Titer and distribution of little cherry virus 2 in Prunus avium.

Little cherry virus 2 (LChV-2) is a causal agent of little cherry disease, which produces small, misshapen fruit with poor color and taste. As LChV-2 symptoms are only present near harvest, molecular detection is essential for effective control. Therefore, we determined the titer and distribution of this virus in infected trees over time. While initial infections were found to be basipetal, in field trees, early-stage infection was characterized by uneven distribution and low titer, concentrated in woody stems. In contrast, established infections were systemic, and detection was consistent across tissues. These data provide improved sampling recommendations for the detection of LChV-2.

Quantitative distribution of Citrus yellow mosaic badnavirus in sweet orange (Citrus sinensis) and its implication in developing disease diagnostics.

Citrus yellow mosaic badnavirus (CMBV) is the etiologic agent of citrus yellow mosaic disease, which has caused serious economic losses to Indian citrus industry. CMBV is a quarantined pathogen that is geographically restricted to India. To prevent unintentional movement of the virus to other major citrus-growing countries in fruits, root stocks or grafted citrus plants and facilitate trade, a sensitive, validated diagnostic tool is needed. In the present study, we developed a SYBR Green real-time PCR-based method to detect and quantify CMBV in different tissues of infected Mosambi sweet orange (Citrus sinensis) and compared its sensitivity to conventional PCR protocols. Primers were designed to recognize a portion of the CMBV capsid protein gene. Conventional and real-time PCR were performed on several different tissues: shoot tips, leaves displaying typical CMBV symptoms, asymptomatic leaves, senescent leaves, thorns, green stems and feeder roots. The detection limit of CMBV by conventional PCR was 2.5 × 104 copies per 5 ng of total genomic DNA, while the detection limit of real-time PCR was found to be 4.6 × 102 virus copies per 5 ng of viral DNA. The viral load varied between different tissues. The highest concentration occurred in feeder roots (3.5 × 108 copies per 5 ng of total genomic DNA) and the lowest in thorns (1 × 106 copies per 5 ng of total genomic DNA). The variation in viral load within different tissues suggests movement of the virus within an infected plant that follows the path of photo-assimilates via the phloem. In symptomatic leaves, the CMBV concentration was highest in the lamella followed by midrib and petiole, suggesting that virus resides inside these sections of a leaf and side by side symptoms develop. On the other hand, in asymptomatic leaves, the petiole contained higher virus load than the lamella and midrib suggesting that the pathogen gets established from the stem through the phloem into petiole then infects the lamella and midrib. In addition to information on virus movement, the distribution of CMBV in different tissues helps with the selection of tissues with relatively higher viral load to sample for early and sensitive diagnosis of the disease, which will be useful for better management of the disease in endemic areas.

Two and three dimensional characterization of Zucchini Yellow Mosaic Virus induced structural alterations in Cucurbita pepo L. plants.

Infection of plants by Zucchini Yellow Mosaic Virus (ZYMV) induces severe ultrastructural changes. The aim of this study was to investigate ultrastructural changes during ZYMV-infection in Cucurbita pepo L. plants on the two and three dimensional (2D and 3D) level and to correlate these changes with the spread of ZYMV throughout the plant by transmission electron microscopy (TEM) and image analysis. This study revealed that after inoculation of the cotyledons ZYMV moved into roots [3 days post inoculation (dpi)], then moved upwards into the stem and apical meristem (5 dpi), then into the first true leaf (7 dpi) and could finally be found in all plant parts (9 dpi). ZYMV-infected cells contained viral inclusion bodies in the form of cylindrical inclusions (CIs). These CIs occurred in four different forms throughout the cytosol of roots and leaves: scrolls and pinwheels when cut transversely and long tubular structures and bundles of filaments when cut longitudinally. 3D reconstruction of ZYMV-infected cells containing scrolls revealed that they form long tubes throughout the cytosol. The majority has a preferred orientation and an average length and width of 3 µm and 120 nm, respectively. Image analysis revealed an increased size of cells and vacuoles (107% and 447%, respectively) in younger ZYMV-infected leaves leading to a similar ratio of cytoplasm to vacuole (about 1:1) in older and younger ZYMV-infected leaves which indicates advanced cell growth in younger tissues. The collected data advances the current knowledge about ZYMV-induced ultrastructural changes in Cucurbita pepo.

