A dsRNA mycovirus causes hypovirulence of Fusarium graminearum to wheat and maize

The association of Fusarium graminearum isolate China-9 with the dsRNA mycovirus FgV-ch9 was evaluated for hypovirulence-related traits. Single conidia-originating cultures of China-9 isolate can be associated either with high, medium or low amounts of the viral dsRNAs. At high and medium dsRNA levels, China-9 isolates exhibit reduced mycelia growth rate and conidiation capacity, abnormal colony morphology, disorganized cytoplasm, as well as reduced virulence for wheat and maize plants. At low dsRNA levels the fungus shows no symptoms, however, the RNA segments can be detected by RT-PCR. Transfection of the virulent F. graminearum PH-1 isolate with purified Virus-like Particles (VLPs) of FgV-ch9 reduced its conidiation capacity, perithecia formation, and pathogenicity for wheat and maize several folds. These results further demonstrate that FgV-ch9 is associated with hypovirulence of F. graminearum.     

侵染苘麻的菜豆金色黄花叶病毒的鉴定及基因组结构分析

为明确引起我国山西晋中地区苘麻叶片表现皱缩和花叶症状的病原物及其基因组分子特征, 本研究利用双生病毒简并引物扩增获得病毒基因组部分序列,经测序、比对后设计特异性引物扩增病毒基因组序列, 进而通过生物信息学方法构建系统发育树并进行序列分析。结果表明:引起苘麻叶片皱缩、花叶的病原物为番茄黄化曲叶病毒(tomato yellow leaf curl virus, TYLCV), 将该分离物命名为TYLCV-Abu, GenBank登录号为OP293347, 但未扩增到β卫星。该病毒DNA-A基因组全长为2 782 bp, 含有6个开放阅读框。TYLCV-Abu分离物与TYLCV茄子分离物KSQ1-3(GenBank登录号KC428753)的核苷酸序列一致性最高, 为98.99%, 其中C4和V2编码的蛋白变异较大。重组结果分析显示,分离物TYLCV-Abu是由TYLCV-F(GenBank登录号KY971326)和TYLCV-KSQ1-3重组得到, 重组区域为其基因组2 617－2 782 nt区域。这是首次从苘麻样品中扩增到TYLCV全基因组序列并进行分析。     

Molecular and biological characterization of Potato mop‐top virus (PMTV,Pomovirus) isolates from the potato‐growing regions of Colombia

Potato mop‐top virus (PMTV) causes necrotic flecks inside and on tubers in temperate countries. In South America, these symptoms have not been observed, although the presence of the virus has been confirmed in the Andes and in Central America. To characterize PMTV isolates from the Andes, soil samples were taken from the main potato‐producing regions in Colombia and virus was recovered by planting Nicotiana benthamiana as bait plants. The complete genomes of five isolates were sequenced and three of the isolates were inoculated to four different indicator plants. Based on sequence comparisons, three types of RNA‐CP (RNA2) and RNA‐TGB (RNA3) were found. The isolates from the centre of the country (CO3 and CO4) were similar to isolates from Europe. The genomes of CO1, CO2 and CO5 differ from other PMTV isolates, placing them in a separate clade in phylogenetic trees. The three Colombian isolates (CO1, CO2 and CO5) only induced slightly different symptoms in the indicator plants. However, the isolate from the northwest of the country (CO1) induced stronger symptoms in N. benthamiana including severe stunting. A correlation between the genotype of the isolates and the symptoms they induced on indicator plants was not found.     

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.     

Development and use of detection methods specific for <Emphasis Type="Italic">Cucumber vein yellowing virus</Emphasis> (CVYV)

Two methods for the detection of Cucumber vein yellowing virus (CVYV) on infected plants were developed, based on the information provided by cDNA clones covering the 3-end of the genome of a Spanish isolate (CVYV-AILM). The sequenced portion of the CVYV-AILM genome showed a 96.6% aminoacid identity with that of a reported sequence of another CVYV isolate from Israel (Lecoq et al., 2000). The first detection method used a RNA specific probe for hybridization with nucleic acids extracted from infected plants. The probe was complementary to a portion of the CVYV genome including the C-terminal part of the NIb and most of the coat protein (CP) coding regions. The second detection method employed polyclonal antisera raised against recombinant viral CP expressed in bacteria. The specific antibodies were used to detect the presence of virus particles in plant extracts. Both procedures resulted in a highly specific detection of CVYV in plants infected with different isolates of the virus. No interference was observed with other cucurbit-infecting viruses. Sensitivities achieved were sufficient for routine diagnosis of the presence of the virus in plants.     

