Leafroll Research Articles

Developing a mealybug pheromone monitoring tool to enhance IPM practices in New Zealand vineyards

Mealybugs are phloem-feeding insects found on many crops worldwide. In New Zealand vineyards, they transmit the economically important Grapevine leafroll-associated virus 3 (GLRaV-3). For some mealybug species, synthetic sex pheromones have been commercialised, and are used as monitoring tools. The mealybugs Pseudococcus longispinus and Pseudococcus calceolariae are major pests in many New Zealand vineyards. We present work on the development of a combined P. longispinus and P. calceolariae pheromone lure. The optimal dose for monitoring P. longispinus was found to be 10 µg of the (S)-(+)-enantiomer, either alone or in the racemic mixture. Addition of the corresponding alcohol did not improve trap catch of P. longispinus. Both the P. longispinus and the P. calceolariae pheromone lures remained active in the field for 90 days. Combining the 2 species’ pheromones had no negative effects on male mealybug trap catch for either species. We conclude that the pheromone ester alone is the best lure for the male P. longispinus. Combining the two mealybug species’ pheromones into a single lure provides the New Zealand viticultural industry with an efficient monitoring tool. Late-vintage deployment of baited lures will provide information on mealybug abundance and local distribution that will inform the scope of future insecticide programmes, to target areas based on need rather than an area-wide application by default.

Spatial Roguing Reduces the Incidence of Leafroll Disease and Curtails Its Spread in a Finger Lakes Cabernet franc Vineyard

<h3>Abstract</h3> Leafroll is one of the most economically important viral diseases of grapevines worldwide. Grapevine leafroll-associated virus 1 (GLRaV-1) and grapevine leafroll-associated virus 3 (GLRaV-3) are prevalent in New York vineyards in which low-density grape mealybug populations reside. A five-year experiment was performed in a commercial ‘Cabernet franc’ vineyard in the Finger Lakes region of New York to test the influence of spatial roguing, i.e., the elimination of virus-infected vines and their two immediate within-row neighbors on each side, on the annual incidence of GLRaV-1 and GLRaV-3. In a second treatment, spatial roguing was combined with insecticides. Vines eliminated in both spatial roguing treatments were replaced by clean vines derived from virus-tested stocks. The objective of this study was to reduce temporal virus incidence to less than 1% over two consecutive years and limit virus spread. In both spatial roguing treatments, virus incidence was reduced from 5% in 2016 to less than 1% in 2020-2021. Among vines in the insecticide-free, non-rogued control treatment, virus incidence increased from 5% to 16% from 2016 to 2021. Insecticides applied in 2016-2021 helped significantly reduce grape mealybug populations to near zero annually, while populations in the untreated control vines were 57 to 257-fold higher during the same period. However, insecticides contributed relatively little to limit the number of newly infected vines. Together, these findings highlight the salient contribution of roguing to an overall leafroll disease management response in a vineyard with low disease incidence and low grape mealybug abundance. To our knowledge, this is the first report on the effectiveness of spatial roguing at reducing the annual incidence of leafroll disease in a vineyard.

Elimination of Eight Viruses and Two Viroids from Preclonal Candidates of Six Grapevine Varieties (Vitis vinifera L.) through In Vivo Thermotherapy and In Vitro Meristem Tip Micrografting.

