Search

Citrus tristeza virus (CTV) is a causal agent of diseases that have challenged the global citrus production for more than a century. The disease named ‘tristeza’, which means ‘sadness’ in Portuguese and Spanish, has spread by aphids and vegetative propagation, resulting in costly pandemics that reshaped the world citrus production by forcing the adaption of disease‐tolerant rootstocks. Furthermore, management of the second major CTV‐induced disease, stem pitting, became a unique example of the use of cross‐protection on a large nationwide scale, allowing many citrus growing regions to control the disease that could not be managed through horticultural practices. The information gathered in this review commemorates a hundred years of research on the virus and the respective diseases, which began with classical horticultural approaches and advanced toward the cutting‐edge molecular biology studies. In this regard, it is proper to mention that CTV research greatly benefited from close international collaboration between research institutions and scientists from the affected and nonaffected citrus areas. Moreover, despite the considerable initial losses caused by tristeza, the world's fresh fruit and juice concentrate citrus industries reemerged as highly productive following the research advancements, a situation that changed throughout Florida and Brazil in the past two decades or so with the outbreak of the devastating citrus greening (Huanglongbing) bacterial disease. This review encompasses past and recent advances in the CTV research positioning the citrus‐CTV pathosystem as a pivotal model system for investigating virus interactions with perennial woody hosts. The review will also serve as an updated version of the respective section on CTV in the Description of Plant Viruses that the Association of Applied Biologists manages. We dedicated this review to the 85th Birthday celebration of Prof. Moshe Bar‐Joseph, a world‐renowned plant pathologist whose half‐a‐century‐long career devoted to citrus diseases yielded many important, pioneering discoveries on CTV and other closteroviruses. Many of those are highlighted in this review. Prof. Bar‐Joseph retired from the Volcani Center, Agricultural Research Organization in Israel several years ago, yet he continues to be actively engaged in research and provides his expertise to citrus pathologists around the world in the present days. [ABSTRACT FROM AUTHOR]

Studies of the virome of olive trees with symptoms of leaf mottling by high-throughput sequencing (HTS) revealed the presence of a new virus. Full coding genome sequences of two isolates were determined and consisted of a single RNA segment of 16,516 nt and 16,489, respectively. The genomic organization contained 10 open reading frames (ORFs) from 5′ to 3′: ORF1a, ORF1b (RdRp), ORF2 (p22), ORF3 (p7), ORF4 (HSP70h), ORF5 (HSP90h), ORF6 (CP), ORF7 (p19), ORF8 (p12), ORF9 (p23) and ORF10 (p9). Phylogenetic analyses clustered this virus in the genus Olivavirus, family Closteroviridae, with the closest species being Olivavirus flaviolae, commonly named olive leaf yellowing-associated virus (OLYaV). However, amino acid sequences of all taxonomically relevant proteins showed, in all cases, a divergence higher than 25% between OLYaV and the new virus, indicating that it represents a new species in the genus Olivavirus for which the common name of olive leaf mottling virus (OLMV) is proposed. This study represents an advance in the genus Olivavirus and provides new insights into the olive virome. [ABSTRACT FROM AUTHOR]

Studies of the virome of olive trees with symptoms of leaf mottling by high-throughput sequencing (HTS) revealed the presence of a new virus. Full coding genome sequences of two isolates were determined and consisted of a single RNA segment of 16,516 nt and 16,489, respectively. The genomic organization contained 10 open reading frames (ORFs) from 5′ to 3′: ORF1a, ORF1b (RdRp), ORF2 (p22), ORF3 (p7), ORF4 (HSP70h), ORF5 (HSP90h), ORF6 (CP), ORF7 (p19), ORF8 (p12), ORF9 (p23) and ORF10 (p9). Phylogenetic analyses clustered this virus in the genus Olivavirus, family Closteroviridae, with the closest species being Olivavirus flaviolae, commonly named olive leaf yellowing-associated virus (OLYaV). However, amino acid sequences of all taxonomically relevant proteins showed, in all cases, a divergence higher than 25% between OLYaV and the new virus, indicating that it represents a new species in the genus Olivavirus for which the common name of olive leaf mottling virus (OLMV) is proposed. This study represents an advance in the genus Olivavirus and provides new insights into the olive virome.

