Prunus necrotic ringspot virus Early Invasion and Its Effects on Apricot Pollen Grain Performance

ABSTRACT The route of infection and the pattern of distribution of Prunus necrotic ringspot virus (PNRSV) in apricot pollen were studied. PNRSV was detected both within and on the surface of infected pollen grains. The virus invaded pollen during its early developmental stages, being detected in pollen mother cells. It was distributed uniformly within the cytoplasm of uni- and bicellular pollen grains and infected the generative cell. In mature pollen grains, characterized by their triangular shape, the virus was located mainly at the apertures, suggesting that PNRSV distribution follows the same pattern as the cellular components required for pollen tube germination and cell wall tube synthesis. PNRSV also was localized inside pollen tubes, especially in the growth zone. In vitro experiments demonstrated that infection with PNRSV decreases the germination percentage of pollen grains by more than half and delays the growth of pollen tubes by approximately 24 h. However, although PNRSV infection affected apricot pollen grain performance during germination, the presence of the virus did not completely prevent fertilization, because the infected apricot pollen tubes, once germinated, were able to reach the apricot embryo sacs, which, in the climatic conditions of southeastern Spain, mature later than in other climates. Thus, infected pollen still could play an important role in the vertical transmission of PNRSV in apricot.     

The location of virus-like particles in the male gametophyte of birch, walnut and cherry naturally infected with cherry leaf roll virus and its relevance to vertical transmission of the virus

Virus-like particles (VLPs) in close-packed paracrystalline arrays were observed in cells forming the anther walls and in the sperm cell cytoplasm of immature pollen grains developing within cherry leaf roll virus (CLRV)-infected birch ( Betula pendula Roth.). VLPs within tubules, that were in some instances multiple and membrane bound, were also observed in anther cells and in pollen grains of CLRV-infected walnut ( Jugians regia L.). VLPs rarely coated the outer surfaces of developing grains. Washings from intact freshly collected pollen did not contain infective agents but pollen triturates were infectious after 12 months storage at-70°C. Purified CLRV (concentration 6·4 ng/ml) was readily detected by enzyme-linked immunosorbent assay (ELISA). CLRV-specific antigens (detected by ELISA) and VLPs (detected on grids coated with an antiserum prepared against CLRV) were readily removed by washing intact pollen grains from infected birch, walnut and cherry ( Prunus avium L. cv. F12/1). The antigen was less tenaciously held to the surfaces of anemophilous (birch and walnut) than entomophilous (cherry) pollen. Treatment of grains before ELISA testing with CLRV-specific γ globulin virtually eliminated the antigenicity of pollen washings whereas γ globulin from a pre-immune serum had no such effect. When anti-CLRV γ globulin-treated pollen grains were disrupted, CLRV-specific antigens were liberated. VLPs trapped on CLRV-antiserum coated grids to which pollen washings or extracts from disrupted grains had been applied were identified by decoration; a halo of antibody molecules enveloped VLPs treated with CLRV-antiserum but not those treated with antiserum prepared against poplar mosaic virus.     

First report of raspberry yellows disease caused by raspberry bushy dwarf virus in Japan

Severe yellowing of leaves was observed on red raspberry in Akita Prefecture. When inoculated with sap from symptomatic raspberry leaves, Chenopodium quinoa plants developed chlorotic ringspot and mottling that are typical of raspberry bushy dwarf virus (RBDV) infection. In western blot analysis, an antibody to the coat protein (CP) of RBDV reacted against ca. 30-kDa protein specific to the diseased trees. In RT-PCR testing for RBDV, single DNA fragments were amplified from total RNA samples of the diseased trees. The nucleotide sequences of the DNA fragments covering the entire CP region revealed 87–97?% identities with those of RBDV isolates.     