Viruses and virus-like organisms are a major problem in viticulture worldwide. They cannot be controlled by standard plant protection measures, and once infected, plants remain infected throughout their life; therefore, the propagation of healthy vegetative material is crucial. In vivo thermotherapy at 36–38 °C for at least six weeks, followed by meristem tip micrografting (0.1–0.2 mm) onto in vitro-growing seedling rootstocks of Vialla (Vitis labrusca × Vitis riparia), was successfully used to eliminate eight viruses (grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine Pinot gris virus (GPGV), grapevine fanleaf virus (GFLV), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine fleck virus (GFkV), grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus-1 (GSyV-1), and raspberry bushy dwarf virus (RBDV)), as well as two viroids (hop stunt viroid (HSVd) and grapevine yellow speckle viroid 1 (GYSVd-1)) from preclonal candidates of six grapevine varieties (Vitis vinifera L.). A half-strength MS medium including vitamins supplemented with 30 g/L of sucrose and solidified with 8 g/L of agar, without plant growth regulators, was used for the growth and root development of micrografts and the subsequently micropropagated plants; no callus formation, hyperhydricity, or necrosis of shoot tips was observed. Although the overall regeneration was low (higher in white than in red varieties), a 100% elimination was achieved for all eight viruses, whereas the elimination level for viroids was lower, reaching only 39.2% of HSVd-free and 42.6% GYSVd-1-free vines. To the best of our knowledge, this is the first report of GPGV, GRVFV, GSyV-1, HSVd, and GYSVd-1 elimination through combining in vivo thermotherapy and in vitro meristem tip micrografting, and the first report of RBDV elimination from grapevines. The virus-free vines were successfully acclimatized in rockwool plugs and then transferred to soil.

Regression Modeling Strategies to Predict and Manage Potato Leaf Roll Virus Disease Incidence and Its Vector

The potato leaf roll virus (PLRV) disease is a serious threat to successful potato production and is mainly controlled by integrated disease management; however, the use of chemicals is excessive and non-judicious, and it could be rationalized using a predictive model based on meteorological variables. The goal of the present investigation was to develop a disease predictive model based on environmental responses viz. minimum and maximum temperature, rainfall and relative humidity. The relationship between epidemiological variables and PLRV disease incidence was determined by correlation analysis, and a stepwise multiple regression was used to develop a model. For this purpose, five years (2010–2015) of data regarding disease incidence and epidemiological variables collected from the Plant Virology Section Ayub Agriculture Research Institute (AARI) Faisalabad were used. The model exhibited 94% variability in disease development. The predictions of the model were evaluated based on two statistical indices, residual (%) and root mean square error (RMSE), which were ≤±20, indicating that the model was able to predict disease development. The model was validated by a two-year (2015–2017) data set of epidemiological variables and disease incidence collected in Faisalabad, Pakistan. The homogeneity of the regression equations of the two models, five years (Y = −47.61 − 0.572x1 + 0.218x2 + 3.78x3 + 1.073x4) and two years (Y = −28.93 − 0.148x1 + 0.510x2 + 0.83x3 + 0.569x4), demonstrated that they validated each other. Scatter plots indicated that minimum temperature (5–18.5 °C), maximum temperature (19.1–34.4 °C), rainfall (3–5 mm) and relative humidity (35–85%) contributed significantly to disease development. The foliar application of salicylic acid alone and in combination with other treatments significantly reduced the PLRV disease incidence and its vector population over control. The salicylic acid together with acetamiprid proved the most effective treatment against PLRV disease incidence and its vector M. persicae.

A novel framework for potato leaf disease detection using an efficient deep learning model

Potato disease management plays a valuable role in the agriculture field as it might cause a significant loss in crops production. Therefore, timely recognition and classification of potato leaves diseases are necessary to minimize the loss, however, it is time taking task and requires human efforts. Thus, an accurate automated technique for timely detection and classification is needed to cope with the aforementioned challenges.There exist techniques grounded on machine learning and deep learning procedures that use the existing dataset i.e., ‘The Plant Village Dataset’ and perform classification into only two classes in potato leaves. Therefore, this article proposes a technique based on an improved deep learning algorithm that uses the potato leaf visual features to classify them into five classes i.e., Potato Late Blight (PLB), Potato Early Blight (PEB), Potato Leaf Roll (PLR), Potato Verticillium_wilt (PVw) and Potato Healthy (PH) class. The propose model is trained on the existing dataset i.e., “The Plant Village” that comprises of images having two ailments such as Early Blight (EB) and Late Blight (LB), and a Healthy class for potato leaves. Additionally, we have gathered the data for classes i.e., Potato Leaf Roll (PLR), Potato Verticillium_wilt (PVw) and Potato Healthy (PH) manually. A pre-trained Efficient DenseNet model has been employed utilizing an extra transition layer in DenseNet-201 to classify the potato leave diseases efficiently. Moreover, the usage of the reweighted cross-entropy loss function makes our proposed algorithm more robust as the training data is highly imbalanced. The dense connections with regularization power help to minimize the overfitting during the training of small training sets of potato leaves samples. The proposed algorithm is a novel and first technique to address and report the successful implementation for the detection and classification of four diseases in potato leaves. The algorithm’s performance was evaluated on the testing set and gave an accuracy of 97.2%. Various experiments have been performed to confirm that our proposed algorithm is more consistent and proficient to detect and classify potato leaves diseases than existing models.

