Use of Degenerate Primers in a RT-PCR Assay for the Identification and Analysis of Some Filamentous Viruses, with Special Reference to Clostero- and Vitiviruses of the Grapevine

RT-PCR with degenerate primers was used for the screening of the genome of some members of the Closterovirus, Vitivirus and Trichovirus genera. Two sets of primers, targeted to conserved sequences of the heat shock protein 70 homologue of closteroviruses or to the RNA dependent RNA polymerase genes of tricho- and vitiviruses, amplified the expected fragments from total RNA extracts or double-stranded RNAs of infected plants. Amplified cDNAs were cloned, sequenced and phylogenetically analyzed. Results support the allocation of grapevine viruses A, B, D and heracleum latent virus (HLV) in the genus Vitivirus, whereas, the detection of a HSP70 homologue in grapevine leafroll-associated viruses agrees with their assignment in the genus Closterovirus. The use of degenerate primers for the identification of grapevine viruses belonging to Vitivirus and Closterovirus genera is envisaged.     

Aphid-borne viruses infecting cultivated watermelon and squash in Spain: Characterization of a variant of cucurbit aphid-borne yellows virus (CABYV)

Aphid-borne viruses are responsible for major cucurbit diseases and hamper the sustainability of crop production. Systematic monitoring can reveal the occurrence and distribution of these viruses, in addition to unadvertised viruses, facilitating the control of diseases. For three consecutive (2018–2020) seasons, the presence of aphid-borne viruses was monitored from a total of 292 samples of watermelon and squash plants that showed yellowing symptoms in three major cucurbit-producing areas (Castilla La-Mancha, Alicante, and Murcia) in Spain. We observed that cucurbit aphid-borne yellows virus (CABYV) was the most common virus found (29%) in the plants from both crops. Likewise, except for squash samples from Castilla La-Mancha and Alicante, watermelon mosaic virus (WMV) was also found (23%) with a relatively high frequency. Furthermore, we observed the exacerbation of bright yellowing symptoms in watermelon plants that was often accompanied by considerable fruit abortion. CABYV was the only causative agent for this new yellowing disease, and two infectious cDNA clones (one from watermelon, CABYV-LP63, and another from melon, CABYV-MEC12.1) were constructed to further compare and characterize this CABYV disease. Based on the full-length genome, both isolates were grouped phylogenetically together within the Mediterranean clade. However, the Koch's postulates tests were only successfully completed for the LP63 isolate, which also showed several amino acid changes and two potential recombination events, as compared to MEC12.1. Remarkably, the LP63 isolate caused more severe symptoms and showed higher RNA accumulation than MEC12.1 in five cucurbit plant species. These results suggest that a novel CABYV variant that causes severe yellowing symptoms may be causing outbreaks in cucurbit crops.     

Yellow vein-affected blackberries and the presence of a novel Crinivirus

During the last 5 years, blackberry plants in Arkansas and North and South Carolina exhibited virus-like symptoms of vein yellowing and mosaic, followed in some cases by death. Diagnostic tests for known blackberry viruses failed to identify a causal agent. Double-stranded RNA was extracted from affected plants and cloned. A new member of the Closteroviridae was identified and designated Blackberry yellow vein associated virus (BYVaV). Molecular and immunological assays have been developed for BYVaV, and examination of plants with symptoms revealed a close association of disease symptoms with the presence of BYVaV, although the virus was also found in symptomless plants. Molecular characterization of isolates from plants exhibiting different degrees of disease severity indicated that sequence diversity is probably not the cause of the observed phenotypic differences.     

浙江省马铃薯病毒病检测分析

马铃薯病毒病是影响马铃薯产量和品质的主要因素之一,其症状表现为花叶、黄化和卷曲等。2019年4月-5月,在浙江省湖州、杭州、绍兴、宁波、金华、台州和丽水等7个地市的主要马铃薯产区采集了具有典型病毒病症状的马铃薯样品,采用转录组测序和RT-PCR验证的方法进行病毒检测,共筛查并验证出6种马铃薯病毒,包括马铃薯X病毒Potato virus X(PVX)、马铃薯Y病毒Potato virus Y(PVY)、马铃薯S病毒Potato virus S(PVS)、马铃薯H病毒Potato virus H(PVH)、马铃薯M病毒Potato virus M(PVM)以及马铃薯奥古巴花叶病毒Potato aucuba mosaic virus(PAMV)。检测结果表明,在调查的这7个地市中每个地市都具有2种以上的病毒发生,其中PVH作为一种新鉴定的麝香石竹潜隐病毒属成员在除宁波以外的各地区都有发生;此外杭州市、湖州市以及绍兴市均检测出了PAMV。对检测到的PVS、PVH、PVM和PAMV外壳蛋白(coat protein,CP)序列进行系统发育分析表明,这些病毒均存在地域差异性。本研究为浙江省马铃薯病毒的防控奠定了重要基础。     