High cannabidiol‐containing plants of Cannabis sativa (high‐CBD) growing in farms in Israel displayed foliar symptoms of interveinal chlorosis and yellowing, brittleness and occasionally necrosis. These symptoms, which were more apparent in older leaves, resembled those caused by the crinivirus lettuce chlorosis virus (LCV). However, this virus was not detected by reverse transcription (RT)‐PCR using specific primer sets. High‐throughput sequencing of viral RNA extracted from symptomatic leaves revealed the presence of cucurbit chlorotic yellows virus (CCYV), a crinivirus in the Closteroviridae family. The complete viral genome sequence was obtained using RT‐PCR followed by Sanger sequencing. The two CCYV RNA genomic segments shared 99.5%–99.85% nucleotide sequence identity with CCYV isolates from the GenBank. The virus was transmitted from symptomatic cannabis leaves to healthy plants of cannabis and Cucumis sativus 'King Star' (cucumber) by the whitefly Bemisia tabaci Middle Eastern Asia Minor 1 (MEAM1) species, causing disease symptoms identical to those of the donor plants. Cannabis‐CCYV was also transmitted between infected cucumber plants and cannabis seedlings of unknown genotype. Severe disease symptoms of yellowing and leaf‐edge necrosis were observed on high‐CBD and high Δ9‐tetrahydrocannabinol‐containing (high‐THC) flowering cannabis plants and were associated with mixed infections of LCV and CCYV. To the best of our knowledge, this is the first report of CCYV infecting C. sativa plants. [ABSTRACT FROM AUTHOR]

Grapevine leafroll disease (GLRD) is the most globally prevalent and destructive disease complex responsible for significant reductions in grape yield and quality as well as wine production. GLRD is associated with several positive-strand RNA viruses of the family Closteroviridae, designated as grapevine leafroll-associated viruses (GLRaVs). However, the specific etiological role of any of these GLRaVs in GLRD has not been demonstrated. Even though GLRaV-3 is considered the chief GLRD agent, little is known about the molecular, cellular, and pathological properties of this virus. Such a knowledge gap is due to multiple factors, including the unavailability of biologically active virus cDNA clones and the lack of reliable experimental systems for launching grapevine infection using such clones. In this work, we tested four methods for inoculating tissue-cultured grapevine plantlets with cDNA clones of GLRaV-3: (i) vacuum agro-infiltration; (ii) agro-pricking; (iii) agro-drenching; and (iv) agro-injection. We showed that vacuum agro-infiltration was the most effective of these methods. Furthermore, we examined the impacts of different experimental conditions on the survival and infectivity rate of grapevines after infiltration. To verify the infectivity rate for different treatments, we used RT-PCR, RT-qPCR, and Western blotting. We found that humidity plays a critical role in the survival of plantlets after agro-infiltration and that the use of RNA silencing suppressor and dormancy treatment both had strong effects on the infection rates. To our knowledge, the experimental protocol reported herein is the most effective system for launching the infection of grapevine using cDNA clones of grapevine viruses featuring up to a 70% infection rate. This system has strong potential to facilitate grapevine virology research including the fulfillment of Koch's postulates for GLRD and other major virus diseases as well as identifying the molecular, cellular, and pathological properties of GLRaVs and, potentially, other important grapevine viruses. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus (GLRaV) infections are accompanied by symptoms influenced by host genotype, rootstock, environment, and which individual or combination of GLRaVs is present. Using a dedicated experimental vineyard, we studied the responses to GLRaVs in ripening berries from Cabernet Franc grapevines grafted to different rootstocks and with zero, one, or pairs of leafroll infection(s). RNA sequencing data were mapped to a high-quality Cabernet Franc genome reference assembled to carry out this study and integrated with hormone and metabolite abundance data. This study characterized conserved and condition-dependent responses to GLRaV infection(s). Common responses to GLRaVs were reproduced in two consecutive years and occurred in plants grafted to different rootstocks in more than one infection condition. Though different infections were inconsistently distinguishable from one another, the effects of infections in plants grafted to different rootstocks were distinct at each developmental stage. Conserved responses included the modulation of genes related to pathogen detection, abscisic acid (ABA) signalling, phenylpropanoid biosynthesis, and cytoskeleton remodelling. ABA, ABA glucose ester, ABA and hormone signalling-related gene expression, and the expression of genes in several transcription factor families differentiated the effects of GLRaVs in berries from Cabernet Franc grapevines grafted to different rootstocks. These results support that ABA participates in the shared responses to GLRaV infection and differentiates the responses observed in grapevines grafted to different rootstocks.