Occurrence and distribution of Raspberry bushy dwarf virus in commercial Rubus plantations in England and Wales

Serological surveys for Raspberry bushy dwarf virus (RBDV) made between 1995 and 1997 and covering ≈ 10% of the commercial farms growing Rubus (red raspberry and hybrid berries) in England and Wales showed that this virus was present on approximately one-quarter of all farms and in approximately one-sixth of all plots tested. RBDV was found in all of the four main raspberry cultivars being grown at that time (Autumn Bliss, Glen Moy, Glen Prosen and Leo), in Loganberry and in Tayberry. Fifteen RBDV genotypes (including two that appeared to be mixed) were identified using RT-PCR/RFLPs, but the majority of genotypes were found only rarely. Of the RBDV isolates tested, two genotypes each comprised 12·5% and another 46·4%. None of the three most common genotypes was associated solely with single Rubus cultivars and vice versa . It is suggested that two separate outbreaks of RBDV are occurring in England and Wales. One outbreak comprises the most frequent genotype combined with one of the moderately frequent genotypes; this outbreak is largely confined to the main growing areas and is either spreading between farms or coming from multiple local sources. Circumstantial evidence suggests that these isolates (and hence this first outbreak) are of the RB pathotype. The second outbreak consists of the other moderately frequent genotype and those genotypes which are less common. These genotypes appear to be more scattered across England and Wales and seem more likely to be coming from local sources and not to be spreading naturally between commercial farms.     

Pollen grains infected with apple stem grooving virus serve as a vector for horizontal transmission of the virus

Journal of General Plant Pathology - Here, Nicotiana benthamiana plants became infected with apple stem grooving virus (ASGV) after pollination with pollen grains produced by ASGV-infected apple...     

Characterization of Synergy Between Cucumber mosaic virus and Potyviruses in Cucurbit Hosts

ABSTRACT Mixed infections of cucurbits by Cucumber mosaic virus (CMV) and potyviruses exhibit a synergistic interaction. Zucchini squash and melon plants coinfected by the potyvirus Zucchini yellow mosaic virus (ZYMV) and either Fny-CMV (subgroup IA) or LS-CMV (subgroup II) displayed strong synergistic pathological responses, eventually progressing to vascular wilt and plant death. Accumulation of Fny- or LS-CMV RNAs in a mixed infection with ZYMV in zucchini squash was slightly higher than infection with CMV strains alone. There was an increase in CMV (+) strand RNA levels, but no increase in CMV (-) RNA3 levels during mixed infection with ZYMV. Moreover, only the level of capsid protein from LS-CMV increased in mixed infection. ZYMV accumulated to similar levels in singly and mixed infected zucchini squash and melon plants. Coinfection of squash with the potyvirus Watermelon mosaic virus (WMV) and CMV strains increased both the Fny-CMV RNA levels and the LS-CMV RNA levels. However, CMV (-) strand RNA3 levels were increased little or not at all for CMV on coinfection with WMV. Infection of CMV strains (LS and Fny) containing satellite RNAs (WL47-sat RNA and B5*-sat RNA) reduced the accumulation of the helper virus RNA, except when B5*-sat RNA was mixed with LS- CMV. However, mixed infection containing ZYMV and the CMV strains with satellites reversed the suppression effect of satellite RNAs on helper virus accumulation and increased satellite RNA accumulation. The synergistic interaction between CMV and potyviruses in cucurbits exhibited different features from that documented in tobacco, indicating there are differences in the mechanisms of potyvirus synergistic phenomena.     

New host for raspberry bushy dwarf virus: arctic bramble (Rubus arcticus)

Raspberry bushy dwarf virus (RBDV) was detected in three new host plants inRubus species,i.e., arctic bramble (R. arcticus ssp.arcticus), Alaskan arctic bramble (R. arcticus ssp.stellatus) and their hybrid (R. arcticus L. nothosubsp.stellarcticus G. Larsson). The virus was identified as RBDV by the symptoms elicited in the test plantsChenopodium quinoa andC. amaranticolor, by sedimentation profile in sucrose density gradient, by RNA banding pattern in agarose gel electrophoresis, by protein analysis of the purified viruses in SDS-polyacrylamide gel electrophoresis, and by Western blotting. There was a high incidence of RBDV-infected plants in the experimental plots. The presence of the virus in arctic bramble did not always induce foliar symptoms. However, yellowing of the leaves around central and lateral veins was quite frequently observed, especially in spring and autumn.     

