banana-growing states of southern Nigeria in which virus symptoms had been reported. Of 996 Musa plants sampled, 641 (66.4%) were infected with CMV, as determined by dot blot immunoassay and ELISA. CMV occurred in 64 out of 84 villages and in all the states sampled. Incidences of CMV in the leaf samples were variable in survey years, ranging between 16.7 and 100%. Major virus-induced symptoms were interveinal chlorosis and chlorotic streaks, observed in 29.3 and 26.7% of the total number of surveyed Musa plants. Low incidences of general leaf chlorosis, leaf crinkling/puckering, vein thickening and chlorotic flecking between 1.4 and 10.5% were also observed. Multiple viral infections in the total number of surveyed Musa plants accounted for 5.4% while 7.9% were asymptomatic. CMV induced some of the viral infections on Musa plants in southern Nigeria.     

Induction of systemic resistance against Cucumber mosaic virus by Penicillium simplicissimum GP17‐2 in Arabidopsis and tobacco

The plant growth‐promoting fungus, Penicillium simplicissimum GP17‐2, was evaluated for its ability to induce resistance against Cucumber mosaic virus (CMV) in Arabidopsis thaliana and tobacco plants. Treatment with barley grain inoculum (BGI) of GP17‐2 significantly enhanced fresh weight, dry weight and leaf number of A. thaliana and tobacco plants 6 weeks after planting. Two weeks after CMV inoculation, all plants treated with BGI of GP17‐2 or its culture filtrate (CF) showed a significant reduction in disease severity compared with non‐treated control plants, which exhibited severe mosaic symptoms by the end of the experiment. The enzyme‐linked immunosorbent assay (ELISA) demonstrated that CMV accumulation was significantly reduced in plants treated with GP17‐2 or its CF relative to control plants. Based on RT‐PCR, plants treated with GP17‐2 (BGI or CF) also exhibited increased expression of regulatory and defence genes involved in the SA and JA/ET signalling pathways. These results suggested that multiple defence pathways in A. thaliana and tobacco were involved in GP17‐2‐mediated resistance to CMV, although neither the transgenic NahG line, nor the npr1, jar1 or ein3 mutants disrupted the response in A. thaliana. This is the first report to demonstrate the induction of systemic resistance against CMV by GP17‐2 or its CF.     

侵染云南鸡蛋果的病毒种类鉴定及CMV cp 基因序列分析

Viral diseased Passiflora edulis samples showing mosaic, shrinking, chlorosis, and malformation were collected from Yuxi, Wenshan, Dehong, and Xishuangbanna in Yunnan Province. Forty diseased samples were detected by RT-PCR/PCR using degenerate primers of members in genera of Umbravirus, Tobamovirus, Luteovirus, Orthotospovirus, Begomovirus, Badnavirus, Potyvirus and specific primers of cucumber mosaic virus (CMV). Results showed that CMV, telosma mosaic virus (TeMV) and papaya leaf curl Guangdong virus (PaLCuGdV) were detected in the diseased P. edulis samples. CMV showed the highest detection rate of 27.5 %, while TeMV and PaLCuGdV had the rates of 20.0% and 7.5% respectively among the P. edulis samples. Mixed infections of PaLCuGdV+CMV, PaLCuGdV+TeMV and CMV+TeMV were also found in these samples. The 657 nucleotide (nt) CMV full-length coat protein (CP) gene was cloned and sequenced from two diseased samples in Yuxi (YYXi-JDG, Acc. No. MW495062) and Xishuangbanna (YXSBN-JDG, Acc. No. MW495063), respectively. These two CMV isolates shared 97.3% identity of nt sequence with each other, while had 76.9%-97.7% and 77.0%-98.2% nt identity with other CMV isolates, respectively. Phylogenetic tree showed that the two CMV isolates from P. edulis belonged to CMV subgroup Ⅰ, and no obvious geographic differentiation and host specificity relationship was found among the selected 33 CMV isolates worldwide.     

Survey of viruses infecting open‐field pepper crops in Côte d'Ivoire and diversity of Pepper veinal mottle virus and Cucumber mosaic virus

