This study was conducted to molecular diagnostics of Grapevine fanleaf virus (GFLV) in leaf samples of grapevine were collected from the grapevine yards in Salahuddin governorate, where grapevines ...are commonly grown in Iraq. The virus was detected in the samples by using SuperScriptTM III RT kit and pair of specific primers GFLV2231F and GFLV2533R was used to amplify fragments of the coat protein gene (CP) in rapid direct one tube RT-PCR.
The ectoparasitic nematode
transmits grapevine fanleaf virus (GFLV) during feeding on grapevine roots, causing fanleaf degeneration in the plant. Hence, resistance breeding is a key to develop novel ...rootstocks to overcome such threats. In past years, various grapevine species were screened, and a few candidates with partial resistance were identified. However, they were hardly sufficient for viticulture because of their many agronomical defects. To develop reliably resistant rootstocks applicable in viticulture, multiple
spp. genotypes were analyzed using root inoculation with nematodes in glass vials as an early and easy evaluation test. Resistance levels were evaluated 35 days after inoculation based on nematode reproduction factors, focusing on juveniles and eggs. Infection of grapevines with GFLV was analyzed after inoculation with viruliferous
. With this fast screening system, putative candidates with resistances against
have been identified for future breeding programs. Particularly, genotypes with the genetic background of
and
were found to be nematode-resistant.
Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently, and ...shown to be highly effective in plants, including grapevine, but the underlying mechanism is unknown. Here we present the high-resolution cryo electron microscopy structure of the GFLV–Nb23 complex, which provides the basis for molecular recognition by the Nb. The structure reveals a composite binding site bridging over three domains of one capsid protein (CP) monomer. The structure provides a precise mapping of the Nb23 epitope on the GFLV capsid in which the antigen loop is accommodated through an induced-fit mechanism. Moreover, we uncover and characterize several resistance-breaking GFLV isolates with amino acids mapping within this epitope, including C-terminal extensions of the CP, which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Nb-mediated resistance to vector transmission. Together, these data provide insights into the molecular mechanism of Nb23-mediated recognition of GFLV and of virus resistance loss.
Grapevine fanleaf virus (GFLV) is the main causal agent of fanleaf degeneration, the most damaging viral disease of grapevine. GFLV is included in most grapevine certification programs that rely on ...robust diagnostic tools such as biological indexing, serological methods, and molecular techniques, for the identification of clean stocks. The emergence of high throughput sequencing (HTS) offers new opportunities for detecting GFLV and other viruses in grapevine accessions of interest. Here, two HTS-based methods,
, RNAseq and smallRNAseq (focusing on the 21 to 27 nt) were explored for their potential to characterize the virome of grapevine samples from two 30-year-old GFLV-infected vineyards in the Champagne region of France. smallrnaseq was optimal for the detection of a wide range of viral species within a sample and RNAseq was the method of choice for full-length viral genome assembly. The implementation of a protocol to discriminate between low GFLV titer and
contamination (intra-lane contamination due to index misassignment) during data processing was critical for data analyses. Furthermore, we compared the performance of semi-quantitative DAS-ELISA (double antibody enzyme-linked immunosorbent assay), RT-qPCR (Reverse transcription-quantitative polymerase chain reaction), Immuno capture (IC)-RT-PCR, northern blot for viral small interfering RNA (vsiRNA) detection and RNAseq for the detection and quantification of GFLV. While detection limits were variable among methods, as expected, GFLV diagnosis was consistently achieved with all of these diagnostic methods. Together, this work highlights the robustness of DAS-ELISA, the current method routinely used in the French grapevine certification program, for the detection of GFLV and offers perspectives on the potential of HTS as an approach of high interest for certification.
Detecting and identifying viral infections in perennial plants, such as grapevines, can be challenging. Therefore, the aim of this study was to perform a real-time RT-PCR (RT-qPCR) high-resolution ...melting (HRM) curve analysis to detect and differentiate Brazilian variants of grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine fanleaf virus (GFLV) in 74 and 10 infected plants, respectively, maintained in a collection block of grapevines. A single amplification curve was generated for each sample by RT-qPCR. Considering the amplified region of genomes of these two viruses, it was possible to identify and distinguish different variants of GLRaV-3 and of GFLV, which showed significantly different melting temperature (Tm) values between themselves, reflecting differences in the nucleotide sequences of the respective amplicons, and allowing discriminating variants and assess the viral diversity in grapevine accessions. The HRM analysis was validated by sequencing and nucleotide comparisons among Brazilian isolates of GLRaV-3 and GFLV.
Detectar e identificar infecções virais em plantas perenes, como videiras, pode ser um desafio. Portanto, o objetivo deste estudo foi realizar uma análise da curva de dissociação de alta resolução (HRM) por RT-PCR em tempo real (RT-qPCR) para detectar e diferenciar variantes do vírus do enrolamento foliar tipo 3 (GLRaV-3) e do vírus do urticado ou nó-curto (GFLV) em 74 e 10 plantas infetadas, respectivamente, mantidas em blocos de coleções de videiras. Uma única curva de amplificação foi gerada para cada amostra por RT-qPCR. Considerando a região amplificada dos genomas dos dois vírus, foi possível identificar diferentes variantes de GLRaV-3 e GFLV, que apresentaram valores de temperatura de dissociação (Tm) significamente diferentes entre si, refletindo diferenças nas sequências de nucleotídeos dos respectivos DNA amplificados e, assim, constituindo uma forma simplificada de diferenciar variantes e avaliar a diversidade viral em acessos de videiras. A análise de HRM foi validada pelo sequenciamento e comparação de nucleotídeos de isolados brasileiros de GLRaV-3 e GFLV.