利用RT-PCR检测库尔勒香梨苹果茎痘病毒的研究

 以库尔勒香梨新鲜、冷藏、冷冻叶片和皮层为材料,对提取双链RNA (dsRNA)的2种方法和提取总RNA的3种方法进行了分析比较,并对总RNA的提取方法进行了改进,获得了纯度较高、完整性较好的dsRNA和总RNA,在此基础上进行了反转录(RT)和PCR扩增。在国内首次完成了对苹果茎痘病毒(ASPV)的RT-PCR检测,建立了ASPV有效RT-PCR反应体系。用此体系扩增到ASPV一个长约316 bp的片段。实验表明以dsRNA和总RNA为模板均能成功进行RT-PCR检测,且dsRNA优于总RNA。     

Effect of viruses on pollen morphology

Many pollen grains from Chenopodium quinoa plants infected with sowhane mosaic sobemovirus (SMV) were collapsed, grooved and had sunken opercula, whereas those from the first flowers of virus-free plants were smooth, rounded and with protuberant opercula. However, pollen grains from later flowers of virus-free plants were similar in appearance to those from the virus-infected plants. Similar but less obvious symptoms were found in pollen of Plantago lanccoiata infected with either ribgrass mosaic tobamovirus or broad bean wilt virus. No symptoms were found in pollen of Hordeum vulgare cv. clipper, H. sponiaitcum or Triticum acstivum infected with barley suripe mosaic hordeivirus, nor in pollen of a Cardaminc sp. naturally infected with a strain of turnip yellow mosaic tymovirus. The symptoms, even those shown by pollen from SMV-infected C quinoa, seemed not to be sufficiently characteristic for diagnosis of virus infection.     

A pollen test to detect ACCase target‐site resistance within Alopecurus myosuroides populations

This study was conducted to determine a suitable medium for in vitro germination of Alopecurus myosuroides pollen and to develop a reliable test for the rapid screening of ACCase target site‐resistant plants within populations. The assay is based upon germination of pollen in a medium supplemented with ACCase inhibitors. A 0.25% agar medium, containing 200 mg L^–1 CaNO₃, 100 mg L^–1 H₃BO₃, 200 g L^–1 sucrose, was selected as a suitable medium for in vitro pollen germination. At 25 °C, this medium supported a mean germination rate of 85% within two hours. Plants highly resistant (Rh) to aryloxyphenoxypropionate (APP), owing to the expression of an insensitive ACCase, were found to express this resistance in their pollen. In contrast, plants moderately resistant (Rm) to APP herbicides, owing to an enhanced capacity to detoxify herbicides, did not exhibit this resistance in their pollen. Concentrations of 120 μM fenoxaprop and 1000 μM clodinafop were selected as the best for reliable discrimination of the target‐site‐resistant biotypes. At these concentrations there was more than 50% germination of the Rh pollen grains whereas less than 10% of the S and Rm pollen grains germinated. This test, using haploid material, may also permit distinction between homozygous‐ and heterozygous‐resistant individuals.     

应用RT-PCR检测温州蜜柑萎缩病毒

温州蜜柑萎缩病是以日本为主的少数亚洲国家柑橘上的重要病毒病害,多数柑橘品种隐症带毒,不易发现,对其早期准确快速检测尤为重要.以毒源植株的叶、枝皮为材料,对提取的总RNA和总核酸进行反转录和PCR扩增,通过SDV特异引物的设计与筛选,反应体系与反应程序的建立与优化,扩增得到一个251 bp的特异片段,测序结果与日本Iwanami报道的SDV序列同源性为99.6%.RT-PCR检测体系的灵敏度为100ng,同时应用该检测体系可以全年检测到毒源植株嫩叶、嫩皮、老叶、老皮中的SDV.     

