Use of in vitro cultures of Nicotiana benthamiana for the purification of grapevine virus A

Seedlings of Nicotiana benthamiana were inoculated with grapevine virus A (GVA). Three weeks later, upon systemic symptom expression, cultures were established in vitro using single nodes from these plants. GVA was purified from these cultures and from leaves of GVA-infected N. benthamiana plants maintained in the greenhouse. More virus was obtained from the proliferating in vitro node cultures than from the leaves. The use of in vitro cultures for virus purification represents a valuable practical application of plant tissue culture techniques.     

Use of Optimised PCR Methods for the Detection of GLRaV3: A Closterovirus Associated with Grapevine Leafroll in Tunisian Grapevine Plants

We report a modification and optimisation of a previously published procedure (Minafra and Hadidi, 1994) for the detection of GLRaV3 in infected grapevine plants. GLRaV3 RNA was successfully detected not only in total crude nucleic acid extracts of infected grapevine tissues but also in viruliferous mealybug extracts by IC-RT-PCR. This detection was rapid, sensitive and specific without occurrence of any background. A comparative ELISA, RT-PCR and IC-RT-PCR assays were carried out and revealed the greater sensitivity and specificity of PCR techniques.     

Detection of Grapevine Viruses in Poland

During a 3‐year study, grapevines from 23 vineyards in Poland were surveyed for virus diseases and tested to determine the prevalence of the most economically important viruses by RT‐PCR. The rate of positive samples was 2.2% for grapevine leafroll‐associated virus 1 (GLRaV‐1), 1.9% for grapevine leafroll‐associated virus 2 (GLRaV‐2), 1.5% grapevine leafroll‐associated virus 3 (GLRaV‐3), 1.9% for grapevine virus A (GVA), 0.2% for grapevine virus B (GVB), 0.2% for grapevine virus E (GVE), 0.65% for grapevine fanleaf virus (GFLV), 20.4% for grapevine fleck virus (GFkV) and 71.9% for grapevine rupestris stem pitting‐associated virus (GRSPaV). These viruses were found to occur as single or mixed infections of different combinations in individual grapevines. The overall viral infection rate in the surveyed grapevines was 82.6%. GRSPaV is the most widely distributed virus of all the viruses currently detected in the region. DNA sequencing confirmed the identification of the viruses in selected samples, and analysis indicated that the Polish isolates shared a close molecular identity with the corresponding isolates in GenBank. To our knowledge, this is the first detection of GLRaV‐1, ‐2, ‐3, GVA, GVB, GVE, GFLV, GFkV and GRSPaV in Poland.     

Agrobacterium rhizogenes—transformed plant roots as a source of grapevine viruses for purification

Agrobacterium rhizogenes-mediated transformation was applied toVitis spp. andNicotiana spp. infected by different grapevine phloem-limited viruses (grapevine fleck virus, grapevine virus A, grapevine virus B) to obtain root cultures for virus purification. All plant species were successfully transformed, and several clones were established in liquid culture. Transformed grapevine roots contained as much virus as non transformed roots and more than leaves, as assessed by ELISA and thin sectioning. Likewise, transformed roots ofNicotiana benthamiana Domin. contained in average more GVA than leaves, especially those at the base and the top of the plant, whereas withNicotiana occidentalis wheel., GVB was apparently less concentrated than in leaves.Nicotiana root grew faster than those ofVitis. All viruses multiplied and persisted in root cultures, which were successfully used for purification. Virus yields were the same (GFkV and GVB) or higher (GVB) than those reported in the literature. Grapevine roots may prove useful for culturing and purifying other non-mechanically transmissible grapevine viruses.     

Transmission of Grapevine virus A and Grapevine leafroll‐associated viruses 1 and 3 by Planococcus ficus and Planococcus citri fed on mixed‐infected plants

The Grapevine virus A (GVA) and Grapevine leafroll‐associated viruses 1 and 3 (GLRaV‐1 and GLRaV‐3) are associated with grapevine diseases that induce severe reductions in yield and berry quality. These three viruses are known to coexist in both grapevine and insect vectors, but their cotransmission has been poorly characterised so far. This study investigates the acquisition and transmission of GLRaV‐1, GLRaV‐3 and GVA by Planococcus ficus and Planococcus citri (Hemiptera: Pseudococcidae) following feeding on multiple‐infected plants. The retention and load of the three viruses in the two insect species were analysed. After feeding onto GVA, GLRaV‐1 and GLRaV‐3 mixed‐infected grapevines, nymphs of P. ficus and P. citri showed similar virus acquisition rates and retained low quantities of viruses until the third post‐acquisition day. Despite the similar acquisition patterns, the two vectors differed in transmission efficiency: P. ficus showed a higher efficiency in transmitting GVA and GLRaV‐3, whereas P. citri transmitted GLRaV‐1 more efficiently. When focusing on the virus cotransmission, it appears that GVA could be transmitted to grapevine without GLRaV‐1 and/or GLRaV‐3 and that the transmission of both GLRaVs could take place in the absence of GVA. This comparative study involving different viruses and vector species improves the current knowledge of the semi‐persistent transmission of these three viruses and contributes to the understanding of grapevine virus epidemiology.     