Molecular biology and epidemiology of dianthoviruses.

The genus Dianthovirus is one of eight genera in the family Tombusviridae. All the genera have monopartite positive-stranded RNA genomes, except the dianthoviruses which have bipartite genomes. The dianthoviruses are distributed worldwide. Although they share common structural features with the other Tombusviridae viruses in their virions and the terminal structure of the genomic RNAs, the bipartite nature of the dianthovirus genome offers an ideal experimental system with which to study basic issues of virology. The two genomic RNAs seem to use distinct strategies to regulate their translation, transcription, genome replication, genome packaging, and cell-to-cell movement during infection. This review summarizes the current state of our knowledge of the dianthoviruses, with its main emphasis on the molecular biology of the virus, including the viral and host factors required for its infection of host plants. The epidemiology of the virus and the possible viral impacts on agriculture and the environment are also discussed.

Within plant distribution of Potato Virus Y in hairy nightshade (Solanum sarrachoides): an inoculum source affecting PVY aphid transmission.

Potato virus Y (PVY) is vectored by several potato-colonizing and non-colonizing aphid species in a non-persistent manner and has a wide host range. It occurs naturally in several plant families. Myzus persicae and Macrosiphum euphorbiae are the most efficient potato-colonizing aphid vectors of PVY. Rhopalosiphum padi, a cereal aphid that migrates in large numbers through potato fields during the middle of the growing season, does not colonize potato plants but can transmit PVY. Hairy nightshade, Solanum sarrachoides, a prevalent annual solanaceous weed in the Pacific Northwest (PNW) of the United States, is an alternative host for PVY and a preferred host for M. persicae and M. euphorbiae. Hence, hairy nightshade plants might play an important role as an inoculum source in the epidemiology of PVY. We looked at titre accumulation and distribution of PVY(O), PVY(N:O) and PVY(NTN) in S. sarrachoides and potato after aphid inoculation with M. persicae and studied the transmission of PVY(O) and PVY(NTN), by M. persicae, M. euphorbiae and R. padi from hairy nightshade to potato plants. Virus titre at different positions on the plant was similar in S. sarrachoides and potato plants with strains PVY(O) and PVY(N:O). Titres of PVY(NTN) were similar in S. sarrachoides and potato but differences in titre were observed at different positions within the plant depending on the plant phenology. Percentage transmission of PVY(NTN) by M. persicae and M. euphorbiae was twice as high (46 and 34%, respectively) from hairy nightshade to potato than from potato to potato (20 and 14%). Percentage transmission of PVY(O) by M. persicae and M. euphorbiae was not affected by the inoculum source. No effect of the inoculum source was observed in the transmission of either PVY strain by R. padi. These results show that hairy nightshade may be an equal or better virus reservoir than potato and thus, important in the epidemiology of PVY.

A real-time RT-PCR assay for detection and absolute quantitation of Citrus tristeza virus in different plant tissues.

A real-time RT-PCR assay using SYBR Green was developed for specific and reliable quantitative detection of Citrus tristeza virus (CTV) in infected plants. A general primer set designed from conserved sequences in ORFs 1b and 2 enabled amplification of the genomic RNA (gRNA) while excluding most subgenomic and defective RNAs. Single RT-PCR products of 204 bp (isolate T36) or 186 bp (other isolates) were obtained with no primer-dimer or non-specific amplifications detected. Melting curve analysis revealed distinct melting temperature peaks (T(m)) for severe and mild isolates. External standard curves using RNA transcripts of the selected target allowed a reproducible quantitative assay, with a wide dynamic range of detection starting with 10(2) gRNA copies and with very low variation coefficient values. This protocol enabled reliable assessments of CTV accumulation in different tissues and from different citrus species, grown in the greenhouse or under field conditions, and infected with CTV isolates differing in their pathogenicity. CTV accumulation was higher in bark and fruits than in roots or leaves and showed minimal differences among several susceptible citrus species, but it was significantly lower in sour orange. This quantitative detection assay will be a valuable tool for diagnosis and molecular studies on CTV biology.

