Cherry Leaf Roll Virus Research Articles

On the presence and distribution of olive viruses in Lebanon

A survey for viruses was carried out in 2003 in the main olive‐growing areas of Lebanon (South Lebanon, North Lebanon, Mount Lebanon and Bekaa). A total of 300 samples was collected in 31 different locations in 76 different commercial orchards and checked by RT‐PCR for the presence of Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Strawberry latent ringspot virus (SLRV), Olive latent virus 1 (OLV‐1) and Olive leaf yellowing‐associated virus (OLYaV), using virus‐specific primers reported in the literature. About one third (31%) of the trees were infected. In particular, the closterovirus OLYaV was the most widespread, as it was detected in 23.7% of the samples, followed by the necrovirus OLV‐1 (8.3%), the two nepoviruses CLRV (2%) and ArMV (0.3%), and the sadwavirus SLRV (0.3%). A high variability was detected in the HSP70 gene of Lebanese and Italian OLYaV isolates, for at least nine different patterns were obtained when this genomic region was subjected to single‐strand conformation polymorphism (SSCP) analysis.

DISTRIBUTION OF OLIVE TREE VIRUSES IN ITALY AS REVEALED BY ONE-STEP RT-PCR

We have used a one-step RT-PCR protocol to detect and identify each of the eight viruses most commonly found in olive trees namely: Arabis mosaic virus (Ar- MV), Cherry leaf roll virus (CLRV), Cucumber mosaic virus (CMV), Olive leaf yellowing associated virus (OLYaV), Olive latent ring spot virus (OLRSV), Olive latent virus-1 (OLV-1), Olive latent virus-2 (OLV-2), and Strawberry latent ring spot virus (SLRSV). We have assayed 345 samples collected from olive fields in Italian areas in which national and local olive tree cultivars and selections are grown. The average percentage virus infection of olive trees was 32.8%. Among the eight viruses assayed, only OLYaV (20.9%), SLRSV (7.8%) and CLRV (4.9%) were detected. The most common virus was OLYaV, which was found at a high percentage in southern Italy. SLRSV was the main virus in central Italy whereas CLRV was detected in five samples from Latium, Umbria and Sicily and in all analyzed trees from Abruzzo.

EVALUATION OF RESISTANCE TO PHYTOPHTHORA CITRICOLA AMONG DIVERSE CLONES OF PARADOX HYBRID ROOTSTOCKS

In two 2004 experiments, 10 clones of diverse walnut hybrids from black (maternal, M) and English (paternal) parents were evaluated for resistance to Phytophthora citricola, a virulent and prevalent crown and root pathogen affecting California walnuts. The clones were selected from seed families for one or more traits, including putative tolerance to P. citricola, Cherry Leaf Roll Virus, or Pratylenchus vulnus; or unique parentage. The selections were propagated as microshoots, rooted, acclimatized and grown in a greenhouse in 4 x 20 cm cones (3 months), subjected to dormant chilling (6 °C, 3 to 5 months), and allowed to resume growth in a greenhouse (2 to 3 months) before inoculation with P. citricola. The plants were transferred from the cones into 2-liter pots filled with non-infested soil (6 plants per clone) or soil artificially infested with P. citricola (12 per clone). Starting 2 weeks after transplanting, all plants received biweekly 48-h periods of soil flooding. Three months after transplanting, each plant was washed free from soil and rated for severity of root and crown rot. Severity of crown rot was affected by interactions of inoculation x clone (P<0.0001) and experiment x inoculation x clone (P=0.05). Across experiments, non-inoculated controls developed little disease (means 0 to 5% crown length rotted), but inoculated clones developed moderate to severe crown rot. Highly susceptible clones (means 52 to 90% of crown length rotted) included AX-1 (M=J. californica); GZ-1 and PX-1 (M=J. hindsii); and WIP3 (M=J. hindsii x regia). Moderately tolerant clones (20 to 43% crown length rotted) included AZ-2, AZ-3, and NZ-1 (M=[J. major x hindsii] x nigra); JX-2 and VX-211 (M=J. hindsii); and RX-1 (M=J. microcarpa). The results, obtained under conditions highly conducive to the disease, suggest that the moderately tolerant clones have potential as improved Paradox rootstocks in orchards infested with P. citricola.

