Variability among turnip mosaic potyvirus isolates

ABSTRACT Eight turnip mosaic potyvirus (TuMV) isolates from the Campania region of Italy were characterized. Experiments based on host range and symptomatology indicated that the isolates were biologically different. In addition, the isolates, with the exception of ITA1 and ITA3, were distinguished from each other by using a combination of monoclonal antibodies recognizing the coat protein. Single-strand conformation polymorphism (SSCP) analysis of the coat protein gene revealed that each isolate produced a specific SSCP profile, except for isolates ITA1 and ITA3. This study indicates that (i) even in a small geographical region, there is a great deal of variation in TuMV isolates; (ii) the use of a set of four differential hosts does not always specify the same pathotype in different environments; (iii) the TuMV isolates with the same pathotype on Brassica napus test lines can still differ in host range, symptoms, serology, and SSCP; and (iv) there was perfect correlation between the panel of antibodies and SSCP in differentiating among the isolates; ITA1 and ITA3 were indistinguishable by either assay.     

Serotypic variation in turnip mosaic virus

A panel of 30 monoclonal antibodies (MAbs) was produced against four isolates of turnip mosaic virus (TuMV). The panel was tested in plate-trapped antigen ELISA tests against 41 TuMV isolates (with different host and geographical origins and of differing pathotypes). The antibodies were also tested against four other potyviruses (bean common mosaic virus, bean common mosaic necrosis virus, lettuce mosaic virus and zucchini yellow mosaic virus). The reactions were assessed quantitatively (using multivariate analysis) and qualitatively (using the standard deviation obtained against healthy leaf material). The MAbs recognized 16–17 TuMV epitopes that were not present in the other potyviruses and a further two potyvirus epitopes. The isolates were grouped into three serotypes. Only one isolate did not fit this grouping. The classification of seven isolates in coat protein amino acid sequence homology groups correlated with serotypes. There was no correlation between serotype and pathotype, or between reactions to individual MAbs and single lines. There was therefore no evidence that the epitopes recognized by the MAbs are elicitors for the resistance genes present in the Brassica napus lines. However, the sensitivity and specificity of the MAbs will be useful for both routine detection of TuMV and fundamental studies on plant–virus interactions.     

TuMV management for brassica crops through host resistance: retrospect and prospects

Turnip mosaic virus (TuMV), the only potyvirus known to infect brassicas, is a devastating virus threatening many economically important brassica crops, including cabbage, Chinese cabbage, oilseed rape and mustard. TuMV disease, which was first discovered in the United States, is now found worldwide, especially in Europe, Asia and North America. TuMV results in a yield loss of up to 70% and has a wide host range, infecting most cruciferous plants, as well as many non-cruciferous species. This virus is also characterized by high pathotype diversity because of its highly variable genome structure and has been divided into 12 pathotypes. These characteristics, as well as its nonpersistent transmission mode by as many as 89 aphid species, mean the disease is difficult to prevent through traditional methods such as the application of chemicals, prompting researchers to seek host resistance for effective control. During the last decade, extensive studies have been conducted to investigate inheritance, mapping and cloning of the TuMV resistance genes, and several NB-LRR- or eIF-encoding loci with divergent molecular mechanisms have been uncovered. These studies have greatly facilitated resistance breeding for brassica crops and have advanced our understanding of virus−host interactions.     

Differences in virulence of <Emphasis Type="Italic">Phytophthora capsici</Emphasis> isolates from a worldwide collection on host fruits

Phytophthora capsici causes root, crown, and fruit rot of vegetable and tropical hosts. Cucumber, zucchini, tomato, and pepper fruits were inoculated using 6-mm-diameter agar plugs of P. capsici, incubated in clear plastic boxes at room temperature (25 ± 2°C and 100% relative humidity), and virulence was estimated by measuring the lesion diameter, pathogen growth diameter, and pathogen sporulation density three (cucumber, zucchini) or four (tomato, pepper) days later. When isolates were grouped by genetic cluster, significant differences in virulence were observed on cucumber and zucchini, with isolates belonging to genetic cluster five causing larger lesions than isolates from genetic cluster six. On tomato, no significant differences were observed for isolates grouped by genetic cluster, but isolates from vegetable crops were generally more virulent than isolates from tropical hosts. Isolates from fabaceous hosts sporulated better on cucumber fruits than isolates from solanaceous hosts. Isolates from vegetable hosts sporulated better on zucchini than isolates from tropical hosts. No significant differences in lesion diameter were noted on pepper when isolates were grouped by host family of origin or genetic cluster, but differences in pathogen sporulation were apparent by host family. Our findings suggest that isolate characteristics such as host family of origin and genetic cluster membership may be used to guide initial isolate selection for cucurbit fruit resistance screening. Final isolate selection should incorporate the phenotypic and genetic diversity of P. capsici, including isolates with differing virulence to the host organ of interest.     