Abutilon mosaic virus DNA B component supports mechanical virus transmission, but does not counteract begomoviral phloem limitation in transgenic plants.

Different Nicotiana benthamiana lines stably transformed with Abutilon mosaic virus (AbMV) dimeric DNA B were capable of systemically spreading complete bipartite AbMV genomes, following agroinoculation of DNA A alone. Constitutively expressed viral movement protein (BC1) did not induce any persistent disease phenotype, but plants developed transient morphological abnormalities such as radially symmetric leaves after kanamycin withdrawal. Systemic AbMV infection produced symptoms and virus titers indistinguishable from those in non-transgenic plants. In systemically invaded leaves, the begomovirus remained phloem-limited, whereas the plants' susceptibility to mechanical transmission of AbMV was enhanced by a factor of three to five, as compared to non-transgenic controls. Hence, DNA B-encoded movement functions can complement local movement to the phloem after mechanical transmission, but fail to support viral invasion of non-phloem cells in systemically infected organs, indicating that the phloem restriction of AbMV does not result predominantly from a lack of transport competence in mesophyll tissues.

Bioimprinted QCM sensors for virus detection-screening of plant sap.

Surface imprinting techniques on polymer-coated quartz-crystal microbalances (QCM) have been used to detect tobacco mosaic viruses (TMV) in aqueous media. Molecularly imprinted polymers (MIP), tailor-made by self organisation of monomers around a template (TMV), were generated directly on the gold electrodes. Imprinted trenches on the polymer surface mimicking the shape and surface functionality of the virus serve as recognition sites for re-adsorption after washing out of the template. The sensors are applicable to TMV detection ranging from 100 ng mL(-1) to 1 mg mL(-1) within minutes. Furthermore, direct measurements without time-consuming sample preparation are possible in complex matrices such as tobacco plant sap.

Detection of melon necrotic spot virus in water samples and melon plants by molecular methods.

Melon necrotic spot virus (MNSV) is a water and soil-borne pathogen affecting species of the Cucurbitaceae family both in hydroponic and soil crops. Molecular methods for detecting MNSV in water samples, nutrient solutions and melon plants were developed. For this purpose, water samples from a water source pool of a hydroponic culture or from the recirculating nutrient solution were concentrated by ultracentrifugation or PEG precipitation followed by RT-PCR analysis. Both concentration methods were suitable to allow the detection of MNSV and represent, as far as we know, the first time that this virus has been detected in water samples. A non-isotopic riboprobe specific for MNSV was obtained and used to detect the virus in plant tissue. Different parts of mechanically infected plants were examined including the roots, stems, inoculated cotyledons and young leaves. Excluding the inoculated cotyledons, the tissues showing the highest accumulation levels of the virus were the roots. The potential inclusion of such tools in management programs is discussed.

P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA unction.

The molecular basis for virus-induced disease in plants has been a long-standing mystery. Infection of Arabidopsis by Turnip mosaic virus (TuMV) induces a number of developmental defects in vegetative and reproductive organs. We found that these defects, many of which resemble those in miRNA-deficient dicer-like1 (dcl1) mutants, were due to the TuMV-encoded RNA-silencing suppressor, P1/HC-Pro. Suppression of RNA silencing is a counterdefensive mechanism that enables systemic infection by TuMV. The suppressor interfered with the activity of miR171 (also known as miRNA39), which directs cleavage of several mRNAs coding for Scarecrow-like transcription factors, by inhibiting miR171-guided nucleolytic function. Out of ten other mRNAs that were validated as miRNA-guided cleavage targets, eight accumulated to elevated levels in the presence of P1/HC-Pro. The basis for TuMV- and other virus-induced disease in plants may be explained, at least partly, by interference with miRNA-controlled developmental pathways that share components with the antiviral RNA-silencing pathway.