Biological and serological procedures to detect three nepoviruses in fruit trees

Cherry leaf roll virus (CLRV), Myrobalan latent ringspot virus (MLRSV) and Strawberry latent ringspot virus (SLRSV) were transferred by budding to woody trees, hybrid Ishtara, peach cv. GF 305 and cv. Lesiberian. Three buffers with antioxidants and stabilisers: 0.01M phosphate with 1% caffeine; 0.007M phosphate-0.01M veronal with 0.01M cysteine hydrochloride and 0.007 EDTA; 0.015M phosphate with 1% nicotine and 0.066M phosphate buffer without additives were compared for their efficiency in mechanical transmission from woody sources to herbaceous hosts (Chenopodium quinoa and C. amaranticolor). 0.007M phosphate-0.01M veronal buffer with 0.01M cysteine hydrochloride, and 0.007 EDTA and 0.015M phosphate buffer with 1% nicotine were found to be the best buffers for the three nepoviruses. Both biological transmission to herbaceous assay hosts and detection by ELISA in the investigated tree are necessary to reliably detect the three nepoviruses. Biological detection is reliable from April to June, and in September and October. ELISA detection is also more difficult in July and August. The suitability of C. quinoa and C. amaranticolor to maintain CLRV, MLRSV and SLRSV was compared. C. amaranticolor plants were found to be more suitable for CLRV and SLRSV, infected plants grow over 6 months after mechanical inoculation by the nepoviruses. C. quinoa plants proved to be most suitable for maintenance of MLRSV, while C. amaranticolor is a symptomless host of MLRSV. Reinoculation with the nepoviruses is recommended in intervals of 4 to 6 months.

Detection and Differentiation of Comoviridae Species using a Semi‐nested RT‐PCR and a Phylogenetic Analysis based on the Polymerase Protein

AbstractA nested reverse transcription polymerase chain reaction (RT‐PCR) method is described that allows detection and identification of Comoviridae species and possibly other bipartite plant picorna‐like viruses. It involves an RT‐PCR, in which a combination of degenerate deoxyinosine (dI)‐substituted primers amplified part of the RNA‐dependent RNA polymerase (RdRp) domain, followed by a semi‐nested PCR amplification that increased detection sensitivity. The PCR amplicons can be easily cloned and sequenced to obtain initial sequence information of Comoviridae genomes for virus species differentiation and characterization. The method can be used on total RNA extracts from infected plants, when viral genomic double‐stranded RNA (dsRNA) or the viral RNA is unavailable. Previously uncharacterized RdRp domains of Potato black ringspot virus (PBRSV), Cherry leafroll virus (CLRV) and Arracacha virus B (AVB) were amplified and sequenced. Neighbour‐joining trees calculated from the homologous partial RdRp amino acid sequences were used to establish the taxonomic relationships between members of plant picorna‐like viruses, revealing lineages that classified all species within their respective genera and clearly distinguished each genus from the other genera. Phylogenetic analysis confirmed the classification of PBRSV and CLRV within the genus, Nepovirus whereas AVB was placed into a cluster different from the Nepovirus, Comovirus and Fabavirus lineages.

LONG-TERM PERFORMANCE OF OWN-ROOTED 'CHANDLER' WALNUT COMPARED TO 'CHANDLER' WALNUT ON PARADOX ROOTSTOCK

LONG-TERM PERFORMANCE OF OWN-ROOTED 'CHANDLER' WALNUT COMPARED TO 'CHANDLER' WALNUT ON PARADOX ROOTSTOCK

Virus-diseased Ulmus laevis in Eastern Germany

Virus-like leaf symptoms and dieback were observed on elm trees in a public park near Potsdam. Infection with Cherry leaf roll virus (CLRV), Elm mottle virus (EMV), Arabis mosaic virus (ArMV) and Tobacco ringspot virus (TRSV), well known viruses to infected elm trees was excluded by bioassays and serological tests. Poty- or carlavirus- like flexible particles of approximately 750 nm in length were isolated repeatedly from diseased elms. The particles were transmissible to diverse Chenopodium species, a herbaceous indicator. The virus was not a member of the Potyviridae family, based on an ELISA and an RT-PCR assay using a potyvirus genus-specific broad-spectrum polyclonal antibody and family-specific primers, respectively. Also no potyvirus-like pinwheel inclusions were found in leaf cells of infected indicator plants in electron microscopic studies. Further molecular characterization of these virus isolates is under way.