First Report of Grapevine Virus L Infecting Grapevine in Southeast France.

Grapevine virus L (GVL) is a recently described vitivirus (family Betaflexiviridae) with a positive-sense single-stranded RNA genome. It has so far been reported from China, Croatia, New-Zealand, the United States and Tunisia (Debat et al. 2019; Diaz-Lara et al. 2019; Alabi et al. 2020; Ben Amar et al. 2020). It has significant genetic variability (up to 26% of nucleotide divergence between isolates) and the existence of four phylogroups has been proposed (Alabi et al. 2020). In the frame of a project investigating the possible links between grapevine trunk diseases and grapevine virome, viral high throughput sequencing (HTS)-based testing was performed on symptomatic and asymptomatic grapevines collected in July 2019 in vineyards of four areas in France (Bourgogne, Charentes, Gard, Gironde) corresponding to five cultivars of Vitis vinifera (Cabernet franc, Cabernet Sauvignon, Chardonnay, Sauvignon, Ugni blanc). Total RNAs were purified from powder of 105 trunk wood samples using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and RNA-seq libraries were prepared using Zymo-Seq RiboFree Total RNA Library Prep Kit (Ozyme, Saint Cyr l'Ecole, France). HTS was performed on a S4 lane of Illumina NovaSeq 6000 using a paired-end read length of 2x150 bp. The trimmed sequence reads obtained from Chardonnay plants CH30-75M (99.9 M) and CH37-19S (114 M) from a vineyard in Gard were analyzed using CLC Genomics Workbench v21 (Qiagen, Courtaboeuf, France) and revealed complex mixed infections. Besides contigs representing a complete GVL genome (average scaffold coverage: 6,197x and 2,970x, respectively), contigs from grapevine rupestris stem pitting virus (1,697x ; 1,124x), grapevine virus A (82x ; 95x), grapevine pinot gris virus (1,475x ; 866x), grapevine leafroll-associated virus 3 (5,122x ; 1,042x), hop stunt viroid (13,783x ; 29,514x) and grapevine yellow speckle viroid 1 (690x ; 1158x) were also identified. Plant CH37-19S was also co-infected by grapevine rupestris vein feathering virus (164x). The GVL contigs integrated respectively 320,000 and 152,000 reads (corresponding to 0.32% and 0.11% of filtered/trimmed reads, respectively). The GVL genomic sequences from each sample (7,616 nt) have been deposited in GenBank (Accession nos. OK042110 and OK042111, respectively). The two contigs are nearly identical (99.9% nt identity) and share respectively 97.5% and 95.9% with GVL-KA from the USA (MH643739) and GVL-RS from China (MH248020), the closest isolates present in GenBank. To confirm the presence of GVL, the original grapevines were resampled in the field and total RNAs were extracted as described above from cambial scrappings and leaves. Total RNAs were used for RT-PCR tests using primers targeting a 279-bp fragment corresponding to the 3' end of the coat protein gene and part of the nucleic acid binding protein gene (Debat et al. 2019). The Sanger-derived sequences from the amplicons shared 100% nt identities with the corresponding sequences of the HTS assembled genomes, confirming the presence of GVL in both tissues of both grapevine samples. To our knowledge, this represents the first report of the occurrence of GVL in vineyards in France. Given the complex mixed infection present in the two analyzed grapevines, no conclusions can be drawn on the pathogenicity of GVL. Further efforts are needed to better understand GVL distribution and its potential pathogenicity to grapevine. References Alabi, O J., et al. 2020. Arch. of Virol. 165:1905-1909. Ben Amar, A., et al. 2020. Plant disease 104:3274. Debat, H., et al. 2019. Eur J Plant Pathol. 155:319. Diaz-Lara, A., et al. 2019. Arch. of Virol. 164:2573. Acknowledgments The authors are grateful to the "Plan National Dépérissement du Vignoble" (Mycovir project) for the financial support.