Mealybugs cause mechanical damage and diseases to plants. Through their feeding activities, they reduce the yield, quality and productivity of crops. This review discusses mealybug vectors of plant viruses, the economic losses they cause, mealybug species and their hosts. Among the numerous mealybug species, Planococcus species are the most effective vector of plant viruses, transmitting many Ampeloviruses. Diverse methods for the control and regulation of mealybugs are also discussed. Physical, cultural and biological control methods are labor-intensive but environmentally friendly compared to chemical methods. However, chlorpyrifos are one the active ingredients of insecticides effective against several mealybug species. Using plant products such as neem oil as a biocontrol method has been effective, similar to other insecticides. Notwithstanding, the biological method of controlling mealybugs is effectively slow but safe and highly recommended. The Anagyrus species have the highest success rate amongst other natural parasites of mealybugs. Also, farm sanitation and pruning as cultural methods help reduce mealybug populations.

Grapevine leafroll disease (GLD) is a globally important disease that affects the metabolic composition and biomass of grapes, leading to a reduction in grape yield and quality of wine produced. Grapevine leafroll-associated virus 3 (GLRaV-3) is the main causal agent for GLD. This study aimed to identify protein-protein interactions between GLRaV-3 and its host. A yeast two-hybrid (Y2H) library was constructed from litis vinifera mRNA and screened against GLRaV-3 open reading frames encoding structural proteins and those potentially involved in systemic spread and silencing of host defense mechanisms. Five interacting protein pairs were identified, three of which were demonstrated in planta. The minor coat protein of GLRaV-3 was shown to interact with 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 02, a protein involved in primary carbohydrate metabolism and the biosynthesis of aromatic amino acids. Interactions were also identified between GLRaV-3 p20A and an 18.1 -kDa class I small heat shock protein, as well as MAP3K epsilon protein kinase 1. Both proteins are involved in the response of plants to various stressors, including pathogen infections. Two additional proteins, chlorophyll a-b binding protein CP26 and a SMAX1-LIKE 6 protein, were identified as interacting with p20A in yeast but these interactions could not be demonstrated in planta. The findings of this study advance our understanding of the functions of GLRaV-3-encoded proteins and how the interaction between these proteins and those of V. vinifera could lead to GLD. [ABSTRACT FROM AUTHOR]

Citrus tristeza virus (CTV) can be transmitted by several aphid species in a semi-persistent mode, with Toxoptera citricida being the most efficient vector. In Brazil, mild CTV isolates are used for pre-immunization of citrus trees against severe isolates. We aimed to determine the capacity and efficiency of T. citricida in separating the viral complex into haplotypes from three well-characterized CTV isolates (PIAC, CS1, and Forte Rolândia). Single-aphid transmission assays were conducted to determine CTV transmission efficiency. The results showed that T. citricida transmitted only haplotypes from the PIAC and CS1 isolates, with efficiencies of 8 and 4%, respectively. Both isolates caused mild CTV symptoms in Brazil. However, isolate Forte Rolândia, which causes severe symptoms in citrus trees, was not transmitted by T. citricida. The detection of CTV haplotypes from PIAC and CS1 isolates in sweet orange (Citrus sinensis) plants after a single aphid transmission occurred at different time points. The first haplotype observed was from PIAC 150 days after citrus was challenged by a single aphid, followed by CS1 at day 210 after transmission. In addition, differences in the single-strand conformation polymorphism patterns between the CTV isolates and CTV haplotypes were determined, suggesting that an aphid can acquire and transmit only one CTV haplotype to citrus plants during phloem feeding. The study of the mechanism of transmission by the vector can increase our knowledge of the interactions among hosts, vectors, and pathogens, which are often neglected.