Effect of the Host and Temperature on the Formation of Defective RNAs Associated with Broad bean mottle virus Infection

ABSTRACT Previously, we demonstrated that Broad bean mottle virus (BBMV), a member of the genus Bromovirus, could accumulate RNA 2-derived defective interfering (DI) RNAs during infection. In this work, we study how host and environmental factors affect the accumulation of DI RNAs. Serial passages of BBMV through selected plant species reveal that, with low-multiplicity inocula, some systemic hosts (Vicia faba, Nicotiana clevelandii, and N. tabacum cv. Samsum) support DI RNA accumulation after the first passage cycle but other hosts (Phaseolus vulgaris, Pisum sativum, and Glycine max) do not. However, several passages with the high-multiplicity inocula can generate DI RNAs in pea plants. Local lesion hosts (Chenopodium quinoa, C. amaranticolor, and C. murale) remain free of the DI RNA components. The size of the de novo-formed DI RNAs depends on the host and on environmental conditions. For instance, broad bean plants cultivated in a greenhouse or in a growth chamber at 20 degrees C accumulated DI RNAs of 2.4 or 1.9 kb in size, respectively. A reverse trend was observed in pea plants. Lower temperatures greatly facilitated the formation of DI RNAs in broad bean and pea hosts after the first passage. The importance of these findings for the studies on DI RNAs are discussed.     

Transmission of tobacco streak virus by Thrips tabach a new method of plant virus transmission

When adults or nymphs of Thrips tabaci were mixed with virus-carrying pollen from Lycopersicon esculentum infected with tobacco streak virus and then placed on Chenopodium amaranticolor test seedlings, the virus was regularly transmitted. The virus was also regularly transmitted when virus-carrying pollen was placed on the leaves of C amaranticolor test seedlings and the thrips then introduced. No transmission occurred when test seedlings were exposed to virus-carrying pollen in the absence of the thrips or to the thrips without pollen. Further, no transmission occurred when the thrips were fed on virus-infected leaves and then transferred to test seedlings in the absence of virus-carrying pollen. The evidence indicates that the transmission of tobacco streak virus by Thrips tabaci depends on the presence of pollen-borne virus, which presumably infects via wounds made by the thrips. This method of virus transmission has not previously been reported.     

Effect of temperature on RNA silencing of a negative‐stranded RNA plant virus: Citrus psorosis virus

Citrus psorosis virus (CPsV), genus Ophiovirus, causes a bark scaling disease of citrus. CPsV virions are kinked filaments with three negative‐stranded RNA molecules (vRNA) and a 48 kDa coat protein. The effect of temperature on symptom expression, virus accumulation and RNA silencing was examined in sweet orange seedlings (Citrus sinensis) graft‐inoculated with three different CPsV isolates and grown in a glasshouse at 26/18°C or 32/26°C (day/night). Most plants kept in the cooler glasshouse showed a shock reaction in the first flush with shoot necrosis, and then moderate to intense chlorotic flecking and spotting in young leaves, whereas plants incubated at 32/26°C did not exhibit shoot necrosis, and young leaf symptoms were milder. Virus titre estimated by ELISA and by northern and dot blot hybridization paralleled symptom intensity, with significantly higher virus accumulation in plants incubated at 26/18°C. The amount of CPsV‐derived small RNAs (CPsV‐sRNAs) slightly increased at 32/26°C, with the ratio of CPsV‐sRNA/vRNA being higher at 32/26°C than at 26/18°C. These results suggest that (i) CPsV infection induces RNA silencing in citrus plants, (ii) symptom intensity is associated with virus accumulation, and (iii) temperature increase enhances the RNA silencing response of citrus plants and decreases virus accumulation.     

Location of Prunus Necrotic Ringspot Ilarvirus Within Pollen Grains of Infected Nectarine Trees: Evidence from RT-PCR, Dot-blot and in situ Hybridisation

Prunus necrotic ringspot Ilarvirus (PNRSV) is a pollen and seed-borne ilarvirus affecting most Prunus spp. The location of the virus in infected nectarine pollen grains was investigated by molecular hybridisation, RT-PCR and in situ hybridisation. The first two approaches revealed an internal location of the virus. In situ hybridisation demonstrated the virus in the bicellular pollen grain, where it was present in the cytoplasm of the vegetative cell but not in the generative cell. This result seems to indicate that the sperm cells, formed by the mitosis of the generative cell, are not involved in virus transmission to seed. Other possible mechanisms are discussed.     