潍坊萝卜红心病病原鉴定

 本文用生物学、血清学和分子生物学证据证明,引起潍坊萝卜红心病的病原为芜菁花叶病毒(Turnip mosaic virus,TuMV).该病毒可系统侵染曼陀罗、油菜、咸阳黄瓜、丝瓜、大白菜和普通烟,局部侵染苋色藜和假酸浆,不侵染豌豆.病毒粒体弯曲线状,长约700 nm,可由蚜虫传播,在红心病组织内形成风轮状和片层凝集状内含体,它在SDS-琼脂糖凝胶免疫双扩散试验中可与TuMV的抗血清形成明显的沉淀线.该病毒的CP基因共867个核苷酸,编码288个氨基酸,分子量为32.98 kD.该序列与国内外20个TuMV分离物的CP氨基酸序列比较结果表明,这些分离物可以分为5组,其中引起萝卜红心病的病毒与日本的H1J、KYD8lJ、意大利的ITA7同属一组.     

Biological and molecular variation amongst Australian Turnip mosaic virus isolates

Turnip mosaic virus (TuMV) causes crop losses worldwide. Eight Australian TuMV isolates originally obtained from five different species in two plant families were inoculated to 14 plant species belonging to four families to compare their host reactions. They differed considerably in virulence in Brassicaceae crop species and virus indicator hosts belonging to three other families. The isolates infected most Brassica species inoculated, but not Raphanus sativus, usually causing systemic mosaic symptoms, so they resembled TuMV biological host type [B]. Whole genome sequences of seven of the Australian isolates were obtained and had lengths of 9834 nucleotides (nt). When they were compared with 37 non‐recombinant TuMV genomes from other continents and another whole genome from Australia, six of them formed an Australian group within the overall world‐B phylogenetic grouping, while the remaining new genome sequence and the additional whole genome from Australia were part of the basal‐B grouping. When the seven new Australian genomes and the additional whole genome from Australia were subjected to recombination analysis, six different recombination events were found. Six genomes contained one or two recombination events each, but one was non‐recombinant. The non‐recombinant isolate was in the Australian grouping within the overall world‐B group while the remaining recombinant isolates were in the basal‐B and world‐B phylogenetic groups.     

Identification and characterization of a potyvirus causing chlorotic spots on <Emphasis Type="Italic">Phalaenopsis</Emphasis> orchids

A putative virus-induced disease showing chlorotic spots on leaves of Phalaenopsis orchids was observed in central Taiwan. A virus culture, phalaenopsis isolate 7-2, was isolated from a diseased Phalaenopsis orchid and established in Chenopodium quinoa and Nicotiana benthamiana. The virus reacted with the monoclonal antibody (POTY) against the potyvirus group. Potyvirus-like long flexuous filament particles around 12–15 × 750–800 nm were observed in the crude sap and purified virus preparations, and pinwheel inclusion bodies were observed in the infected cells. The conserved region of the viral RNA was amplified using the degenerate primers for the potyviruses and sequence analysis of the virus isolate 7-2 showed 56.6–63.1% nucleotide and 44.8–65.1% amino acid identities with those of Bean yellow mosaic virus (BYMV), Beet mosaic virus (BtMV), Turnip mosaic virus (TuMV) and Bean common mosaic virus (BCMV). The coat protein (CP) gene of isolate 7-2 was amplified, sequenced and found to have 280 amino acids. A homology search in GenBank indicated that the virus is a potyvirus but no highly homologous sequence was found. The virus was designated as Phalaenopsis chlorotic spot virus (PhCSV) in early 2006. Subsequently, a potyvirus, named Basella rugose mosaic virus isolated from malabar spinach was reported in December 2006. It was found to share 96.8% amino acid identity with the CP of PhCSV. Back-inoculation with the isolated virus was conducted to confirm that PhCSV is the causal agent of chlorotic spot disease of Phalaenopsis orchids in Taiwan. This is the first report of a potyvirus causing a disease on Phalaenopsis orchids.     