Mealybugs cause mechanical damage and diseases to plants. Through their feeding activities, they reduce the yield, quality and productivity of crops. This review discusses mealybug vectors of plant viruses, the economic losses they cause, mealybug species and their hosts. Among the numerous mealybug species, Planococcus species are the most effective vector of plant viruses, transmitting many Ampeloviruses. Diverse methods for the control and regulation of mealybugs are also discussed. Physical, cultural and biological control methods are labor-intensive but environmentally friendly compared to chemical methods. However, chlorpyrifos are one the active ingredients of insecticides effective against several mealybug species. Using plant products such as neem oil as a biocontrol method has been effective, similar to other insecticides. Notwithstanding, the biological method of controlling mealybugs is effectively slow but safe and highly recommended. The Anagyrus species have the highest success rate amongst other natural parasites of mealybugs. Also, farm sanitation and pruning as cultural methods help reduce mealybug populations. [ABSTRACT FROM AUTHOR]

Olive trees (Olea europea L.) are constantly threatened by many viruses, such as the olive leaf yellowing-associated virus (OLYaV), that belong to the Olivavirus genus, family Closteroviridae. In this work, the OLYaV incidence in different regions of Italy and Spain, which represent the two most important European areas for olive production, was evaluated through the development of a real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for reliable and sensitive OLYaV detection. The specificity and accuracy of the developed real-time RT-LAMP assay were determined; the assay showed that potential cross-reactivity with other viruses belonging to the Closteroviridae family was excluded. The LAMP assay detected OLYaV with a higher sensitivity than conventional end-point RT-PCR, detecting a total of 1.34 × 10−2 genome copies. A total of 80 and 120 plants of different olive cultivars from Spain (Comunitat Valenciana, Andalusia) and Italy (Sicily, Calabria, Apulia, Lazio, and Umbria) regions were tested, respectively. The percentage of infected plants was 46.25% and 30% for Spain and Italy, respectively, while the most susceptible cultivars were "Serrana Espadán" and "Villalonga" from Comunitat Valenciana and Andalusia regions (Spain) and "Ogliarola barese" from Apulia region (Italy). In addition, the survey demonstrated that the real-time RT-LAMP showed good sensitivity for OLYaV-positive sample detection, especially on asymptomatic olive trees. For this reason, the developed assay could be very suitable for phytopathological laboratories as a reliable and efficient method for a rapid and sensitive routine test on olive samples. [ABSTRACT FROM AUTHOR]

Grapevine leafroll disease is one of the most important virus diseases of grapevines and occurs in every major grape-growing region of the world. The vector-transmission mechanisms of the causative agent, Grapevine leafroll-associated virus 3 (GLRaV-3), remain poorly understood. We show that the vine mealybug, Planococcus ficus, feeds through a membrane feeding system on GLRaV-3 viral purifications from both V. vinifera and N. benthamiana and transmits the virus to test plants from plants from both species. Building on this strategy, we used an immunofluorescence approach to localize virions to two retention sites in P. ficus mouthparts. Assays testing molecules capable of blocking virus transmission demonstrated that GLRaV-3-transmission by P. ficus could be disrupted. Our results indicate that our membrane feeding system and transmission-blocking assays are a valid approach and can be used to screen other candidate blocking molecules.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses of grapevine but, despite this, there remain several gaps in our understanding of its biology. Because of its narrow host range - limited to Vitis species - and because the virus is restricted to the phloem, most GLRaV-3 research has concentrated on epidemiology and the development of detection assays. The recent discovery that GLRaV-3 can infect Nicotiana benthamiana, a plant model organism, makes new opportunities available for research in this field. We used RNA-seq to compare both V. vinifera and P1/HC-Pro N. benthamiana host responses to GLRaV-3 infection. Our analysis revealed that the majority of DEGs observed between the two hosts were unique although responses between the two hosts also showed several shared gene expression results. When comparing gene expression patterns that were shared between the two hosts, we observed the downregulation of genes associated with stress chaperones, and the induction of gene families involved in primary plant physiological processes. This is the first analysis of gene expression profiles beyond Vitis to mealybug-transmitted GLRaV-3 and demonstrates that N. benthamiana could serve as a useful tool for future studies of GLRaV-3-host interactions.