Shape-dependent leaf manipulation in the leaf rolling weevil Phymatapoderus pavens (Coleoptera: Attelabidae)

Abstract Plant leaves are utilized by various shelter-building insects. Some insects are constrained in their host plant use by the difficulty in modifying leaves into shelters, while others may cope through different construction methods. Here, we show that the leaf-rolling weevil Phymatapoderus pavens (Coleoptera, Attelabidae) constructs two types of leaf rolⅼ on Boehmeria silvestrii (Urticaceae) according to the shape of the leaf. Female weevils make leaf rolls using the whole leaf when the leaf is small and weakly lobed, whereas they use only one of the lateral lobes when the leaf is large and deeply lobed. Both types were found within a population and each functioned as a site for the growth of offspring. Weevils bite the leaf veins differently when constructing each of these leaf rolls, suggesting that the leaf roll type is determined before the leaf is rolled. Compared with another leaf-rolling weevil species, Apoderus praecellens (Coleoptera, Attelabidae), which makes leaf rolls on similarly lobed leaves of Isodon umbrosus var. hakusanensis (Lamiaceae), P. pavens utilizes a wider range of leaf shapes because these weevils are able to construct leaf rolls on the lateral lobes of the leaves. Differences in leaf-rolling behaviour, leaf venation pattern, and the relative size of weevils and leaves are factors affecting the results.

Climate variability and aridity modulate the role of leaf shelters for arthropods: A global experiment.

Current climate change is disrupting biotic interactions and eroding biodiversity worldwide. However, species sensitive to aridity, high temperatures, and climate variability might find shelter in microclimatic refuges, such as leaf rolls built by arthropods. To explore how the importance of leaf shelters for terrestrial arthropods changes with latitude, elevation, and climate, we conducted a distributed experiment comparing arthropods in leaf rolls versus control leaves across 52sites along an 11,790km latitudinal gradient. We then probed the impact of short- versus long-term climatic impacts on roll use, by comparing the relative impact of conditions during the experiment versus average, baseline conditions at the site. Leaf shelters supported larger organisms and higher arthropod biomass and species diversity than non-rolled control leaves. However, the magnitude of the leaf rolls' effect differed between long- and short-term climate conditions, metrics (species richness, biomass, and body size), and trophic groups (predators vs. herbivores). The effect of leaf rolls on predator richness was influenced only by baseline climate, increasing in magnitude in regions experiencing increased long-term aridity, regardless of latitude, elevation, and weather during the experiment. This suggests that shelter use by predators may be innate, and thus, driven by natural selection. In contrast, the effect of leaf rolls on predator biomass and predator body size decreased with increasing temperature, and increased with increasing precipitation, respectively, during the experiment. The magnitude of shelter usage by herbivores increased with the abundance of predators and decreased with increasing temperature during the experiment. Taken together, these results highlight that leaf roll use may have both proximal and ultimate causes. Projected increases in climate variability and aridity are, therefore, likely to increase the importance of biotic refugia in mitigating the effects of climate change on species persistence.

A survey of ‘Candidatus Phytoplasma pyri’ isolates in the Czech Republic based on imp gene genotyping