New Australian record for Xiphinema vuittenezi on Vitis vinifera

Xiphinema vuittenezi is reported for the first time in Australia in soil associated with roots of declining grapevines (Vitis vinifera) in a Barossa Valley (SA) vineyard. This nematode may be able to transmit some strains of Cherry leaf roll virus and is the putative vector of Grapevine chrome mosaic virus; hence its finding may have potential significance for the spread of some nepoviruses in grapes and other hosts.

First Report of a Disease of Peony Caused by Alfalfa mosaic virus.

During the summers of 2001 and 2002, Japanese peony (Paeonia albiflora Pall., synonym P. lactiflora, family Paeoniaceae) plants, cultivated in the Botanical Garden of the University of Parma (Emilia Romagna Region of northern Italy), were found affected by a disease with virus-like symptoms. The oldest leaves showed yellow, mosaic, oak-like arabesques and line-patterns; the remaining leaves and pink flowers were symptomless. A disease of peony, known as peony ring spot disease, has been reported worldwide (Europe, United States, Japan, and New Zeland) for several years and is associated with strains of Tobacco rattle virus (TRV) (1). Electron microscopic observations of peony leaf sap (leaf dip preparations stained with uranyl acetate and phospotungstic acid) did not show the presence of any rod-shaped virus particles, including TRV. Mechanical inoculations of sap from symptomatic leaves caused symptoms typical of Alfalfa mosaic virus (AMV) on Chenopodium amaranticolor Coste & Reyn. (local chlorotic and necrotic lesions and systemic periveinal line-patterns), Ocimum basilicum L. (yellow mosaic), Vigna unguiculata (L.) Walp. (red, local necrotic lesions), and Nicotiana tabacum cv. Samsun (white, necrotic lesions, systemic leaf malformation, and mosaic), and N. glutinosa L. (systemic leaf variegation). Symptomatic leaves of peony and infected herbaceous plants were analyzed for virus presence by protein A sandwich enzyme-linked immunosorbent assay (PAS-ELISA). The polyclonal antisera tested were those to AMV (PVAS 92, American Type Culture Collection, Manassas, VA), AMV-Vinca minor L. (DiSTA collection, Italy), and the nepoviruses Strawberry latent ringspot virus, Tomato ringspot virus, and Cherry leaf roll virus. PAS-ELISA revealed only the presence of AMV. Immunoelectron microscopy and gold-labeled decoration confirmed the identity of the virus. In 2001, five symptomless peony plants (provided by a commercial grower and previously analyzed for AMV and TRV presence) were grafted with shoots from peony showing yellow mosaic on the leaves and maintained in a greenhouse under aphid-proof cage. During the summer of 2002, one of the grafted plants showed a mild mosaic on the leaves; PAS-ELISA revealed this peony was infected by AMV. To our knowledge, this is the first report of AMV in peony. Reference: (1) Chang et al. Ann. Phytopathol. Soc. Jpn. 42:325, 1976.

Simultaneous and co-operational amplification (Co-PCR): a new concept for detection of plant viruses

A new and highly sensitive method for the amplification of viral RNA targets from plant material has been developed and patented. This technique called Co-operational amplification (Co-PCR) can be carried out easily in a simple tetraprimer reaction based on the simultaneous action of four primers. The reaction process consists of the simultaneous reverse transcription of two different fragments from the same target, one containing the other; the production of four amplicons by the combination of the two pair of primers, one pair external to other; and the co-operational action of amplicons for the production of the largest fragment. The technique was used successfully, both in metal block and capillary air thermal cyclers for the detection of plant RNA viruses ( Cherry leaf roll virus, Strawberry latent ringspot virus, Cucumber mosaic virus, Plum pox virus and Citrus tristeza virus). The sensitivity observed is at least 100 times higher than that achieved with RT-PCR and similar to nested RT-PCR. Colorimetric detection was coupled with this methodology facilitating its introduction for routine indexing programs and for phytosanitary selection of virus-free plant material.