Characterisation of a novel ilarvirus causing grapevine angular mosaic disease

Unusual symptoms were observed on ‘Baresana’ x ‘Baresana’ Vitis vinifera hybrid vines in the Grapevine Variety Collection of the Grapevine Institute, Athens. The affected vines showed sharp angular mosaic on leaves, along the veins and in vein angles, malformations, abortive flowers or very few berries with smaller, wrinkled and non-germinating seeds, as well as gradual decline, severe stunting and death of the vine. Serological tests on diseased vines for the presence of 13 known grapevine viruses gave negative results. An infectious agent was transmitted mechanically to several herbaceous indicator plants. Koch’s Postulates were fulfilled, and the agent, proven to be a virus, was named Grapevine angular mosaic virus (GAMV). Serological tests have been developed for the virus. The most conserved polymerase region showed significant similarity of GAMV with members of subgroup 1 of the Ilarvirus genus; however ML phylogenetic analysis could not support its clustering within this subgroup. GAMV differs serologically and in particle morphology from Grapevine line pattern virus (GLPV) a putative member of the Ilarvirus genus that infects grapevine. It is proposed that GAMV is a novel member of the Ilarvirus genus.     

Phylogenetic relationships within the family potyviridae: wheat streak mosaic virus and brome streak mosaic virus are not members of the genus rymovirus

ABSTRACT The complete nucleotide sequence of wheat streak mosaic virus (WSMV) has been determined based on complementary DNA clones derived from the 9,384-nucleotide (nt) RNA of the virus. The genome of WSMV has a 130-nt 5' leader and 149-nt 3'-untranslated region and is polyadenylated at the 3' end. WSMV RNA encodes a single polyprotein of 3,035 amino acid residues and has a deduced genome organization typical for a member of the family Potyviridae (5'-P1/HC-Pro/P3/6K1/CI/6K2/VPg-NIa/NIb/CP-3'). Because WSMV shares with ryegrass mosaic virus (RGMV) the biological property of transmission by eriophyid mites, WSMV has been assigned to the genus Rymovirus, of which RGMV is the type species. Phylogenetic analyses were conducted with complete polyprotein or NIb protein sequences of 11 members of the family Potyviridae, including viruses of monocots or dicots and viruses transmitted by aphids, whiteflies, and mites. WSMV and the monocot-infecting, mite-transmitted brome streak mosaic virus (BrSMV) are sister taxa and share a most recent common ancestor with the whitefly-transmitted sweet potato mild mottle virus, the type species of the proposed genus "Ipomovirus." In contrast, RGMV shares a most recent common ancestor with aphid-transmitted species of the genus Potyvirus. These results indicate that WSMV and BrSMV should be classified within a new genus of the family Potyviridae and should not be considered species of the genus Rymovirus.     

Yellowing disease in zucchini squash produced by mixed infections of Cucurbit yellow stunting disorder virus and Cucumber vein yellowing virus

Zucchini squash is host to Cucurbit yellow stunting disorder virus (CYSDV), a member of the genus Crinivirus, and Cucumber vein yellowing virus (CVYV), a member of the genus Ipomovirus, both transmitted by the whitefly Bemisia tabaci. Field observations suggest the appearance of new symptoms observed on leaves of zucchini squash crops when both viruses were present. When infected during controlled experiments with CYSDV only, zucchini plants showed no obvious symptoms and the virus titer decreased between 15 and 45 days postinoculation (dpi), after which it was no longer detected. CVYV caused inconspicuous symptoms restricted to vein clearing on some of the apical leaves and the virus accumulated progressively between 15 and 60 dpi. Similar accumulations of virus followed single inoculations with the potyvirus Zucchini yellow mosaic virus (ZYMV) and plants showed severe stunting, leaf deformation, and mosaic yellowing. However, in mixed infections with CYSDV and CVYV, intermediate leaves showed chlorotic mottling which evolved later to rolling, brittleness, and complete yellowing of the leaf lamina, with exception of the veins. No consistent alteration of CVYV accumulation was detected but the amounts of CYSDV increased ≈100-fold and remained detectable at 60 dpi. Such synergistic effects on the titer of the crinivirus and symptom expression were not observed when co-infected with ZYMV.     