‘Candidatus Phytoplasma spp.’ are pathogenic bacteria that infect many plant species. ‘Candidatus Phytoplasma pyri’, one of the members of the 16SrX group causes pear decline disease that adversely affects pear crops. To describe the prevalence of ‘Ca. P. pyri’ genotypes in the Czech Republic, 143 pear samples were collected from 41 locations including commercial orchards as well as trees along roads. Phytoplasma was detected by PCR in 115 samples, and it was possible to determine imp gene genotype in 84 samples. The most frequent genotypes were A1, B1, and C, which were identified in 71% of phytoplasma positive samples. ‘Ca. P. pyri’ was present either alone or as a mix of two populations in 88% of genotyped samples, and in another 6% of samples it was found in a mixed infection with ‘Ca. Phytoplasma mali’. A sole infection with ‘Ca. Phytoplasma mali’ was observed in 6% of samples. As for symptoms, 19% of symptomatic samples were found to be phytoplasma negative, and 74% of asymptomatic samples proved to be phytoplasma positive; leaf roll was more often observed in phytoplasma positive samples, while leaf narrowing rather indicated the absence of phytoplasma. The mildest symptoms were observed in samples infected with ‘Ca. P. pyri’ of the A1 imp genotype.

Detection of Ampelovirus and Nepovirus by Lab-on-a-Chip: A Promising Alternative to ELISA Test for Large Scale Health Screening of Grapevine.

The Ampelovirus Grapevine leafroll-associated virus 3 (GLRaV-3) and the Nepovirus Grapevine fanleaf virus (GFLV) are pathogens reported in many grapevine-growing areas all over the world, main causal agents of grapevine leafroll disease and grapevine fanleaf disease, respectively. Prevention of virus spread thanks to rapid diagnosis of infected plants is a key factor for control of both diseases. Although serological (e.g., enzyme-linked immunosorbent assay-ELISA test) and molecular methods are available to reveal the presence of the viruses, they turn out to be quite expensive, time-consuming and laborious, especially for large-scale health screening. Here we report the optimization of a lab-on-a-chip (LOC) for GLRaV-3 and GFLV detection, based on an electrochemical transduction and a microfluidic multichamber design for measurements in quadruplicate and simultaneous detection of both targets. The LOC detect GLRaV-3 and GFLV at dilution factors more than 15 times higher than ELISA, providing a higher sensitivity in the detection of both viruses. Furthermore, the platform offers several advantages as easy-to-use, rapid-test, portability and low costs, favoring its potential application for large-scale monitoring programs. Compared to other grapevine virus biosensors, our sensing platform is the first one to provide a dose-dependent calibration curve combined with a microfluidic module for sample analysis and a portable electronics providing an operator-independent read-out scheme.

Complete genome sequence of peony leafroll-associated virus, a novel ampelovirus in subgroup I.

The complete genome sequence of peony leafroll-associated virus (PLRaV) was determined by deep sequencing of ribosomal-RNA-depleted total RNA extracted from a peony plant exhibiting leafroll symptoms. Further PCR and RACE analysis showed that the PLRaV genome consists of 15,406 nucleotides and contains 10 putative open reading frames, with an organization typical of members of the genus Ampelovirus, family Closteroviridae. Amino acid sequence comparisons showed that the viral heat shock protein 70 homolog (HSP70h) shared the highest sequence identity (41.7%) with the corresponding region of grapevine leafroll-associated virus 1, and the coat protein (CP) and RNA-dependent RNA polymerase (RdRp) shared the highest sequence identity (32.1% and 52.3%, respectively) with grapevine leafroll-associated virus 13. Phylogenetic analysis of the HSP70h, CP, and RdRp aa sequences showed that PLRaV clustered with members of subgroup I of the genus Ampelovirus.

Manipulation of Vector Host Preference by Pathogens: Implications for Virus Spread and Disease Management.

Some plant pathogens manipulate the behavior and performance of their vectors, potentially enhancing pathogen spread. The implications are evolutionary and epidemiological but also economic for pathogens that cause disease in crops. Here we explore with models the effects of vector manipulation on crop yield loss to disease and on the economic returns for vector suppression. We use two frameworks, one that simulates the proportional occurrence of the pathogen in the vector population with the option to eliminate vectors by a single insecticidal treatment, and one that includes vector population dynamics and the potential for multiple insecticidal sprays in a season to suppress vectors. We parameterize the models with published data on vector manipulation, crop yields as affected by the age of the plant at infection, commodity prices and costs of vector control for three pathosystems. Using the first framework, maximum returns for treating vectors are greater with vector manipulation than without it by approximately US$10 per acre (US$24.7/ha) in peas infected by Pea enation mosaic virus and Bean leaf roll virus, and approximately US$50 per acre (US$124/ha) for potatoes infected by Potato leaf roll virus. Using the second framework, maximum returns for controlling the psyllid vectors of Candidatus Liberibacter solanacearum are 50% greater (approximately US$400/acre, US$988/ha) but additional returns for multiple weekly sprays diminish more with vector manipulation than without it. These results suggest that the economics of vector manipulation can be substantial and provide a framework that can inform management decisions.