Tentative description of Hippeastrum latent virus in Hippeastrum hybridum plants and differentiation from Hippeastrum mosaic virus

In mosaic-diseased plants ofHippeastrum hybridum two viruses were found. One virus with a normal length of 706 nm caused local lesions onHyoscyamus niger test plants and mosaic symptoms in the leaves ofH. hybridum. This virus was identified with theHippeastrum mosaic virus (HMV) (*/*∶*/*∶E/E∶S/*) and had a dilution end point between 10^?3 and 10^?4, a thermal inactivation point between 55–60°C and a longevity at room temperature of 28–32 hours. The second virus had a normal length between 584 and 611 nm depending on the method used. It caused local lesions onGomphrena globosa andChenopodium quinoa leaves, and after inoculation ofH. hybridum was found to be present without showing symptoms. It was readily purified from inoculated leaf tissue ofC. quinoa andNicotiana clevelandii by differential centrifugation and ofH. hybridum by density-gradient centrifugation. Purified virus had an absorption minimum at 242 nm, a maximum at 262 nm and a 260/280 absorption ratio of 1.19. The dilution end point was between 10^?3 and 10^?4, the thermal inactivation point between 70 and 80°C and the longevity in vitro at room temperature 28–32 hours. Although no direct comparisons have been made with other members of the potexvirus group, the virus seems to be a new one now namedHippeastrum latent virus. Both viruses were not seed-borne.     

瓜类褪绿黄化病毒山东分离物全基因组序列扩增及分析

为明确分离自山东省寿光市甜瓜上的瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus,CCYV)分离物的全基因组序列信息和遗传变异情况,利用毛形病毒属Crinivirus简并引物进行RTPCR检测,利用RACE技术结合RT-PCR方法克隆CCYV山东分离物2条RNA链的全基因组序列,通过与GenBank中其它地区CCYV分离物的全长序列进行比对分析其同源性,并基于CP基因序列构建系统进化树分析其遗传变异情况。结果表明,山东分离物经RT-PCR检测和测序后确定为CCYV。CCYV山东分离物与其它CCYV分离物的RNA1链和RNA2链的全基因组序列一致性的平均值分别为99.82%和99.88%,且2条链的5′末端均比较保守,没有碱基突变的情况发生;RNA1链3′末端存在2个碱基变异,RNA2链3′末端存在1个碱基变异。CCYV不同地区分离物主要分为3个簇群,其中山东分离物和中国其它地区分离物、日本分离物、苏丹分离物、黎巴嫩分离物和塞浦路斯分离物聚类在一起。研究表明CCYV基因组序列比较保守,该病毒的分化可能与地理来源存在一定的相关性。     

Hippeastrum mosaic virus and another filamentous virus in Eucharis grandiflora

Two viruses were found in mosaic-diseased plants ofEucharis grandiflora in a glasshouse of the laboratory. One virus with a normal particle length of 733 nm caused local lesions onHyoscyamus niger and mosaic symptoms in leaves of healthy-lookingEucharis andHippeastrum plants. On the basis of its host range, physical properties and serology it was identified asHippeastrum mosaic virus, a member of the potyvirus group. This was confirmed by the presence of spherical nuclear inclusions and pinwheels in different tissues of diseasedEucharis plants. The second virus with a normal particle length of 598 nm was present in both healthy-looking and mosaic-diseasedEucharis plants, and it inconsistently induced local lesions onGomphrena globosa. According to its morphology and its reaction onGomphrena, it might be identical or related toHippeastrum latent virus. Crystal-like inclusions were observed in the cytoplasm of cells of both healthy-looking and mosaic-showingEucharis leaves. As no virus-free seedlings ofEucharis were available, the virus nature of these inclusions could not be established.     