Localization of Transmissible and Nontransmissible Viruses in the Vector Nematode Xiphinema americanum

ABSTRACT The inner lining of the food canal of nematodes that transmit plantinfecting viruses is regarded as the retention region of viruses. To characterize the location of transmissible and nontransmissible viruses in the vector nematode Xiphinema americanum, three nepoviruses, Tobacco ringspot virus (TRSV), Tomato ringspot virus(TomRSV), and Cherry leaf roll virus(CLRV), and one non-nematode-transmissible virus, Squash mosaic virus (SqMV), were evaluated for transmission efficiency and localization sites in the nematode. Transmission trials showed highest transmission efficiency for TomRSV (38% with 1 and 100% with 10 nematodes, respectively), intermediate efficiency for TRSV (27% with 1 and 65% with 10 nematodes, respectively), and no transmission for CLRV and SqMV. Electron microscopy and immunofluorescent labeling revealed that TRSV was primarily localized to the lining of the lumen of the stylet extension and the anterior esophagus, but only rarely in the triradiate lumen. Within a nematode population, particles of TRSV were no longer observed in these three regions 10 weeks after acquisition, and it is assumed that there was gradual and random loss of the virus from these areas. The percentage of nematodes that were labeled by virus-specific immunofluorescent labeling in a population of viruliferous nematodes decreased gradually after TRSV acquisition when the nematodes were placed on a nonhost of the virus, and the loss of immunofluorescent labeling paralleled the decrease in the ability of the nematode population to transmit the virus. TomRSV was localized only in the triradiate lumen based on thin-section electron microscopy. No virus-like particles were observed in any part of the food canal of nematodes that had fed on CLRV-infected plants. Virus-like particles that appeared to be partially degraded were observed only in the triradiate lumen of nematodes that had fed on SqMV-infected plants. These results clarified the status of localization of two nontransmissible viruses in X. americanum and presented evidence that two nematode-transmissible viruses, TRSV and TomRSV, are localized in different regions of the food canal of X. americanum.

DEVELOPMENT OF A NESTED PCR PROTOCOL FOR DETECTION OF OLIVE-INFECTING VIRUSES IN CRUDE EXTRACTS

A reverse transcription nested polymerase chain reaction (RT-n-PCR) was developed and applied to the detection of Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Olive latent virus-1 (OLV-1), Olive latent virus-2 (OLV-2), Olive latent ringspot virus (OLRSV) and Strawberry latent ringspot virus (SLRV) in naturally infected olive cuttings. The method was more sensitive and reliable than conventional RT-PCR. This increased sensitivity allowed virus detection in samples that were negative to the first round of RT-PCR amplification. Coupled with simple sample preparation from cortical scrapings of olive cuttings, this protocol should be useful in routine testing for olive viruses.

Single-step multiplex RT-PCR for simultaneous and colourimetric detection of six RNA viruses in olive trees

A single-step multiplex RT-PCR was developed for the simultaneous and colourimetric detection of six RNA viruses ( Cucumber mosaic virus, Cherry leaf roll virus, strawberry latent ringspot virus, Arabis mosaic virus, Olive latent-1 virus and Olive latent-2 virus) which infect olive trees. Six compatible primer set for one-step RT-PCR amplification in a single closed-tube and 3′ digoxigenin labelled probes were designed in optimal, specific and conserved regions. The method has been assessed with 195 Spanish field olive trees, suggesting that ∼1.5% of the tested material was infected by Cucumber mosaic virus and 0.5% by Cherry leaf roll virus. This method saves time and reagent costs compared with monospecific RT-PCR which needs several reactions for the same number of tests. Using colourimetric detection, it is possible to analyse many samples, it increases sensitivity 10-fold, and whilst facilitating the interpretation of results, it avoids the use of gels and the toxic ethidium bromide. The method could be used routinely for sanitary and certification programmes.