Grapevine viruses are found throughout the viticultural world and have detrimental effects on vine productivity and grape and wine quality. This report provides a comprehensive and up-to-date review on grapevine viruses in Australia with a focus on "Shiraz Disease" (SD) and its two major associated viruses, grapevine virus A (GVA) and grapevine leafroll-associated virus 3 (GLRaV-3). Sensitive grapevine cultivars like Shiraz infected with GVA alone or with a co-infection of a leafroll virus, primarily GLRaV-3, show symptoms of SD leading to significant yield and quality reductions in Australia and in South Africa. Symptom descriptors for SD will be outlined and a phylogenetic tree will be presented indicating the SD-associated isolates of GVA in both countries belong to the same clade. Virus transmission, which occurs through infected propagation material, grafting, and naturally vectored by mealybugs and scale insects, will be discussed. Laboratory and field-based indexing will also be discussed along with management strategies including rogueing and replanting certified stock that decrease the incidence and spread of SD. Finally, we present several cases of SD incidence in South Australian vineyards and their effects on vine productivity. We conclude by offering strategies for virus detection and management that can be adopted by viticulturists. Novel technologies such as high throughput sequencing and remote sensing for virus detection will be outlined.

The complete genome sequence of a previously uncharacterized sugarcane‐infecting virus of the genus Ampelovirus was obtained by high‐throughput sequencing (HTS) and rapid amplification of cDNA ends. This genome consists of 13,114 nucleotides (nt) and harbours seven open reading frames. Phylogenetic and pairwise identity analyses with the complete or near‐complete genome sequences of 16 isolates revealed that they all belong to a new Ampelovirus species, family Closteroviridae. These 16 isolates were distributed in three phylogenetic groups and potential recombinant events were identified for only one of these isolates. An immunocapture‐reverse transcription‐PCR (IC‐RT‐PCR) assay was developed with antibodies previously produced against a clostero‐like virus that had not yet been recognized by the International Committee on Taxonomy of Viruses but for which the name sugarcane mild mosaic virus (SCMMV) had been proposed. The new ampelovirus was detected by IC‐RT‐PCR, and Sanger‐sequenced amplicons confirmed the identity of the virus based on HTS data. SCMMV was detected in 65 of 350 samples of quarantined sugarcane germplasm using RT‐PCR targeting the hsp70 gene. These samples originated from Africa, the Caribbean, North America, South America and South‐east Asia, thus revealing that SCMMV is present in many sugarcane‐growing regions of the world. The complete or near‐complete sequences reported here represent the first genomic sequences for SCMMV. Furthermore, the newly developed RT‐PCR diagnostic assay will facilitate the understanding of SCMMV epidemiology and will help improve management practices. [ABSTRACT FROM AUTHOR]

Grapevine leafroll disease (GLRD) is the most globally prevalent and destructive disease complex responsible for significant reductions in grape yield and quality as well as wine production. GLRD is associated with several positive-strand RNA viruses of the family Closteroviridae, designated as grapevine leafroll-associated viruses (GLRaVs). However, the specific etiological role of any of these GLRaVs in GLRD has not been demonstrated. Even though GLRaV-3 is considered the chief GLRD agent, little is known about the molecular, cellular, and pathological properties of this virus. Such a knowledge gap is due to multiple factors, including the unavailability of biologically active virus cDNA clones and the lack of reliable experimental systems for launching grapevine infection using such clones. In this work, we tested four methods for inoculating tissue-cultured grapevine plantlets with cDNA clones of GLRaV-3: (i) vacuum agro-infiltration; (ii) agro-pricking; (iii) agro-drenching; and (iv) agro-injection. We showed that vacuum agro-infiltration was the most effective of these methods. Furthermore, we examined the impacts of different experimental conditions on the survival and infectivity rate of grapevines after infiltration. To verify the infectivity rate for different treatments, we used RT-PCR, RT-qPCR, and Western blotting. We found that humidity plays a critical role in the survival of plantlets after agro-infiltration and that the use of RNA silencing suppressor and dormancy treatment both had strong effects on the infection rates. To our knowledge, the experimental protocol reported herein is the most effective system for launching the infection of grapevine using cDNA clones of grapevine viruses featuring up to a 70% infection rate. This system has strong potential to facilitate grapevine virology research including the fulfillment of Koch’s postulates for GLRD and other major virus diseases as well as identifying the molecular, cellular, and pathological properties of GLRaVs and, potentially, other important grapevine viruses.