An unusual isolate of turnip mosaic potyvirus fromAbutilon theophrasti in Piedmont, Italy

An isolate of the poty virus turnip mosaic virus (TuMV-Ab) showing severe mosaic symptoms inAbutilon theophrasti from Piedmont (northwestern Italy) in 1993, has been found to be of an unusual pathotype and serotype. The isolate was easily transmitted byAphyis gossiypii and Myzus persicae and was not seed-transmitted inA. Theophrasti. The host range of TuMV-Ab was different from that of another Piedmont isolate of TuMV fromAlliaria officinalis and from a TuMV isolate fromBrassica napus. TuMV-Ab was characterized using the reactions on the fourB. Napus lines S4, R4, 165 and S1 as the rare pathotype 7, found only once previously in Europe. Tests with polyclonal antisera indicated that TuMV-Ab was only distantly related to the two other TuMV isolates. Serological characterization with a panel of 30 monoclonal antibodies showed that TuMV-Ab belonged to one of the less common serotypes (JPN).     

Molecular characterisation of <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> isolates from Brassicaceae weeds

Eight provinces of Iran were surveyed during 2003–2008 to find Brassicaceae reservoir weed hosts of Turnip mosaic virus (TuMV). A total of 532 weed samples were collected from plants with virus-like symptoms. The samples were tested for the presence of TuMV by enzyme-linked immunosorbent assay using specific antibodies. Among those tested, 340 samples (64%) were found to be infected with TuMV. Rapistrum rugosum, Sisymberium loeselii, S. irio and Hirschfeldia incana were identified as the Brassicaceae weed hosts of TuMV, and the former two plant species were found to be the most important weed hosts for the virus in Iran. The full-length sequences of the genomic RNAs of IRN TRa6 and IRN SS5 isolates from R. rugosum and S. loeselii were determined. No evidence of recombination was found in both isolates using different recombination-detecting programmes. Phylogenetic analyses of the weed isolates with representative isolates from the world showed that the IRN TRa6 and IRN SS5 isolates fell into an ancestral basal-Brassica group. This study shows for the first time the wide distribution and phylogenetic relationships of TuMV from weeds in the mid-Eurasia of Iran.     

Different Classes of Resistance to Turnip Mosaic Virus in Brassica rapa

Pathotype-specific and broad-spectrum resistance to turnip mosaic virus (TuMV) have been identified in the diploid A genome brassica species Brassica rapa. The pathotype-specific resistance is effective against pathotype 1 isolates of TuMV, which are the most common in Europe. It is almost identical in its specificity to that of a mapped resistance gene (TuRB01) present in the A genome of the amphidiploid species Brassica napus. A mutant of a pathotype 1 isolate of TuMV (UK 1M) that is able to overcome TuRB01 also overcame the B. rapa resistance. This, combined with the fact that a single-nucleotide mutation in the cylindrical inclusion gene of TuMV that has been shown to induce a change from avirulence to virulence against TuRB01, had an identical effect on the B. rapa resistance, suggest that the two resistances are conditioned by the same gene. A second source of resistance in B. rapa prevented systemic spread of all TuMV isolates tested. A third source of resistance that appears to provide immunity to, or severely restrict replication of most isolates of TuMV has been characterised. This resistance source also prevented systemic spread of all TuMV isolates tested. Prior to this study, no resistance to pathotype 4 or pathotype 12 isolates of TuMV had ever been identified. For each of these three resistance sources, plant lines that are not segregating for some of the resistance phenotypes and that are presumably homozygous for the genes controlling these phenotypes have been generated. Strategies for further characterising and deploying these resistances in different Brassica species are described.     

Programmed cell death pathways induced by early plant‐virus infection are determined by isolate virulence and stage of infection