Responses of white yam (Dioscorea rotundata) cultivars to inoculation with three viruses

The responses of 24 white yam ( Dioscorea rotundata ) cultivars to mechanical and vector transmission with each of three viruses infecting yams were assessed through enzyme-linked immunosorbent assay (ELISA) and symptom development. The viruses were Dioscorea alata virus (DAV), genus Potyvirus ; Dioscorea alata bacilliform virus (DaBV), genus Badnavirus ; and Cucumber mosaic virus (CMV), genus Cucumovirus . Only TDr 95-128, a landrace cultivar from Nigeria, developed symptoms of infection with CMV and DaBV following mechanical and vector transmission, respectively. PAS-ELISA showed that nine genotypes remained uninfected by DAV and 11 were uninfected by CMV or DaBV. Genotypes TDr 747 and TDr 1640 both showed resistance to all three viruses.     

侵染藠头的大蒜潜隐病毒分子鉴定及部分序列分析

 藠头(Allium chinense G. Don),为百合科葱属鳞球茎多年生植物,是一种高产、高营养和具有食疗作用的蔬菜类作物,也常作为中药材加以利用和研究。国内外学者对发生在葱属植物上的病毒病原鉴定和分类做了深入研究,但针对藠头病害的发生特点和病原鉴定研究不多, 自Chen等^[1]在我国发病野生藠头样品中发现2种病毒即藠头花叶病毒(Scallion mosaic virus,ScaMV)和藠头X病毒(Scallion virus X,ScaVX)后,国内外没有更多有关藠头病毒病的研究报道。由于长期无性繁殖及多年连作,极易导致藠头病毒扩散及积累,使病毒病害日趋严重,种苗出现严重衰退。前期调查发现,藠头感病后出现明显的矮缩或叶片皱缩、扭曲,有的出现黄绿斑驳呈花叶状或褪绿条纹,病株地下鳞茎变小、分蘖减少,还首次发现洋葱黄矮病毒(Onion yellow dwarf virus,OYDV)侵染,以及多种病毒复合感染现象^[2]。     

The Natural Occurrence of Two Distinct Begomoviruses Associated with DNAbeta and a Recombinant DNA in a Tomato Plant from Indonesia

ABSTRACT Two begomoviruses (Java virus-1 and Java virus-2), two satellite DNAs (DNAbeta01 and DNAbeta02), and a recombinant DNA (recDNA) were cloned from a single tomato plant from Indonesia with leaf curl symptoms, and the role of these satellite DNAs in the etiology of begomovirus disease was investigated. The genome organizations of the two viruses were similar to those of other Old World monopartite begomoviruses. Comparison of the sequences with other begomoviruses revealed that Java virus-1 was a newly described virus for which the name Tomato leaf curl Java virus (ToLCJAV) is proposed. Java virus-2 was a strain of Ageratum yellow vein virus (AYVV) (AYVV-[Java]). ToLCJAV or AYVV-[Java] alone did not induce leaf curl symptoms in tomato plants. However, in the presence of DNAbeta02, both ToLCJAV and AYVV-[Java] induced leaf curl symptoms in tomato plants. In the presence of DNAbeta01, these viruses induced mild leaf curl symptoms in tomato plants. The recDNA had a chimeric sequence, which arose from recombination among ToLCJAV, AYVV-[Java], DNAbeta01, and DNAbeta02; it was replicated only in the presence of AYVV-[Java] in tomato plants.     