The roles of proteins encoded by members of the genus Ampelovirus, family Closteroviridae are largely inferred by sequence homology or analogy to similarly located ORFs in related viruses. This study employed yeast two-hybrid and bimolecular fluorescence complementation assays to investigate interactions between proteins of grapevine leafroll-associated virus 3 (GLRaV-3). The p5 movement protein, HSP70 homolog, coat protein, and p20B of GLRaV-3 were all found to self-interact, however, the mechanism by which p5 interacts remains unknown due to the absence of a cysteine residue crucial for the dimerisation of the closterovirus homolog of this protein. Although HSP70h forms part of the virion head of closteroviruses, in GLRaV-3, it interacts with the coat protein that makes up the body of the virion. Silencing suppressor p20B has been shown to interact with HSP70h, as well as the major coat protein and the minor coat protein. The results of this study suggest that the virion assembly of a member of the genus Ampelovirus occurs in a similar but not identical manner to those of other genera in the family Closteroviridae. Identification of interactions of p20B with virus structural proteins provides an avenue for future research to explore the mechanisms behind the suppression of host silencing and suggests possible involvement in other aspects of the viral replication cycle. [ABSTRACT FROM AUTHOR]

Tomato chlorosis virus (ToCV, genus Crinivirus, family Closteroviridae) is an emerging plant virus first identified in the mid‐1990s affecting tomato in Florida. Today, the virus is present in at least 39 countries and territories around the world, and this number has not stopped growing. ToCV prevalence in tomato has been reported to frequently reach 100%, with a consequent loss in production. Although ToCV infects mainly tomato, it has been reported to infect a wide range of plants, both cultivated and wild, including many economically important crops, such as pepper and potato. To date, 119 species of host plants belonging to 28 families have been recorded. ToCV is transmitted in nature by whiteflies belonging to two genera, namely members of the Bemisia tabaci complex and Trialeurodes vaporariorum and Trialeurodes abutiloneus. This review summarises what is known about ToCV, an example of an emerging plant pathogen, and is the basis of the Association of Applied Biologists' Description of Plant Viruses for ToCV. [ABSTRACT FROM AUTHOR]

Several new full genome sequences of olive viruses came to light recently via high-throughput sequencing (HTS) analysis. In this study, total RNA HTS analysis of two Greek olive trees revealed the presence of an olive virus T (OlVT) isolate and an olive leaf yellowing-associated virus (OLYaV) isolate. The full viral genome of OlVT isolate (50Ch) is composed of 6862 nucleotides encoding for three proteins (replicase, movement protein, and capsid protein) with typical betaflexiviruses' genomic features. However, both sequence and phylogenetic data analysis exhibited high levels of variability between 50Ch and the previously characterized OlVT isolates. In addition, the almost full genome of the Greek OLYaV isolate (OL2) was obtained, which is composed of 16,693 nucleotides encoding for 11 open reading frames (ORFs) and shares common genomic features with the recently characterized OLYaV isolates from Spain and Brazil. Sequence and phylogenetic analysis revealed high similarity between these three isolates. Due to problems encountered with the detection of both viruses, new nested RT-PCR assays were developed and applied. In addition, recombination events were observed in OlVT isolates (50Ch GR-168), thus highlighting the potential role of this mechanism in the evolution of the virus. This study is adding further knowledge to the limited information available about these recently characterized olive infecting viral pathogens and highlights their widespread distribution in Greece, one of the most important olive producing countries of the world. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species. In addition, new divergent variants have been discovered recently around the world. Accurate identification of the virus is an essential component in the management and control of GLRaV-3; however, the diversity of GLRaV-3, coupled with the limited sequence information, have complicated the development of a reliable detection assay. In this study, GLRaV-3 sequence data available in GenBank and those generated at Foundation Plant Services, University of California-Davis, was used to develop a new RT-qPCR assay with the capacity to detect all known GLRaV-3 variants. The new assay, referred to as FPST, was challenged against samples that included plants infected with different GLRaV-3 variants and originating from 46 countries. The FPST assay detected all known GLRaV-3 variants, including the highly divergent variants, by amplifying a small highly conserved region in the 3' untranslated terminal region (UTR) of the virus genome. The reliability of the new RT-qPCR assay was confirmed by an enzyme linked immunosorbent assay (ELISA) that can detect all known GLRaV-3 variants characterized to date. Additionally, three new GLRaV-3 divergent variants, represented by four isolates, were identified using a hierarchical testing process involving the FPST assay, GLRaV-3 variant-specific assays and high-throughput sequencing analysis. These variants were distantly related to groups I, II, III, V, VI, VII and IX, but much similar to GLRaV-3 variants with no assigned group; thus, they may represent new clades. Finally, based on the phylogenetic analysis, a new GLRaV-3 subclade is proposed and named as group X.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses affecting global grape and wine production. GLRaV-3 is the chief agent associated with grapevine leafroll disease (GLRD), the most prevalent and economically destructive grapevine viral disease complex. Response of grapevine to GLRaV-3 infection at the gene expression level is poorly characterized, limiting the understanding of GLRaV-3 pathogenesis and viral-associated symptom development. In this research, we used RNA-Seq to profile the changes in global gene expression of Cabernet franc, a premium red wine grape, analyzing leaf and berry tissues at three key different developmental stages. We have identified 1457 differentially expressed genes (DEGs) in leaves and 1181 DEGs in berries. The expression profiles of a subset of DEGs were validated through RT-qPCR, including those involved in photosynthesis (VvPSBP1), carbohydrate partitioning (VvSUT2, VvHT5, VvGBSS1, and VvSUS), flavonoid biosynthesis (VvUFGT, VvLAR1, and VvFLS), defense response (VvPR-10.3, and VvPR-10.7), and mitochondrial activities (ETFB, TIM13, and NDUFA1). GLRaV-3 infection altered source–sink relationship between leaves and berries. Photosynthesis and photosynthate assimilation were inhibited in mature leaves while increased in young berries. The expression of genes involved in anthocyanin biosynthesis increased in GLRaV-3-infected leaves, correlating with interveinal tissue reddening, a hallmark of GLRD symptoms. Notably, we identified changes in gene expression that suggest a compromised sugar export and increased sugar retrieval in GLRaV-3-infected leaves. Genes associated with mitochondria were down-regulated in both leaves and berries of Cabernet franc infected with GLRaV-3. Results of the present study suggest that GLRaV-3 infection may disrupt mitochondrial function in grapevine leaves, leading to repressed sugar export and accumulation of sugar in mature leaf tissues. The excessive sugar accumulation in GLRaV-3-infected leaves may trigger downstream GLRD symptom development and negatively impact berry quality. We propose a working model to account for the molecular events underlying the pathogenesis of GLRaV-3 and symptom development. [ABSTRACT FROM AUTHOR]