甘蔗类泛素蛋白UBL5应答SCMV侵染及其与SCMV-6K2的互作

<p id="C3">Ubiquitylation plays key roles in the regulation of protein function, growth and development, and response to stress. Ubiquitin-like proteins (UBLs) are the main components of the ubiquitin-proteasome system (UPS). In our previous study, the UBL5 homologue was isolated from sugarcane (<italic>Saccharum </italic>spp. hybrid) by yeast two-hybrid (Y2H) with the 6K2 of <italic>Sugarcane mosaic virus</italic> (SCMV) as bait, and then designated as ScUBL5 with 73 aa in length. In the present study, the interaction of ScUBL5 with the SCMV-6K2 was further confirmed by bimolecular fluorescence complementation assays (BiFC). Bioinformatics analysis showed that ScUBL5 is a stable hydrophilic non-secretory protein without signal peptide or transmembrane domain. Phylogenetic tree analysis showed that <italic>ScUBL5</italic> is species specific. Subcellular localization analysis showed that ScUBL5 is localized in cytoplasm and nucleus. <italic>ScUBL5</italic> gene shows obvious tissue specificity in sugarcane by real-time quantitative PCR analysis. The expression levels of ScUBL5 gene in the established morphogenesis tissues such as the 1st leaf, the 7th leaf, the 8th internode and the root were significantly higher than those in the immature tissues such as leaf roll and the 3rd internode. The expression of<italic> ScUBL5</italic> gene is significantly affected by SCMV infection. <italic>ScUBL5</italic> was significantly upregulated in the early stage of SCMV infection, then downregulated but significantly higher than the control at the late stage of SCMV infection.

First Report of Grapevine Kizil Sapak Virus in Grapevine in China.

Grapevine Kizil Sapak virus (GKSV) is a novel member of the family Betaflexiviridae classified into the proposed genus Fivivirus within the subfamily Trivirinae. It was first discovered in USA from a grapevine originating from Turkmenistan (Al Rwahnih et al. 2019) and later in France from a grapevine accession from Iran (Marais et al. 2020). In October 2019, an asymptomatic grapevine cv. 'Crimson Seedless' (native to USA) was collected from Xinjiang province in China and analyzed by high-throughput sequencing (HTS). Ribosome-depleted RNA preparations were used for library synthesis followed by HTS on an Illumina HiSeq X-ten platform. A total of 29,141,024 cleaned reads were obtained, and 7,878 contigs were generated using CLC Genomics Workbench 9.5 (QIAGEN). One long contig (7,328 bp) showed 88.2% nucleotide (nt) identity with the sequence of GKSV-127 (MN172165) via Blastx, with an average coverage of 284-X. Bioinformatic analysis of the remaining contigs showed the presence of Grapevine leafroll-associated virus 4, Grapevine rupestris vein feathering virus, Grapevine fabavirus, grapevine yellow speckle viroid-1 (GYSVd-1), GYSVd-2 and Hop stunt viroid in the sample. The presence of GKSV was checked by RT-PCR using the primer GKSV-F/R (Al Rwahnih et al. 2019); the 1,240 bp PCR product was cloned using a pTOPO-T vector (Aidlab, China) and sequenced. In pairwise comparison, the obtained nt sequences shared 92.6 to 95.2% identity to the corresponding HTS sequence, confirming the presence of GKSV in the sample. The complete GKSV genome sequence was obtained as two pieces of overlapping DNA sequence using primers GKSV-20A/20B (5'-TAGTCTGGATTTCCCTACCT/5'-CTCCCTAAACTGATTTGATG) and GKSV-25A/25B (5'-GCCACTGGTGAATGAAAAGA/5'-CTAAATGAATGGGCAGGTAT) designed based on the HTS-generated sequence. The 5' and 3' termini were determined by rapid amplification of cDNA ends using SMARTer RACE 5'/3' Kit (Takara, Dalian, China). The complete genome of GKSV isolate CS (MW582898) comprised 7,604 nt (without the polyA tail) and shared 77.8 to 89.2% identities with the other nine reported GKSV isolates, among which it shared the highest nt identity (89.2%) with GKSV-127. In phylogenetic analysis based on complete or nearly complete genome sequences of representative members of Betaflexiviridae, GKSV-CS clustered with the nine known GKSV isolates, forming a subclade with GKSV-127 (Supplementary Fig. 1). To determine the incidence and distribution of GKSV in China, 476 grapevine samples of 75 cultivars were collected from 20 provinces and tested by RT-PCR using primers GKSV-F/R (Al Rwahnih et al. 2019) and Vini-F1/R1 (Marais et al. 2020). The results showed that 0.42% (2 of 476) of the samples tested positive with both primers, including samples GKSV-CS and a 'Black Monukka' grape (native to India) also sampled from Xinjiang. Both PCR products of 'Black Monukka' were cloned and sequenced (MZ311588 to MZ311602) and they showed 85.1 to 88.9% nt identities to the GKSV-CS sequence. This is the first report of GKSV infecting grapevine in China. Although the pathogenicity of GKSV is yet to be determined, it has been found in several countries such as USA (Al Rwahnih et al. 2019), France (Marais et al. 2020) and China (this study). Both positive samples in this study were collected from Nanjiang region in Xinjiang province, indicating the sporadic occurrence of GKSV in this area.