Cucumber mosaic virus isolated from Yucca flaccida

Cucumber mosaic virus (CMV) was isolated fromYucca flaccida. The isolate was identified as CMV by host range, ways of transmission, physical properties and serology. No symptoms appeared on healthyYucca seedlings inoculated with purified virus or virus-containing sap, neither could the virus be recovered from these plants.     

Pathogenetic roles of beet necrotic yellow vein virus RNA5 in the exacerbation of symptoms and yield reduction,development of scab-like symptoms,and Rz1-resistance breaking in sugar beet

Beet necrotic yellow vein virus (BNYVV) generally has a four-segmented positive-sense RNA genome (RNAs 1–4), but some European and most Asian strains have an additional segment, RNA5. This study examined the effect of RNA5 and RNA3 on different sugar beet cultivars using a Polymyxa-mediated inoculation system under field and laboratory conditions. In field tests, the degree of sugar yield served as an index for assessing the virulence of BNYVV strains. Japanese A-II type isolates without RNA5 caused mostly 15%–90% sugar yield reductions, depending on the susceptibility of sugar beet cultivars, whereas the isolates with RNA5 induced more than 90% yield losses in the seven susceptible cultivars, but small yield losses in one Rz1-resistant and Rizor cultivars. However, a laboratory-produced isolate containing RNA5 but lacking RNA3 caused higher yield losses in Rizor than in susceptible plants, and induced scab-like symptoms on the root surface of both susceptible and resistant plants. In laboratory tests, A-II type isolates without RNA5 had low viral RNA accumulation levels in roots of Rizor and Rz1-resistant plants at early stages of infection, but in the presence of RNA5, viral RNA3 accumulation levels increased remarkably. This increased RNA3 accumulation was not observed in roots of the WB42 accession with the Rz2 gene. In contrast, the presence of RNA3 did not affect RNA5 accumulation levels. Collectively, this study demonstrated that RNA5 is involved in the development of scab-like symptoms and the enhancement of RNA3 accumulation, and suggests these characteristics of RNA5 are associated with Rz1-resistance breaking.     

The S RNA segment determines symptom differences on Tetragonia expansa between two Watermelon silver mottle virus isolates

The WS-Y isolate of Watermelon silver mottle virus (WSMoV) causes severe necrosis in Tetragonia expansa. To determine the RNA segment that induces symptoms, genome reassorants between WS-Y and an isolate causing mild mottle, WS-O, were generated. The origin of each segment in the reassortants was identified by RT-PCR and subsequent restriction enzyme analysis of the amplified fragments. Thirty genome reassortants were isolated from co-infected T. expansa plants. The reassortants with the S RNA segment of WS-Y caused severe necrosis, while those with the S RNA segment of WS-O caused a mild mottle; hence, the S RNA determined symptom expression. The incidence of reassortants was disproportional among genotypes. The most frequent genome reassortant possesses the L RNA of WS-Y, the M RNA of WS-O and the S RNA of WS-Y. A similar ratio of genotypes was found in isolates of local lesions on Chenopodium quinoa. These results strongly suggested that competition occurred independently between the individual RNA segments in a co-infected T. expansa plant, not between isolates.     

Characterization of five Indian isolates of the nuclear polyhedrosis virus ofHelicoverpa armigera

Five Indian geographical isolates of the nuclear polyhedrosis virus ofHelicoverpa (Heliothis) armigera were studied for the variation in their size, virulence, and the restriction and electrophoretic patterns of the polyhedrin. The diameter of the polyhedra varied from 1.6 to 2.4 μm (mean 1.933 μm), with no significant differences among the five isolates. The most virulent isolate was that from Ooty (OOT), with the lowest LC₅₀ value of 2.538 × 10³ purified occlusion bodies (POB)/ml, followed by the isolate from Coimbatore (CBE). Genomic variability as determined by the HindIII digestion pattern of the DNA, showed that isolates OOT and CBE were similar and that the three other isolates,viz., APK, GUJ, and RAJ represented another group which had a similar restriction pattern. The SDS-PAGE electrophoretic pattern of the polyhedrin of all the isolates was the same.     