(GFLV) is one of the most severe virus diseases of grapevines, causing fanleaf degeneration that is transmitted by
This paper aims to isolate
species from Tunisian vineyard soil samples and assess ...their ability to acquire and transmit GFLV under natural and controlled conditions. Based on morphological and morphometric analyses, Tunisian dagger nematodes were identified as
and
These results were confirmed with molecular identification tools using species-specific polymerase chain reaction primers. The total RNA of GFLV was extracted from specimens of
and amplified based on real-time polymerase chain reaction using virus-specific primers. Our results showed that
could acquire and transmit the viral particles of GFLV. This nepovirus was not detected in
, under natural conditions; however, under controlled conditions, this nematode was able to successfully acquire and transmit the viral particles of GFLV.
Several grapevine viruses were reported in Algeria and especially in grapevine germplasm collection, therefore it is a great challenge to free these varieties from virus infection before any breeding ...programs. Our study focused on the development of chemotherapy on autochthonous varieties collected in the grapevine germplasm collection of ITAFV. All these varieties were tested by DAS-ELISA and the presence of GLRaV-3 and GFLV was confirmed in all used samples for the sanitation. After 8 weeks of shoot tips
in vitro
culture in a modified M S medium containing ribavirin, DAS-ELISA test revealed that GLRaV-3 was completely eliminated and GFLV to a significant rate.
To determine the occurrence and distribution of prevalent viruses in commercially important vineyards, a survey was carried out in all thirteen wine-growing regions in Germany. Results reveal that ...the recently emerged
Grapevine pinot gris virus
(GPGV) was the most abundant virus with a percentage of 18% prevalence, followed by 13%
Grapevine fleck virus
(GFkV), 9%
Grapevine leafroll-associated virus
1 (GLRaV-1), 4%
Grapevine fanleaf virus
(GFLV), 2%
Raspberry ringspot virus
(RpRSV), 2%
Arabis mosaic virus
(ArMV) and 2%
Grapevine leafroll-associated virus
3 (GLRaV-3). Distribution of some viruses varies greatly between individual regions, thus regional hotspots or gradients were detected. GPGV for example is mostly found in southeastern Germany, while its incidence decreases to the north along the river Rhine. The findings of this survey provide an overview of the allocation of the most prevalent grapevine viruses in Germany and can support regional virus management and national risk assessment especially GPGV.
Purpose. Grapevines (Vitis spp.) are affected by many viral diseases which cause serious pathological problems. GLRaV-3 is among the most widespread leafroll viruses, while Grapevine Fanleaf Virus ...(GFLV) is a destructive pathogen which reduces the lifespan of grapevine. Considering the impact and the spread of these diseases, our objective was to analyse the presence of these two viruses in several grapevine varieties in grapevine collection at ATTC Vlore. Data gathered from plant pathogens serve to better understand and prevent the spread of pathogens, as a mandatory rule for the quality control of certified plant material during vegetative propagation.
Method. The presence of two common viruses were tested using virus specific primers; LC1/LC2 primer pair designed from the hHSP70 gene for detecting Grapevine Leafroll-associated Virus-3 (GLRaV3) and C3390/H2999 primer pair, designed from coat protein coding regions for detecting Grapevine Fanleaf Virus (GFLV), in six varieties; ‘Merlot’, ‘Kallmet’, ‘Shesh i zi’, ‘Shesh i bardhё’, ‘Debinё’, and ‘Pulёz’, provided through a randomised sampling procedure. One Step Reverse Transcription Polymerase Chain Reaction assay was used to detect the viral presence.
Results showed a high (100%) prevalence of GLRaV3 virus in all of analysed samples, as the most frequent among the two pathogens. Analysis for of GFLV virus showed low infection rate, being present in only one sample.
Conclusions. We herein show an efficient, fast and reproducible method for detecting grapevine viruses through one step RT-PCR. Our results suggest that sampling of the infected plant material should be avoided due to the presence of viral infections.
To better understand the distribution and genetic variety of Grapevine fanleaf virus (GFLV) in two organs of the grapevine, the presence of this virus was tested in leaf and pollen samples by ELISA ...and RT-PCR methods. On average, the GFLV is present in the pollen and the leaves with percentages 93 and 36% respectively. Capsid protein sequences were aligned and showed 85-92,1% identity at the nucleotide level for isolates obtained from leaves and 96-99% identity for isolates obtained from pollen. GFLV isolates from pollen are less divergent and phylogenetically closer to each other than those detected in leaves based on the analysis of the 2 CP sequences. GFLV was detected in seeds, berries, peduncles and grape bunches. Pollen appeared to have potential contribution in GFLV transmission by cross-pollination flowers on healthy plants, which leads to the formation of infected seeds and racemes.
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