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。     

Historical and recent tomato black ring virus and beet ringspot virus isolate genomes reveal interspecies recombination and plant health regulation inconsistencies

Tomato black ring virus (TBRV) and beet ringspot virus (BRSV) are closely related but distinct members of subgroup B of the genus Nepovirus. Both viruses have broad host ranges and are transmitted by seed, pollen, and ectoparasitic nematodes. Although 13 TBRV and 3 BRSV genome sequences were already available, no attempt has been made to link sequence data from these recent sequences with those of historical isolates studied in the pre-sequencing era. High-throughput sequencing was used to generate eight new TBRV and BRSV genome sequences from three historical >60-year-old and two >30-year-old isolates, and three more recent isolates. These eight isolates were from the Czech Republic, Germany, and the UK. We compared these with all genomes sequenced previously. Intraspecies recombination (three of four TBRV and two of four BRSV isolates) was frequent amongst the eight new genomes. Interspecies recombination was also present within the RNA1 of TBRV isolates BRSV-3393 SG GB and BRSV-9888 ST GB. No satellite RNAs were associated with the eight new genomes. Two commercial enzyme-linked immunosorbent assay (ELISA) kits used to detect TBRV during routine testing differed in that one detected only TBRV and the other only BRSV, so they are likely to provide incorrect but potentially complementary virus occurrence information. We suggest both ELISA kits, or appropriate molecular tests, be used by biosecurity authorities to avoid this problem. This study illustrates the value of sequencing historical isolates preserved from the pre-sequencing era.     

Pathogenesis of Alfalfa mosaic virus in Soybean (Glycine max) and Expression of Chimeric Rabies Peptide in Virus-Infected Soybean Plants

ABSTRACT Infection of soybean (Glycine max) plants inoculated with particles of Alfalfa mosaic virus (AlMV) isolate 425 at 12 days after germination was monitored throughout the life cycle of the plant (vegetative growth, flowering, seed formation, and seed maturation) by western blot analysis of tissue samples. At 8 to 10 days after inoculation, the upper uninoculated leaves showed symptoms of virus infection and accumulation of viral coat protein (CP). Virus CP was detectable in leaves, stem, roots, seedpods, and seed coat up to 45 days postinoculation (dpi), but only in the seedpod and seed coat at 65 dpi. No virus accumulation was detected in embryos and cotyledons at any time during infection, and no seed transmission of virus was observed. Soybean plants inoculated with recombinant AlMV passaged from upper uninoculated leaves of infected plants showed accumulation of full-length chimeric AlMV CP containing rabies antigen in systemically infected leaves and seed coat. These results suggest the potential usefulness of plants and plant viruses as vehicles for producing proteins of biomedical importance in a safe and inexpensive manner. Moreover, even the soybean seed coat, treated as waste tissue during conventional processing for oil and other products, may be utilized for the expression of value-added proteins.     

Subterranean clover mottle virus: another virus from Australia with encapsidated viroid-like RNA

A new virus, subterranean clover mottle virus (SCMoV) was found at several locations in Western Australia in Trifolium subterraneum L. (subterranean clover) with severe disease symptoms. Paiticles of the virus were shown to share many physical properties with members of the sobemo-virus group. However, in addition to the single-stranded RNA of M_J about 1.5 × 10⁶ which is characteristic of sobemoviruses, SCMoV particles also contained circular and linear viroid-like RNAs of two size classes, one about 400 and the other about 300 nucleotides long. At present it is not clear if the two types of viroid-like RNAs are components of the same SCMoV strain or of closely related strains of the virus which are able to confect subterranean clover.
SCMoV was shown to be serologically related to lucerne transient streak virus (LTSV). However, the relationship was remote and whereas most high-titred antisera to LTSV reacted with SCMoV particles, antisera to SCMoV failed to recogaize those of LTSV. Neither SCMoV nor LTSV are serologically related to velvet tobacco mottle virus (VTMoV) or Solanum nodiflorum mottle virus (SNMV). However, particles of all four viruses have many physical and chemical properties in common, including RNA complements consisting of both virus-like and viroid-like RNAs. Because of their affinities and unique RNA complements, we suggest that SCMoV, LTSV, VTMoV and SNMV may warrant inclusion in a new taxonomic group.