Subcellular localisation of cherry leaf roll virus coat protein and genomic RNAs in tobacco leaves

The in vivo subcellular location of the coat protein and RNAs of cherry leaf roll nepovirus (CLRV) was studied in infected tobacco plants by two different approaches and it was correlated with the cytopathic structures induced by the virus. Subcellular fractions were obtained by differential centrifugation, visualised by electron microscopy and analysed for their viral RNA and coat protein content by Northern blot and Western blot analysis, respectively. Results indicate that viral RNAs accumulated preferentially at the microsomal fraction. Immunocytochemical studies revealed a clear association of the coat protein of CLRV with the virus-induced cytopathological structures. In situ hybridisation studies confirmed the cytoplasmic location of the virus and allowed one to elucidate the distribution of the CLRV genomic RNAs in the different cell types of infected tissue.

Evidence for Participation of RNA 1-Encoded Elicitor in Cowpea mosaic virus-Mediated Concurrent Protection

The cowpea (Vigna unguiculata) line Arlington, inoculated with Cowpea mosaic virus (CPMV), showed no symptoms, and no infectivity or accumulation of capsid antigen was detected at several days after inoculation. Coinoculation, but not sequential inoculation, of CPMV with similar concentrations of another Comovirus; Cowpea severe mosaic virus (CPSMV), resulted in reduced numbers of CPSMV-induced lesions. This apparent, CPMV-mediated reduction in number of CPSMV-induced infection centers was termed concurrent protection. We report results obtained by inoculating two nearly isogenic cowpea lines derived from a CPMV-susceptible cowpea crossed to Arlington, one line CPMV-susceptible and the other resistant. The CPMV virions B and M, encapsidating genomic RNAs 1 and 2, respectively, were extensively purified by gradient centrifugation. In the CPMV-resistant cowpea, either CPMV or CPMV B affected concurrent protection against CPSMV and against two distinct non-Comoviruses: Cherry leafroll virus and Southern bean mosaic virus. Adding CPMV M to the inoculum did not enhance CPMV-B-mediated protection. CPMV B was ineffective in protecting CPMV-susceptible cowpea. We postulate that CPMV-mediated concurrent protection is elicited in CPMV-resistant cowpea by a CPMV RNA-1-encoded factor and acts to reduce accumulation or spread of CPMV and certain coinoculated challenging viruses in or from the inoculated cell. Coinoculated CPMV did not protect CPMV-resistant cowpea against Tomato bushy stunt virus or Cucumber mosaic virus.

Development of a Sensitive Colorimetric-PCR Assay for Detection of Viruses in Woody Plants.

Diagnostic methods employing the polymerase chain reaction (PCR) provide the most sensitive means currently available for detecting viruses in woody plants. A new technique has been tested that does not rely on gel electrophoresis or molecular hybridization to detect virus-specific PCR products. This colorimetric method for detection of PCR products from woody plants was demonstrated to be at least as sensitive as gel analysis. When combined with immunocapture of virions from plant sap, colorimetric detection provides a means to apply PCR technology to a large number of samples. Here, we report on the use of this technique for detection and quantitation of a walnut isolate of cherry leafroll virus (CLRV-W), citrus tristeza virus (CTV), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), and tomato ringspot virus (ToRSV) in woody and herbaceous plants. For purified virus preparations, detection limits ranged from 100 pg/ml to 100 ag/ml. We also describe the colorimetric PCR detection of CTV in pooled samples.

Detection of cherry leaf roll nepovirus (CLRV) in birch, beech and petunia by immuno‐capture RT‐PCR using a conserved primer pair

SummaryViruses in woody forest plants are known to be difficult to detect owing to phenolic compounds in plant extracts and an often irregular distribution of the pathogens within the plants. We describe a modified immunocapture reverse transcription polymerase chain reaction (RT‐PCR) to detect cherry leaf roll virus (CLRV) in forest trees. The application of a conserved primer pair is shown to be suitable for the detection of different isolates of CLRV from birch from distant forest stands in Germany and also in strains of CLRV from other host plants, including beech, walnut and petunia hybrids. The CLRV isolates and strains could be distinguished by direct sequencing of their PCR products. CLRV was easily detectable throughout the year in tissue of male inflorescences, leaf buds, leaves, single seeds, cortical tissues of young twigs and also in single aphids and bugs (Kleidocerys resedae) collected from a CLRV‐infected birch‐ tree.