Programmed cell death (PCD) pathways caused by Turnip mosaic virus (TuMV) infection before symptom appearance were studied by light microscopy and electrolyte leakage following sap inoculation of Brassica carinata (Ethiopian mustard) TZ‐SMN‐44‐6 plants. Leaf responses to inoculation with avirulent (TuMV‐avir) and virulent (TuMV‐vir) isolates, and mock‐inoculation, were compared at 2, 20 and 52 h after inoculation (hai). The phenotypes induced were localized resistance (TuMV‐avir) and systemic susceptibility (TuMV‐vir). No visible TuMV symptoms were recorded in any inoculated plants during the 2–52 hai sampling period, but appeared as chlorotic spots in inoculated leaves at 5 days after inoculation. With TuMV‐vir alone, they were followed by systemic infection (mosaic). Dead cell number, deformation, percentage area and percentage integrated intensity, and conductivity of electrolyte leakage data, were analysed to examine their possible roles in stimulating cell death pathways. At 2 hai, dead cell number and percentage area were significantly greater for TuMV‐avir than TuMV‐vir infection or mock‐inoculation. Overall, isolate TuMV‐vir caused significantly greater cell deformation than TuMV‐avir, whereas wounding by mock‐inoculation had negligible effects. By 52 hai, isolate TuMV‐avir caused significantly greater electrolyte leakage than isolate TuMV‐vir or mock‐inoculation. This suggests both isolates triggered morphological changes consistent with apoptotic‐like PCD and necrosis‐like PCD that depended upon isolate virulence and stage of infection, respectively. These findings highlight how quantification of dead cell deformation and electrolyte leakage offer a new understanding of compatible and incompatible plant responses to early virus infection in plants.     

芜菁花叶病毒欧亚分离物HC-Pro基因的克隆和序列分析

 16个芜菁花叶病毒(Turnip mosaic virus,TuMV)欧亚分离物分别来自奥地利、丹麦、德国、匈牙利、尼泊尔和英国6国。利用免疫捕获反转录PCR(Immunocapture RT-PCR,IC-RT-PCR)对16个分离物的HC-Pro(Helper component pro-teinase)基因进行PCR扩增,扩增产物克隆后进行序列测定,HC-Pro基因序列长度均为1374个核苷酸,编码458个氨基酸。16个分离物的HC-Pro基因核苷酸序列同源性为79.5%~99.8%,所编码的氨基酸同源性为94.1%~99.8%。对16个分离物及GenBank上已报道的其它14个TuMV的HC-Pro基因核苷酸的系统进化树分析表明:在16个TuMV欧亚分离物中,除了来自亚洲的分离物N23属Asian-BR组,其余15个来自欧洲的分离物都属于world-B组,其中分离物H1归属world-wide亚组,另外14个分离物则归属New World亚组。     

豌豆病毒病病原研究

 1986年至1990年,从豌豆田中采集了150余份病毒病样本,鉴定出蚕豆萎蔫病毒(BB-WV)、芜菁花叶病毒(TuMV)、马铃薯Y病毒组分离物、黄瓜花叶病毒(CMV)、莴苣花叶病毒(LMV)、大豆花叶病毒(SMV)、豌豆花叶病毒(PMV)、菜豆黄花叶病毒(BYMV)和苜蓿花叶病毒(AMV)等9种病毒。样本中,BBWV所占的比例最高,达59.2%,其次为CMV,占15.5%。BBWV常与CMV复合侵染豌豆,LMV发生也较普遍。田间调查表明,豌豆病毒病发病率因种植地区及品种不同而有差异,平均发病率为12.4%。     

芜菁花叶病毒对油菜致病力差异及壳蛋白基因序列分析

 2004年春季参试的湖北、安徽2省11个芜菁花叶病毒(Turnip mosaic virus,TuMV)分离物感染4个油菜品种,病情指数幅度为26.2~76.0;秋季参试5个TuMV分离物感染14个油菜品种,病情指数幅度为38.3~55.9,均值方差分析表明,致病力差异分别达到极显著和显著水平。壳蛋白(CP)基因序列分析表明,来源于2省油菜、白菜、红菜薹、芝麻和萝卜的17个TuMV分离物与浙江分离物ZJB3序列同源性在97%以上,同属于MB类群;而另一个萝卜分离物WRS1与ZJR1、CH1和CH2分离物序列同源性在95.4%~98.7%之间,属于MR类群。类群间分离物序列同源性仅为88.0%~92.2%。遗传进化树分析表明,萝卜分离物WRS2在MB类群中单独构成一个分支,可能是MR类群和MB类群发生重组的后代。     

Virus recognition and pathogenicity: Implications for resistance mechanisms and breeding