Olive trees (Olea europea L.) are constantly threatened by many viruses, such as the olive leaf yellowing-associated virus (OLYaV), that belong to the Olivavirus genus, family Closteroviridae. In this work, the OLYaV incidence in different regions of Italy and Spain, which represent the two most important European areas for olive production, was evaluated through the development of a real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for reliable and sensitive OLYaV detection. The specificity and accuracy of the developed real-time RT-LAMP assay were determined; the assay showed that potential cross-reactivity with other viruses belonging to the Closteroviridae family was excluded. The LAMP assay detected OLYaV with a higher sensitivity than conventional end-point RT-PCR, detecting a total of 1.34 × 10−2 genome copies. A total of 80 and 120 plants of different olive cultivars from Spain (Comunitat Valenciana, Andalusia) and Italy (Sicily, Calabria, Apulia, Lazio, and Umbria) regions were tested, respectively. The percentage of infected plants was 46.25% and 30% for Spain and Italy, respectively, while the most susceptible cultivars were “Serrana Espadán” and “Villalonga” from Comunitat Valenciana and Andalusia regions (Spain) and “Ogliarola barese” from Apulia region (Italy). In addition, the survey demonstrated that the real-time RT-LAMP showed good sensitivity for OLYaV-positive sample detection, especially on asymptomatic olive trees. For this reason, the developed assay could be very suitable for phytopathological laboratories as a reliable and efficient method for a rapid and sensitive routine test on olive samples.