Characterization and Detection of Several Filamentous Viruses of Cherry: Adaptation of an Alternative Cloning Method (DOP-PCR), and Modification of an RNA Extraction Protocol

For the identification and analysis of new RNA plant viruses infecting fruit trees, an initial step often involves the laborious procedure of isolation and cDNA synthesis and cloning from purified viral dsRNA. For subsequent RT-PCR detection of these and other viruses from tissue with high phenolic and polysaccharide concentrations, a simple and efficient extraction protocol for viral nucleic acid is also important. A method for rapid cDNA cloning from small amounts of purified dsRNA using a modification of degenerate oligo primed polymerase chain reaction mbox(DOP-PCR), and a modification of a protocol for effective extraction of viral RNA for use in RT-PCR are presented. Both methods were used to analyze a number of mottling diseases described in cherry. The causal agents for two of these diseases have been previously described, Cherry green ring mottle virus, a tentative member of the foveaviruses, and Cherry mottle leaf virus, a member of the trichoviruses. For the diseases cherry rusty mottle and cherry necrotic rusty mottle, data are presented identifying viruses associated with each disease. Viruses associated with cherry rusty mottle, cherry necrotic rusty mottle and European isolates of cherry mottle leaf diseases, are closely related to Cherry green ring mottle virus and can be tentatively included in the foveavirus genus. An additional virus, related to cherry green ring mottle virus, was discovered by RT-PCR cloning and appears to be a common latent virus of cherry. Finally, isolates of cherry necrotic mottle disease could be assayed positive by RT-PCR for a virus     

The effects of beet yellows virus on the growth and physiology of sugar beet (Beta vulgaris)

The effect of beet yellows virus (genus Closterovirus , BYV) on sugar beet growth was studied in a series of field and glasshouse experiments. Infection reduced total plant weight by 20%, primarily through a 25% reduction in storage root growth. Sugar extraction efficiency was depressed by an increase in root impurities. BYV had little effect on above-ground yield or total crop cover but did decrease green cover significantly. Infection did not reduce water extraction depth in field experiments despite decreasing lateral root growth in the glasshouse. The growth reduction in infected plants resulted from both a decrease in net photosynthesis and an increase in the proportion of light intercepted by yellow leaves. Damage to the photosynthetic mechanism at least partly caused the reduction in net photosynthesis.     

Linear-motion tattoo machine and prefabricated needle sets for the delivery of plant viruses by vascular puncture inoculation

Vascular puncture inoculation (VPI) of plant viruses previously has been conducted either manually or by use of a commercial engraving tool and laboratory-fabricated needle arrays. In an effort to improve this technique, a linear-motion tattoo machine driving industry-standard needle arrays was tested as a means of delivering plant viruses into maize and small grain seed embryos. The new method was applied in the successful transmission of maize rayado fino virus (MRFV), the type member of the genus Marafivirus, from an archived sample to maize. Subsequent transfer of MRFV from the sap of an infected plant using the method produced an average infection rate in maize of 70% (range 39–93%). Maize, oat, and triticale were successfully infected with oat blue dwarf virus (OBDV) using the method; similar infection rates were observed between maize seeds inoculated with the tattoo machine and those inoculated with the engraving machine when using prefabricated needle arrays. No infection was obtained in repeated tests with barley yellow dwarf virus (BYDV-PAV) or cereal yellow dwarf virus (CYDV-RPV) using either sap or RNA from infectious cloned cDNA. Replacement of the original engraving-tool with a linear-motion tattoo machine in VPI provides greater flexibility and convenience in a quiet, readily-available instrument, while improving reproducibility through the use of prefabricated needle arrays.     

Survey for viruses infecting onion, garlic and leek crops in Iran

Surveys to identify virus diseases affecting garlic ( Allium sativum ), onion ( Allium cepa ) and Persian leek ( Allium ampeloprasum var. persicum ) were conducted from 1999 to 2002. Surveys covered different regions of Iran (Tehran [different vegetable markets, farmer fields and cultivation areas], Noushahr, Chalous, Roudbar, Sari, Hamadan, Touyserkan, Ghazvin and Jiroft). A total of 2045 (1285 garlic, 525 onion and 230 leek) samples showing symptoms of virus infection were collected and tested by ELISA; and in some cases tests were also confirmed by immunoelectron microscopy (IEM) for the presence of Allium viruses. ELISA results showed that the following viruses were detected: Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV) (genus Potyvirus , family Potyviridae ), Garlic common latent virus (GarCLV), Shallot latent virus (SLV) (genus Carlavirus ), Garlic virus D (GarV-D), Garlic virus B (GarV-B) and Garlic virus C type (GarV-C) (genus Allexivirus ). None of the samples reacted with antibodies to Shallot yellow stripe virus (SYSV) genus Potyvirus , family Potyviridae ), Shallot virus X (ShVX) and Garlic virus A (GarV-A, genus Allexivirus ). GarCLV, SLV, GarV-D, GarV-B and GarV-C are reported for the first time from Allium crops in Iran.     