The DNA-B of the non-phloem-limited bean dwarf mosaic virus (BDMV) is able to move the phloem-limited Abutilon mosaic virus (AbMV) out of the phloem, but DNA-B of AbMV is unable to confine BDMV to the phloem.

Abutilon mosaic virus (AbMV) and bean dwarf mosaic virus (BDMV) are two phylogenetically related bipartite begomoviruses. While AbMV is restricted to phloem, BDMV spreads to non-phloem tissues. Cell-to-cell and long-distance movement of AbMV and BDMV were investigated after replacing the coat protein (CP) gene with the reporter gene encoding the green fluorescence protein (GFP). The DNA-A and DNA-B genomic components of AbMV and BDMV, and their pseudorecombinants (PR), were delivered to bean (Phaseolus vulgaris) seedlings and detached leaves with DNA-coated microprojectiles. Virus-associated fluorescence was observed with the confocal microscope. Delivery of AbMV and BDMV GFP reporters showed that the epidermal tissue was the main recipient of the viral DNA; the DNA-A of the two viruses was unable to move out of the recipient cells. AbMV DNA-A co-inoculated with AbMV DNA-B did not move from cell to cell in the epidermis and did not reach the phloem. However, co-inoculation of AbMV DNA-A with BDMV DNA-B resulted in PR cell-to-cell movement out of the epidermis and long-distance movement in the phloem. In contrast, BDMV DNA-A moved from cell to cell and over a long distance when co-inoculated with either its own DNA-B or with the DNA-B of AbMV. Thus, the DNA-B of the non-phloem-limited BDMV overcame the phloem limitation of AbMV. In the reciprocal case, the DNA-B of the phloem-limited AbMV did not confine the non-phloem limited BDMV to the phloem. Hence, we assume that the DNA-A component of BDMV includes determinants involved in the movement pattern of the virus in addition to the DNA-B-encoded BC1 and BV1 which have previously been shown to be involved in virus movement. The results also confirm that the CP is not necessary for virus movement; however, replacing the CP of AbMV and BDMV with GFP resulted in a decrease in symptom severity. DNA-B was involved in symptom severity; the B component of BDMV produced symptoms more severe than those induced by that of AbMV, whether in wild-type PRs or in PRs with CP-GFP replacement. It is interesting to note that when the GFP gene under the control of the CaMV 35S promoter (35S-GFP) was delivered to the bean tissue, with or without the DNA-B component of BDMV, GFP was expressed but did not move from cell to cell. However, when the 35S-GFP was delivered together with BDMV DNA-A and DNA-B, GFP showed cell-to-cell movement in the epidermis but was restricted to these cells. Hence, infection of cells with a functional bipartite begomovirus may facilitate cell-to-cell movement of macromolecules.

Evidence that soilborne wheat mosaic virus moves long distance through the xylem in wheat.

Soilborne wheat mosaic virus (SBWMV) is a member of the genus Furovirus of plant viruses. SBWMV is transmitted to wheat roots by the plasmodiophorid vector Polymyxa graminis. Experiments were conducted to determine the path for SBWMV transport from roots to leaves. The results of immunogold labeling suggest that SBWMV enters and moves long distance through the xylem. SBWMV may enter primary xylem elements before cell death occurs and then move upward in the plant after the xylem has matured into hollow vessels. There is also evidence for lateral movement between adjacent xylem vessels.

Potyviral helper-component proteinase expressed in transgenic plants enhances titers of Potato leaf roll virus but does not alleviate its phloem limitation.