Diagnosis and typing of plant viruses by immunocapture RT-PCR coupled to a fluorogenic exonuclease assay

A method for the detection of RNA viral and subviral plant pathogens was developed that combines pathogen partial purification by solid-phase adsorbed antibodies, reverse transcriptional-polymerase chain reaction (RT-PCR) and quantitation of the amplified products by fluorescence. The reverse transcription of the RNA is performed directly on the retained material without any previous thermal or chemical disruption of the virus particles. The whole procedure can be carried out in a microtiter plate. Its validity has been succesfully confirmed for the detection of bean yellow mosaic virus, cherry leafroll virus, cucumber mosaic virus, citrus tristeza virus, grapevine fanleaf virus, potato leafroll virus, pepper mild mottle virus, and tomato spotted wilt virus, as well as the satellite RNA of cucumber mosaic virus and potato spindle tuber viroid. In this procedure virus-specific antibodies can be replaced by monoclonal antibodies against double-stranded RNA, thus offering the possibility of detection when no specific virus antibodies are available, or immunological methods are difficult to use (i.e., subviral pathogens like satellite-RNAs or viroids). The method described has the typical sensitivity of assays based on the polymerase chain reaction, it is not more laborious than ELISA, and an equivalent degree of automation is possible.

Spatial Distribution of Acidic Chitinases and Their Messenger RNAs in Tobacco Plants Infected with Cherry Leaf Roll Virus

Cherry leaf roll virus (CLRV) infection in Nicotiana tabacum cv. Xanthi nc plants induces the expression of, among others, basic and acidic chitinases at 3 d.p.i. (days postinoculation) and 8 d.p.i. in inoculated and uninoculated, systemically infected leaves, respectively. The spatial distribution of the acidictobacco chitinases (PR-P and PR-Q) mRNAs in CLRV-infected tobacco leaves has been studied by tissue printing and Northern (RNA) blot analysis. The mRNA was preferentially detected in and around the concentric necrotic rings caused by the viral infection. In addition, the accumulation pattern of the proteins was very similar although a basal signal was detected in nonsymptomatic tissue in uninoculated, systemically infected leaves. These results indicate a positive association of acidic chitinase gene expression with the necrotic tissue, where the virus is mainly localized, in the CLRV-tobacco interaction.

Walnut Rootstock Comparison and Own-rooted `Chandler' vs. `Chandler' on Paradox Rootstock

In a comparison of six walnut rootstocks either nursery-grafted or field-grafted to `Chandler' (Juglans regia), the highest-yielding trees after 9 years are on either seedling or clonal Paradox rootstocks. Trees growing on both Paradox rootstocks had higher yield efficiency than trees on the black rootstocks in both 1995 and 1996. Since 1993, relative tree size based on trunk circumference has not changed: southern California black (J. californica), seedling Paradox and northern California black (J. Hindsii) have remained significantly larger than clonal Paradox, Texas (J. microcarpa) or Arizona (J. major) black rootstocks. The smaller size of clonal as compared with seedling Paradox trees might be explained by a delay in field grafting success. Although both northern and southern California black rootstock trees were significantly larger than clonal Paradox trees, they did not differ significantly in yield and had significantly lower yield efficiency in 1996. Clonal Paradox trees have significantly smaller nut size than northern California black rootstock trees that can be explained by its higher yield efficiency. An adjacent trial planted in 1991 compares micropropagated `Chandler' on its own root vs. `Chandler' on seedling Paradox rootstock. In 1995 and 1996, own-rooted `Chandler' had significantly greater trunk circumference, yield, and yield efficiency than did `Chandler' on Paradox rootstock. Many of the trees on Paradox rootstock are growing very poorly compared to the own rooted trees. This could be due to diversity within the Paradox seed source. If own-rooted `Chandler' trees become commercially available, they may have potential in areas where other rootstocks are undesirable because of hypersensitivity to cherry leafroll virus.