Grapevine Viruses' Detection and Sanitary Selection in Grapevine Germplasm of Calabria (Southern Italy)

A survey of grapevine viruses present in the region of Calabria (southern Italy) was carried out, and the sanitary selection was conducted on various indigenous varieties. Serological (ELISA) and molecular (multiplex RT‐PCR) tests were used to detect the viruses included in the Italian certification programme: Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV), Grapevine leafroll associated virus 1 (GLRaV‐1), Grapevine leafroll associated virus 2 (GLRaV‐2), Grapevine leafroll associated virus 3 (GLRaV‐3), Grapevine virus A (GVA), Grapevine virus B (GVB) and Grapevine fleck virus (GFkV). The frequency with which the above viruses have been detected was 37.4, 32.6, 12.8, 7.7, 7.3, 1.9 and 0.3%, respectively, for GVA, GLRaV‐3, GFLV, GFKV, GLRaV‐1, GLRaV‐2 and GVB. ArMV was never found. The sanitary selection allowed for the detection of 6 putative clones of ‘Arvino’, 2 of ‘Magliocco dolce’ and 2 of the rootstock ‘17–37’ free of the above‐mentioned viruses. The necessary process for the commercialization of these clones as ‘certified’ propagation material was accomplished, and their official approval by the Italian Ministry of Agriculture is currently in progress.     

Partial Sequence Analysis of Geographically Close Grapevine virus A Isolates Reveals their High Regional Variability and an Intra‐Isolate Heterogeneity

To evaluate the genetic diversity of Grapevine virus A (GVA), the genomic region encompassing the partial capsid protein gene and ORF5 was analysed from 10 GVA isolates. Phylogenetic study showed a broad variability of the GVA isolates recovered from a limited geographical area (Slovakia and Czech Republic) and further confirmed the absence of geographical structuration within GVA. Moreover, assessment of structure and intra‐isolate variability revealed that grapevine samples infected with SK13 and SK29 isolates have harboured a population of different sequence variants.     

Use of the Reverse Transcription-Polymerase Chain Reaction for the Detection of Pelargonium Flower Break Carmovirus

A polymerase chain reaction (PCR)-based assay was used to detect pelargonium flower break carmovirus (PFBV) in total RNA extractions made from infected Pelargonium plants. Extracts were reverse transcribed (RT) and the resultant cDNA was amplified by PCR, using oligonucleotide primers specific for 343, 510 and 832 base pair fragments of the RNA-dependent RNA polymerase gene of PFBV.
The specificity and sensitivity of RT-PCR were compared with the enzyme-linked immunosorbent assay (ELISA) for the detection of PFBV in Pelargonium tissues. The virus could be detected efficiently in high dilutions of sap from infected plants and at low concentrations of purified virus. Although ELISA is a powerful tool for virus detection, RT-PCR was over 1000 times more sensitive in detecting PFBV in leaf extracts of infected Pelargonium than was ELISA. The limit of detecting PFBV RNA by RT-PCR was 200 fg, compared with 200 pg of virus by ELISA.     

Highly Efficient Immunodiagnosis of Episomal Banana streak MY virus Using Polyclonal Antibodies Raised Against Recombinant Viral‐Associated Protein

Banana streak MY virus (BSMYV) is the causal agent of viral leaf streak disease of banana, which leads to considerable losses in banana production in most of the banana‐growing regions worldwide. Developing high‐throughput virus detection system is essential for managing viral diseases especially in vegetatively propagated crops like banana. In this study, viral‐associated protein (VAP) coded by ORF II of BSMYV was expressed in Escherichia coli, and polyclonal antibodies were raised against purified recombinant VAP (rVAP) fusion protein in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western blot, immunosorbent electron microscopy (ISEM) and enzyme‐linked immunosorbent assays (ELISAs). In direct antigen‐coated (DAC)‐ELISA, antibodies reacted specifically to BSMYV in crude sap, up to 1 : 8000 dilutions, but not to healthy leaf extracts. Using this antiserum, an immunocapture polymerase chain reaction (IC‐PCR) assay was developed and compared with DAC‐ELISA. VAP antibody‐based IC‐PCR is highly specific and could differentiate episomal virus infection from the integrated endogenous BSV (eBSV) sequences. The recombinant antibodies were validated by testing with a large number of banana germplasm conserved in the field gene bank. Field samples collected during surveys and mother cultures used in tissue culture propagation suggest that antibodies generated against rVAP are sensitive and useful for large‐scale detection of BSMYV. To the best of our knowledge, this is the first report on the production of polyclonal antiserum against recombinant VAP of BSMYV and its suitability for serology‐based testing by ELISA and IC‐PCR. This VAP‐based immunodiagnosis can be applied in quarantine, germplasm exchange and certification programmes.     

Cloning and characterization of VIGG,a novel virus-induced grapevine protein,correlated with fruit quality

We report here the identification and characterization of VIGG, a novel virus-induced grapevine protein. Analysis of VIGG expression in grapevine demonstrated that VIGG was constitutively expressed in leaves and stems in virus-infected grapevine, and that VIGG expression was induced by grapevine virus A (GVA) infection, but not by infection with other viruses. The virus-induced expression profile of VIGG was supported by the finding that virus-free meristem cultures prepared from virus-infected grapevines did not express VIGG. An experiment using GFP–VIGG fusion protein demonstrated that VIGG might be localized in or around the endoplasmic reticulum (ER). Treatment of grapevine cells with ER stress inducers resulted in the induction of VIGG expression. Berries from VIGG-expressing grapevines had higher organic acid and phenolic contents than those from control grapevines that did not express VIGG. Interestingly, fruit composition of a grapevine that was simultaneously infected by GVA and grapevine virus B (GVB), which did not express VIGG, was significantly different from that of GVA-infected grapevines expressing VIGG, suggesting that the effector of fruit composition alteration might be VIGG expression, but not GVA infection. Taken together, VIGG expression might suppress the decrease in organic acid content and increase phenol content in berries. Further investigation of the biological function of VIGG is expected to provide new information on the fruit quality of grapevines.