Coinfection of Nicotiana benthamiana with Potato virus A (PVA, a potyvirus) and Potato leaf-roll virus (PLRV, a luteovirus) induces a synergistic interaction manifested by enhanced titers of PLRV. The helper component proteinase (HC-Pro) of potyviruses is involved in viral vascular movement and suppression of an antiviral defense mechanism in plants. Data of our study showed that accumulation of PLRV in transgenic N. benthamiana expressing the PVA HC-Pro was enhanced on average by 4.5-fold, as compared to a 6.0-fold enhancement in wild-type N. benthamiana plants doubly infected with PVA and PLRV. Enhancement of PLRV accumulation was directly proportional to the concentration of the HC-Pro in leaves. In the HC-Pro-transgenic plants and wild-type plants, PLRV was almost exclusively confined to the phloem, but the HC-Pro-transgenic plants had a fourfold greater number of PLRV-infected cells within the phloem tissues, as revealed by immunohistochemical staining. In the leaves doubly infected with PLRV and PVA, PLRV was found to exit the phloem in 25.0% of the veins, infecting all types of leaf cells, but, on average, PLRV accumulation was not enhanced more than by sixfold at the whole-leaf level. Therefore, potyviral/luteoviral synergism seems to be based on two mechanisms. One of them is mediated by the HC-Pro and increases luteovirus accumulation without allowing detectable egress from vascular tissue. The other mechanism probably depends on additional potyviral proteins and alleviates the normal phloem limitation of PLRV.

Synergistic interactions of a potyvirus and a phloem-limited crinivirus in sweet potato plants.

When infecting alone, Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) and Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus) cause no or only mild symptoms (slight stunting and purpling), respectively, in the sweet potato (Ipomoea batatas L. ). In the SPFMV-resistant cv. Tanzania, SPFMV is also present at extremely low titers, though plants are systemically infected. However, infection with both viruses results in the development of sweet potato virus disease (SPVD) characterized by severe symptoms in leaves and stunting of the plants. Data from this study showed that SPCSV remains confined to phloem and at a similar or slightly lower titer in the SPVD-affected plants, whereas the amounts of SPFMV RNA and CP antigen increase 600-fold. SPFMV was not confined to phloem, and the movement from the inoculated leaf to the upper leaves occurred at a similar rate, regardless of whether or not the plants were infected with SPCSV. Hence, resistance to SPFMV in cv. Tanzania was not based on restricted virus movement, neither did SPCSV significantly enhance the phloem loading or unloading of SPFMV. It is also noteworthy that SPVD is an unusual synergistic interaction in that the potyvirus component is not the cause of synergism but is the beneficiary. It is hypothesized that SPCSV is able to enhance the multiplication of SPFMV in tissues other than where it occurs itself, perhaps by interfering with systemic phloem-dependent signaling required in a resistance mechanism directed against SPFMV.

Relative concentration of plum pox virus in leaves and flowers of some Prunus species and cultivars.

The relative concentration of plum pox virus (PPV) in leaves and flowers of plum, damson, myrobalan, blackthorn, apricot and peach trees was determined by enzyme-linked immunosorbent assay (ELISA) and expressed as the lowest dilution with positive reaction. Significant differences in relative PPV concentration were found in leaves among individual Prunus species naturally or artificially infected with the virus. The highest relative PPV concentration was found in blackthorns (7.81 x 10(-4)), common plum and apricot (1.56 x 10(-3) for the both latters). Wild growing PPV-infected plums and blackthorns can be considered equally important source of sharka infection as PPV-susceptible cultivars of plums, apricots and peaches. High PPV concentration in flowers is of diagnostical value. High variability of relative PPV concentration was observed inside the species among individual cultivars. Susceptible cultivars were characteristic by high relative PPV concentration, e.g. apricot cvs. Vegama (9.8 x 10(-5)) and Velkopavlovická (1.95 x 10(-4)), and peach cvs. Maria Emilia (7.81 x 10(-4)) and Harbinger (1.56 x 10(-3)). On the other hand, cultivars resistant to PPV were characteristic by very low relative PPV concentration, e.g. apricot cv. Stark Early Orange (5 x 10(-2)) and peach cvs. Envoy (5 x 10(-2)) and Favorita Morettini (2.5 x 10(-2)). The highest relative PPV concentration was found in young trees newly infected with PPV.