Molecular characterisation of <Emphasis Type="Italic">Rice stripe necrosis virus</Emphasis> as a new species of the genus <Emphasis Type="Italic">Benyvirus</Emphasis>

The complete nucleotide sequences of RNAs 1 and 2 of Rice stripe necrosis virus (RSNV) were determined and compared to the corresponding genomes of all sequenced, rod-shaped plant viruses. The genome organisation of RSNV RNA1 and RNA2 is nearly identical to that of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV), definitive species of the genus Benyvirus. As demonstrated for BNYVV and BSBMV, the RNA1 of RSNV also encodes a single ORF with putative replicase-associated motifs, which distinguishes benyviruses from all other viruses possessing rod-shaped particles. As described for BNYVV, RNSV RNA-2 also contains six ORFs: the capsid protein gene, the read-through protein gene, a triple gene block gene that codes for three different proteins, and a 17 kDa cysteine-rich protein. RNAs 3 and 4 (or 5 in the case of BNYVV), identified in natural infections of BNYVV and BSBMV, were not detected in any of the 44 RSNV cDNA clones obtained in this investigation. Nevertheless, phylogenetic and amino comparative acid sequence analyses demonstrated that RSNV is more closely related to BNYVV and BSBMV than to any other rod-shaped plant virus characterised to date.     

Characterization of a new potyvirus isolated from peanut (Arachis hypogaea)

During a survey of viruses of peanuts in South Africa a mechanically transmissible virus was isolated from a plant exhibiting chlorotic ringspots and blotches on the leaves. Typical potyvirus-like flexuous particles were detected by electron microscope examination. Pinwheel-shaped and laminated inclusions in ultrathin sections, reaction with a monoclonal antibody directed to a potyvirus common epitope, a single 33 kDa coat protein and aphid transmission using Myzus persicae all confirmed that the virus was a subdivision II member of the Potyviridae. Host range studies suggested that the virus was none of the previously reported potyviruses of peanuts or of subdivision II potyviruses. The serological relationships of the virus were studied using a range of 17 antisera to potyviruses in ELISA and immunosorbent electron microscopy (ISEM). The isolate reacted weakly with antisera to plum pox virus and bean yellow mosaic virus in ISEM only. Nucleotide sequence of a 624 bp DNA product was obtained following immuno-capture with a potyvirus common epitope antiserum, cDNA synthesis and PCR amplification with potyvirus specific primers which amplify the 3' untranslated region and a part of the coat protein gene. The sequence was only distantly related to a number of potyviruses, whether amino acid or nucleotide sequences were used for comparisons. It is proposed that the virus be named peanut chlorotic blotch virus and be accepted as a new member of the genus Potyvirus in the family Potyviridae.     

Evidence for Lettuce big‐vein associated virus as the causal agent of a syndrome of necrotic rings and spots in lettuce

Lettuce big‐vein associated virus (LBVaV, genus Varicosavirus) was shown to be responsible for characteristic necrotic symptoms observed in combination with big‐vein symptoms in lettuce breeding lines when tested for their susceptibility to lettuce big‐vein disease (BVD) using viruliferous Olpidium virulentus spores in a nutrient film technique (NFT) system. Lettuce plants showing BVD are generally infected by two viruses: Mirafiori lettuce big‐vein virus (MiLBVV, genus Ophiovirus) and LBVaV. New mechanical inoculation methods were developed to separate the two viruses from each other and to transfer both viruses to indicator plants and lettuce. After mechanical inoculation onto lettuce plants MiLBVV induced vein‐band chlorosis, which is the characteristic symptom of BVD. LBVaV caused a syndrome of necrotic spots and rings which was also observed earlier in lettuce plants inoculated in the NFT system, resembling symptoms described for lettuce ring necrosis disease (RND). This observation is in contrast with the idea that LBVaV only causes latent infections in lettuce. De novo next‐generation sequencing demonstrated that LBVaV was the only pathogen present in a mechanically inoculated lettuce plant with symptoms, providing evidence that LBVaV was the causal agent of the observed necrotic syndrome and thus fulfilling Koch’s postulates for this virus. The necrotic syndrome caused by LBVaV in lettuce is referred to as LBVaV‐associated necrosis (LAN).