The prevalence of viruses in pepper crops grown in open fields in the different agro‐ecological zones (AEZs) of Côte d'Ivoire was surveyed. Pepper veinal mottle virus (PVMV; genus Potyvirus) and Cucumber mosaic virus (CMV; genus Cucumovirus) were the most frequent viruses among those surveyed, while tobamoviruses (genus Tobamovirus) were detected at low frequency. PVMV showed a high heterogeneity across AEZs, which may be related to climatic, ecological or agronomical conditions, whereas CMV was more homogeneously distributed. The molecular diversity of CMV and PVMV were analysed from partial genome sequences. Despite the low number of CMV isolates characterized, two molecular groups were revealed, one corresponding to subgroup IA and the other to reassortants between subgroups IA and IB. RNAs 1 and 3 of the reassortants clustered with the IB subgroup of CMV isolates, whereas their RNA 2 clustered with the IA subgroup. Importantly, RNA 1 of CMV isolates of the IB subgroup has been shown to be responsible for adaptation to pepper resistance. The diversity of PVMV in the VPg‐ and coat protein‐coding regions revealed multiple clades. The central part of the VPg showed a high level of amino acid diversity and evidence of positive selection, which may be a signature of adaptation to plant recessive resistance. As a consequence, for efficient deployment of resistant pepper cultivars, it would be desirable to examine the occurrence of virulent isolates in the CMV or PVMV populations in Côte d'Ivoire and to follow their evolution as the resistance becomes more widely deployed.     

Defective Long-distance Transport of <Emphasis Type="Italic">Cucumber mosaic virus </Emphasis>in Radish Is Efficiently Complemented by <Emphasis Type="Italic">Turnip mosaic virus</Emphasis>

A strain of Cucumber mosaic virus (CMV-D8) systemically infects Japanese radish (Raphanus sativus), but the Y strain of CMV (CMV-Y) only infects the inoculated leaves. Both of these strains cause severe systemic mosaic on the plants after dual infection with Turnip mosaic virus (TuMV). Synergistic interactions on long-distance transport of CMV-Y and CMV-D8 with TuMV were analyzed using an immunobinding assay. Direct tissue blots probed with either anti-CMV-Y or anti-TuMV antiserum clearly showed that CMV-Y efficiently spread and accumulated in the tissues of noninoculated upper leaves and roots when co-inoculated with TuMV, and that long-distance movement of CMV-D8 was enhanced by the presence of TuMV. Received 16 September 1999/ Accepted in revised form 5 February 2000     

Large subclonal variation in Phytophthora infestans populations associated with Ecuadorian potato landraces

The population of Phytophthora infestans on potato landraces in three provinces (Carchi, Chimborazo and Loja) of Ecuador was analysed. All isolates (n = 66) were of the A1 mating type. Simple sequence repeats (SSR) were used to assess the genetic diversity of the isolates. The P. infestans isolates from the potato landraces grouped in a single clade together with reference isolates belonging to the clonal lineage EC‐1. In the 66 SSR profiles obtained, 31 multilocus genotypes were identified. The 66 isolates constituted 49 different races according to the Solanum demissum differential set ( R1 to R11). The P. infestans population was complex and virulent on 4 to 11 R genes. Analysis showed that the subclonal variation in the Ecuadorian EC‐1 clone is increasing over time and is much larger than clonal variation in lineages in the Netherlands and Nicaragua, suggesting high mutation rates and little or no selection in Ecuador.     

五种烟草病毒TMV、CMV、TEV、PVY及TVBMV的多重RT-PCR同步检测

 我国烟草病毒主要有烟草花叶病毒(TMV)、黄瓜花叶病毒(CMV)、烟草蚀纹病毒(TEV)、马铃薯Y病毒(PVY)和烟草脉带花叶病毒(TVBMV),通常发生复合侵染。本研究对我国5种烟草病毒的外壳蛋白基因部分序列设计引物,通过优化引物和模板浓度,摸索扩增参数,在一个体系中成功对5种病毒复合侵染的烟草材料进行多重RT-PCR扩增,得到237、273、347、456和547 bp共5条特异性条带,建立了能同时检测TMV、CMV、TEV、PVY和TVBMV的多重RT-PCR检测体系。对田间样品检测结果证明,多重RT-PCR体系能够同时检测5种病毒,并且灵敏度高。     

Incidence of viruses in <Emphasis Type="Italic">Alstroemeria</Emphasis> plants cultivated in Japan and characterization of <Emphasis Type="Italic">Broad bean wilt virus-2, Cucumber mosaic virus</Emphasis> and <Emphasis Type="Italic">Youcai mosaic virus</Emphasis>

Alstroemeria plants were surveyed for viruses in Japan from 2002 to 2004. Seventy-two Alstroemeria plants were collected from Aichi, Nagano, and Hokkaido prefectures and 54.2% were infected with some species of virus. The predominant virus was Alstroemeria mosaic virus, followed by Tomato spotted wilt virus, Youcai mosaic virus (YoMV), Cucumber mosaic virus (CMV), Alstroemeria virus X and Broad bean wilt virus-2 (BBWV-2). On the basis of nucleotide sequence of the coat protein genes, all four CMV isolates belong to subgroup IA. CMV isolates induced mosaic and/or necrosis on Alstroemeria. YoMV and BBWV-2 were newly identified by traits such as host range, particle morphology, and nucleotide sequence as viruses infecting Alstroemeria. A BBWV-2 isolate also induced mosaic symptoms on Alstroemeria seedlings.