Occurrence of Grapevine Leafroll-Associated Virus-3 (GLRaV-3), Complete Nucleotide Sequence and Cultivar Susceptibility to a GLRaV-3 Isolate from Shaanxi Province of China

Grapevine (Vitis spp.) is globally one of the most economically important fruit crops. China is the largest grapevine-growing country of the world and Shaanxi province is one of the major grapevine-growing provinces in the country. A survey of GLRaV-3 found it widespread, with 57–100% infection frequencies, in both wine and table grapevine cultivars of three grapevine-growing regions of Shaanxi province. The virus infection frequencies varied with cultivars and regions. In order to obtain the full genomic length of a new GLRaV-3 isolate, GLRaV-3-Sau (accession number MK988555), was sequenced. This isolate has a genome of 18026 nucleotides, and 14 open reading frames (ORFs). The full-genome of the isolate GLRaV-3-Sau shared 85.88% nucleotide identity to GLRaV-3-LN, another isolate found in China. Coat protein (CP) genes of GLRaV-3 isolates were identical (99%) to the Vitis vinifera isolate (accession number HQ185608.1) from the USA. Immunohistochemistry for virus localization found that distribution patterns were similar in red-berried cultivar ‘Cabernet Sauvignon’ and white-berried cultivar ‘Chardonnay’, and GLRaV-3 is restricted in phloem tissue of vascular bundles. Virus transmission by micrografting found virus transmission efficiency was higher in ‘Chardonnay’ and ‘Thompson Seedless’ than in ‘Hunan-1’, indicating that ‘Hunan-1’ was less sensitive to GLRaV-3. As far as we know, these are the most comprehensive comparisons on the genome and CP genes of GLRaV-3 worldwide and the first to have found that the grapevine ‘Hunan-1’ is less susceptible to GLRaV-3.