菜豆普通花叶病毒RT-PCR检测及3′末端序列分析

根据哈尔滨地区豇豆感病植株的症状,初步鉴定感染豇豆的病毒为菜豆普通花叶病毒(Bean common mosaic virus, BCMV)。利用马铃薯Y病毒属通用引物扩增出病毒基因组3′末端序列,经BLAST检索表明该病毒为BCMV,将该序列与GenBank上的21个BCMV分离物的3′末端序列进行比较,显示其核苷酸序列与其他分离物的序列同源性为91.7%～97.3%。系统进化分析显示不同分离物可聚为5个类群,并显示出一定的寄主相关性。哈尔滨分离物BCMV X与2个浙江分离物、1个澳大利亚分离物聚为一支,且该4株分离物的寄主均为豇豆。RNA二级结构分析显示BCMV基因组3′末端非编码区形成4个茎环结构,不同分离物的序列变化并未引起茎环结构的明显变化。     

Cauliflower Mosaic Virus Isolate NY8153 Breaks Resistance in Arabidopsis Ecotype En-2

ABSTRACT Arabidopsis thaliana ecotype En-2 was previously shown to be resistant to cauliflower mosaic caulimovirus (CaMV) isolate CM4-184. In this study, En-2 plants were screened with eight other isolates of CaMV to identify viruses capable of overcoming resistance and to determine if the mechanism of resistance was the same for each virus. En-2 resistance to most CaMV isolates was mediated by the same mechanism, i.e., preventing virus long-distance movement. One CaMV isolate, NY8153, was found that produced a severe systemic infection on En-2 plants. In addition, the CM1841 isolate was able to spread systemically through En-2 plants, to a limited extent, without producing visible symptoms. These data indicate that the resistance shown by En-2 plants is not an all-or-none phenomenon. En-2 plants were susceptible to turnip mosaic potyvirus, suggesting that resistance is specific to CaMV.     

Multi-gene sequence analysis and phenotypic diversity of Phaeoacremonium species isolated from grapevines in Spain

Esca and Petri disease, caused by Phaeoa-cremonium, and other grapevine decline diseases, such as eutypa dieback, are responsible for serious economic losses to the wine industry worldwide. In the present work, 175 isolates of Phaeoacremonium spp. were collected (mostly in the region of Castilla y León, Spain) from vines either asymptomatic or symptomatic for grapevine decline diseases. The sequences of DNA fragments of 30 isolates were analysed. Amplicons of ITS, and partial gene of the β-tubulin, actin, calmodulin and translation elongation factor 1-α [EF1-α], were obtained by PCR. This is the first time that any exon of the EF1-α gene or exons 6–7 of the β-tubulin gene of seven Phaeoacremonium spp. have been amplified. Amplicons of the calmodulin and EF1-α exons were the most informative in terms of species differentiation: only 52.6 % and 56.6 % similarity across six and seven species was respectively seen. Phylogenetic analysis of combined DNA sequences revealed five Phaeoacremonium species isolated from the sampled plants; P. aleophilum was the most common. Four other species were found, however only one isolate each. P. mortoniae and P. iranianum are here reported for the first time on grapevines in the region of Castilla y León and the neighbouring region of La Rioja respectively. In total, 29 nucleotide differences among the studied gene fragments were seen across 23 isolates of P. aleophilum. Network analysis showed these Spanish P. aleophilum isolates to fall into two main groups of genotypes. P. aleophilum genotypes belonging to group 1 were mostly isolated from young grapevines affected by Petri disease but genotypes included in group 2 were isolated from adult plants showing esca or eutypa dieback symptoms.     

Similarity of clover yellow vein virus and pea necrosis virus

There still is confusion concerning the relationships between clover yellow vein virus (ClYVV), pea necrosis virus (PNV) and bean yellow mosaic virus (BYMV). Therefore, three Swedish isolates of ClYVV and its type strain have now been compared with three isolates of PNV. A bean mosaic isolate and three pea necrosis isolates of BYMV have been used for reference. Based on host range tests, serology, and light microscope studies of inclusion bodies, ClYVV and PNV isolates are now considered to be strains of one virus, with the first name having priority. ClYVV (including the original PNV) especially differs from BYMV in its ability to infect white clover, to produce local lesions on cucumber cotyledons (at least two cultivars), to go systemic inChenopodium quinoa (the two local selections used at Wageningen and at Uppsala), to be rather virulent onNicotiana clevelandii, and to provoke extensive nucleolar enlargements in its host cells. Serologically the two viruses are more or less distinct.     