The major method of control of virus diseases in crop plants is breeding for resistance. The genetics of resistance, and of matching virulence (the ability of a virus strain to overcome a specific host resistance gene) have been studied less for viruses than for fungal and bacterial pathogens. This paper draws on a survey of the genetics of resistance to a large number of viruses in cultivated crops, and makes some generalisations and predictions about mechanisms. Most resistance to viruses in crops is monogenic. Dominant alleles are associated with virus-localisation mechanisms, which are induced after infection. The nature of the ‘recognition event’ between plant- and virus-coded functions, which triggers resistance plus a cascade of secondary responses, is not yet known. Gene dosage-dependent alleles tend to be associated with non-localising resistance, which allows some virus spread, but inhibits multiplication. Recessive alleles may involve a negative type of resistance mechanism, whereby the resistant plant lacks some function normally required by the virus for pathogenesis. Such resistance tends to be expressed as complete immunity. Many resistance genes have been overcome by virulent isolates of viruses; only 10 % of the sample of resistance genes have proved exceptionally durable. Virulence may involve different viral functions. The production of infectious cDNA clones, and construction of chimaeric recombinants between clones of virulent and avirulent isolates, is now allowing detailed mapping of virulence determinants. Transformation of plants with ‘novel’ genes for virus resistance, based on coat proteins and viral satellites, may allow construction of more robust resistance systems.     

Causal agent of sharka disease: Plum pox virus genome and function of gene products

Our understanding of the molecular biology of Plum pox virus (PPV) and of the interactions between the virus and its hosts has advanced notably in recent years. Complete genome sequences have been obtained showing up the existence of new subgroups. Functions related to replication, symptom induction, virus movement, and interference with host defense mechanisms have been assigned to the different protein products derived from the proteolytic processing of PPV polyprotein. Moreover, the use of new approaches to define protein–protein interactions between the different viral proteins and between these and the host proteins has begun to yield the first results improving our understanding of how the virus and its host interact during infection.     

Molecular, physiological and pathological characterization of Corynespora leaf spot fungi from rubber plantations in Sri Lanka

The plant pathogenic fungus Corynespora cassiicola causes a severe leaf spot disease on more than 70 host plant species including Hevea brasiliensis . Genetic variability in 32 isolates of C. cassiicola collected from diverse hosts and locations in Sri Lanka and Australia was assessed using restriction fragment length polymorphism (RFLP) analysis of the internal transcribed spacer (ITS) region of ribosomal DNA and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis of total fungal DNA. Amplified ITS fragments from all 32 C. cassiicola isolates exhibited an identical size, and restriction analysis with seven different restriction endonucleases revealed identity in all of the detected DNA fragments. This finding of high genetic relatedness was further supported by the cloning and DNA sequencing of the ITS2 region from one Sri Lankan and one Australian isolate. However, RAPD-PCR profiles generated by 15 oligonucleotide decamer primers revealed significant polymorphism between groups of organisms. Genetic relationships among the isolates were determined by cluster analysis of the RAPD-PCR data and seven different RAPD groups were identified. Isolates showed strong correlations between the assigned RAPD group and the location and host plant genotype from which the isolate was collected. Correlations were also observed between the RAPD group, growth of the isolate and pathogenicity on different plant hosts.     

Susceptibility of different plant species and tomato cultivars to two isolates of <Emphasis Type="Italic">Pepino mosaic virus</Emphasis>

As Pepino mosaic virus has become a pathogen of major importance in worldwide tomato production, information is needed on possible differences between the sensitivity of cultivars towards infection. Furthermore, it is important what hosts other than Solanaceae may be virus reservoirs and are, therefore, threats for tomato cultivation. Two PepMV isolates (PepMV-Sav, E397, a European tomato isolate and PV-0554, a Peruvian pepino isolate) differing in their origin and virulence were used for several experiments to investigate these issues. The response to mechanical inoculation with PepMV was studied using 25 tomato cultivars, seven indicator plant species, and nine other possible horticultural host plants. Symptom development after infection with PepMV was monitored and the virus was detected by DAS-ELISA and IC-RT-PCR. Garlic and broad bean were shown to be additional hosts of PepMV depending on the virus isolate. Nicotiana benthamiana seems to be the most sensitive indicator among all tested indicator plants developing symptoms. Both PepMV isolates infected all tested tomato cultivars. Development of disease symptoms depended on the cultivar and the virus isolate but symptoms were not visible in all cases. None of the cultivars showed tolerance against the two isolates but two responded with a lower susceptibility at an absorbance level of 0.2 (healthy control 0.09). It was observed that some cultivars grown hydroponically showed also lower losses in biomass and yield. Data indicated a correlation between absorbance level in DAS-ELISA and reduction in total tomato growth.