Grapevine leafroll-associated viruses (leafroll disease)

Grapevine leafroll-associated viruses (leafroll disease)

Cherry leaf roll virus (walnut ringspot)

Cherry leaf roll virus (walnut ringspot)

Engineering CRISPR immune systems conferring GLRaV-3 resistance in grapevine.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the causal agents of grapevine leafroll disease (GLD), which severely impacts grapevine production in most viticultural regions of the world. The development of virus-resistant plants is a desirable strategy for the efficient control of viral diseases. However, natural resistant resources have not been reported in the genus Vitis, and anti-GLRaV-3 research has been quite limited in grapevine. In this study, by expressing FnCas9 and LshCas13a, we established a highly effective transgenic construct screening system via an optimized Agrobacterium-mediated transient delivery system in grapevine plantlets. Our study indicated that CRISPR/FnCas9 and LshCas13a caused GLRaV-3 inhibition. Moreover, three vectors—pCR01-CP, pCR11-Hsp70h and pCR11-CP—exhibited the most robust inhibition efficiency compared to those targeting other sites and could be further engineered to generate GLRaV-3-resistant grapevine. In addition, the viral interference efficiency of FnCas9 was dependent on its RNA binding activity. The efficiency of virus inhibition was positively correlated with the level of Cas gene expression. Importantly, we demonstrated that LshCas13a had better interference efficiency against viruses than FnCas9. In summary, this study confirmed that these two RNA-targeting CRISPR mechanisms can confer immunity against viruses in grapevine, providing new avenues to control GLRaV-3 or other RNA viruses in fruit crops.

Effect of Different Treatments on Producing in vitro Healthy Vitis vinifera L. cv. Halwani Free From Grapevine Leafroll-Associated Virus-3

El-Samin, M., K. El-Maari and F. El-Biski. 2022. Effect of Different Treatments on Producing in vitro Healthy Vitis vinifera L. cv. Halwani Free From Grapevine Leafroll-Associated Virus-3. Arab Journal of Plant Protection, 40(4): 334- 339. https://doi.org/10.22268/AJPP-40.4.334339 This study was carried out during the period 2020-2021, to produce virus-free propagation material from Halwani cultivar of grape infected with Grapevine leafroll-associated virus 3 (GLRaV-3), by using the meristem tip culture technique together with thermal and chemical treatments. The meristem tip of 500 micrometers was excised from infected plants and placed on 1/2 MS medium which contains 0.5 mg/l BAP and 30 g/l sucrose. Virus presence or absence was confirmed by the ELISA test (DAS-ELISA). Results showed that the highest Percentage of virus free plantlets (87.67%) was obtained from the combined treatment, whereas the thermal treatment resulted in the lowest percentage of virus-free plants (56.25%). The results obtained also showed the significant superiority of using meristem-tip culture compared with all other treatments in terms of percent growing plants, which reached 98.13%. This study indicated that there were negative effects of all treatments on plant elongation, especially the thermal treatment, which gave the least plant growth elongation (0.33 cm). Keywords: Grapevine leafroll-associated virus, grape, meristem, thermal treatment, chemical treatment, DAS-ELISA

Grapes recovery from viruses during reproduction by biotechnological method

Grape regenerant plants obtained from apical meristems were assessed for the presence of the viruses using PCR and enzyme-linked immunosorbent assay (ELISA). The vast majority of the samples studied for the presence of the most common viruses (Grapevine Leafroll-Associated Virus – 1 (GLRaV-1); Grapevine yellow mosaic virus; Grapevine vein banding; virus; Grapevine Leafroll Virus; Grapevine stemm pitting) by ELISA showed negative results. Testing of regenerant plants of the Augustine variety clones showed that only one plant out of 80 plants showed a positive reaction to the presence of this virus. Analysis of 80 plants of the Moldova variety clones revealed a positive reaction to the yellow mosaic virus (Grapevine yellow mosaic virus) in 1 plant as well as in the original donor plant (Moldova variety). The rest of the regenerated plants (79 pcs.) were absolutely healthy. PCR analysis showed that the spectrum of fragments of the original genotypes of Moldova varieties (lanes 1–4) and Augustin (lanes 5–8) contained DNA fragments of 450 bps, corresponding to Candidatus Phytoplasma vitis Flavescence doree. In Bart variety (lanes 9–11), no pathogen was detected. Analysis of the spectrum of fragments of grape regenerant plants obtained from apical meristems (lanes 1–4) and Augustine (lanes 5–8) showed the absence of the pathogen Candidatus Phytoplasma vitis Flavescence doree.