黄瓜绿斑驳花叶病毒基因组全序列分析及病害的田间调查

为明确近年来在浙江省葫芦科作物上发生的黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)基因组特征及其发生分布情况,从浙江省及上海地区的甜瓜、西瓜和瓠瓜上采集疑似样品进行RT-PCR鉴定,通过分段扩增测序的方法拼接获得基因组全序列并进行系统进化分析,利用特异性引物扩增获得CGMMV外壳蛋白(coat protein,CP)基因序列,制备CGMMV CP抗血清进行Western-blot和Dot-ELISA检测。结果显示,来自甜瓜、西瓜和瓠瓜的3个CGMMV分离物基因组全序列均具有烟草花叶病毒属典型基因组结构特征,全部由6 423 nt构成;3个全序列间的核苷酸同源性高达99.11%~99.67%,编码的CP氨基酸同源性为100%。系统进化分析发现,CGMMV不同分离物形成2个进化相关群体,3个浙江的CGMMV分离物均位于第I组内,与已报道的中国CGMMV分离物和韩国CGMMV分离物亲缘性较高。Western-blot检测表明CGMMV CP抗血清可以与感病植株中的病毒发生特异性反应,可用于CGMMV鉴定;Dot-ELISA检测发现CGMMV在浙江省和上海市的葫芦科作物上普遍存在。     

Serological variability among European isolates of radish mosaic virus

Serological variability of radish mosaic virus (RaMV) isolates from white mustard, winter turnip rape, Camelina sativa and Chinese cabbage, collected in the Czech Republic and Russia, was studied using antisera against the Czech isolate RaMV1. In contrast to previous studies, reasonable serological differences were found between isolates from different locations and hosts, and even between neighbouring mustard plants in the field. Serological variability of European isolates was confirmed in extended experiments involving an Italian isolate, the American Type Strain (ATS) and its homologous antiserum. The results indicate that, in Europe, RaMV isolates may occur which differ both serologically and in host plant response, but no typical strains can be defined with the methods and isolates employed so far. Camelina sativa is reported as a new host of the virus.     

Mite-borne virus isolates from cultivated Allium species,and their classification into two new rymoviruses in the family Potyviridae

While testing several samples of onion and of vegetatively propagated garlic, sand leek and shallot from a number of countries, virus isolates with unusually flexuous particles were obtained by mite (Aceria tulipae) or sap transmissions. No aphid-borne poty-or carlavirus was transmitted by mites, and mite-borne virus isolates could not be transmitted by aphids. The mite-borne isolates did not react with antisera to aphid-borne potyviruses ofAllium spp. or with the Agdia potyvirus group monoclonal. In contrast to the mite-borne onion and garlic mosaic viruses reported in the literature, our mite-borne isolates induced no visible or only very mild symptoms inAllium spp., except isolates from shallot ‘Santé’ which caused diffuse striping. Heavily mite-infested test plants or plant samples showed streaking and malformation due to mite feeding (tangle-top). The mite-borne virus isolates could be classified with test plants and a discriminating antiserum into three groups, representing two viruses and a strain of one of them. They are tentatively named onion mite-borne latent virus (OMbLV), garlic strain of this virus (OMbLV-G), and shallot mite-borne latent virus (SMbLV). Mite transmission, length of virus particles (ca. 700 to 800 nm), and the presence of granular inclusion bodies in infected tissue indicate that the viruses belong to the mite-borne genusRymovirus of the familyPotyviridae. OMbLV from shallot and onion, and OMbLV-G from garlic and sand leek, can be assayed onChenopodium murale but differ in their natural hosts. They are very common. SMbLV, to whichC. murale does not react, was isolated from shallot originating from Asia and Russia.