The genome sequence of a closterovirus (genus Closterovirus, family Closteroviridae), tentatively named Thesium chinense closterovirus 1 (TcCV1), was identified by performing high-throughput RNA-sequencing of the haustoria and root tissues of Thesium chinense, a parasitic plant. The TcCV1 genome was predicted to encode nine proteins, eight of which have orthologs in previously identified closteroviruses. The TcCV1 RNA-dependent RNA polymerase (RdRp) and heat shock protein 70 homolog (Hsp70h) showed 27.8–68.2% and 23.8–55.1% amino acid identity, respectively, to orthologous proteins of known closteroviruses. The putative +1 ribosomal frameshifting site required for producing RdRp was identified as GUUUAGC with UAG stop codon and the skipped nucleotide U. Phylogenetic trees based on RdRp and Hsp70h show that TcCV1 is a novel member of the genus Closterovirus, forming a subclade with a group of known closteroviruses, including mint virus 1 and carnation necrotic fleck virus. The genome sequence of TcCV1 may be useful for studying the genome evolution of closteroviruses. [ABSTRACT FROM AUTHOR]

The viromic profile of Polyscias balfouriana cv. Marginata, a perennial woody and ornamental plant, was determined using ribosomal RNA-depleted total RNA (rRNA-depleted totRNA) sequencing. Five viruses (i.e., polyscias mosaic virus, PoMV; one potential novel rhabdovirus; and three novel viruses of Betaflexiviridae and Closteroviridae) were detected and prevalence-surveyed in Hainan province, China. The genomes of polyscias capillovirus 1 (PCaV-1) and polyscias citrivirus 1 (PCiV-1) of family Betaflexiviridae were completed, and the genomes of polyscias crinivirus 1 (PCrV-1) of Closteroviridae were nearly completed lacking the 5′ and 3′ termini. PCaV-1 shares 68% genome nucleotide (nt) identity and 66% replicase (Rep) amino acid (aa) identity with homologues in apple stem grooving virus (ASGV). PCiV-1 shares 65% genome nt identity and 64% Rep aa identity with homologs in citrus leaf blotch virus (CLBV). Meeting the species demarcation criteria, PCaV-1 and PCiV-1 were considered to be new species in genera Capillovirus and Citrivirus, respectively. PCrV-1 shares high genome nt identity (62%), heat shock protein 70-like protein (HSP70h) and RNA-dependent RNA polymerase (RdRp) aa identity (78–80%) with homologues in tomato chlorosis virus (ToCV). We tentatively consider PCrV-1 to be an unclassified member of the Crinivirus genus. PoMV, PCaV-1, PCiV-1, and PCrV-1 are the prevalent viruses with >73% occurrence in the Xinglong Tropical Botanical Garden, Hainan, China. [ABSTRACT FROM AUTHOR]

Background: the most widely distributed and virulent Grapevine leafroll-associated viruses (GLRaV) that affect grapevine are GLRaV-1 and GLRaV-3, transmitted semi-persistently by different mealybugs and soft scales, mainly causing downward rolling of the leaf margins and interveinal reddening. Methods: the main objectives of this study were to investigate the genetic structure and molecular diversity of GLRaV-1 and GLRaV-3 in 617 samples from 11 autochthonous Sicilian grapevine cultivars, ascertaining their presence and spread. The detection was implemented by serological and molecular analyses and subsequently phylogenetic analyses on selected Sicilian isolates were conducted. Results: in total, 33 and 138 samples resulted positive to GLRaV-1 and GLRaV-3, with an incidence of 5.34% and 22.36%, respectively; 9 out of the 11 cultivars resulted positive, while the presence of both viruses was not found in 'Grillo' and 'Moscato' cultivars. Conclusions: phylogenetic analyses of the coat protein (CP) gene of 12 GLRaV-1 selected sequences showed a close relationship with European isolates; the discrete nucleotide differentiation and positive selection could demonstrate a current increase in population fitness. The phylogenetic analyses of the CP gene of 31 GLRaV-3 Sicilian CP sequences demonstrates a close relationship between Sicilian and different countries isolates; a certain stability of GLRaV-3 in the different cultivars analyzed is suggested by the discrete differentiation nucleotide and negative selection of the Sicilian isolates. [ABSTRACT FROM AUTHOR]

Grapevine leafroll disease is an increasing problem in all grape-growing regions of the world. The most widespread agent of the disease, Grapevine leafroll-associated virus 3 (GLRaV-3), has never been shown to infect species outside of the genus Vitis. Virus transmission to several plant species used as model systems was tested using the vine mealybug, Planococcus ficus. We show that GLRaV-3 is able to infect Nicotiana benthamiana. Working with GLRaV-3 infected N. benthamiana revealed distinct advantages in comparison with its natural host Vitis vinifera, yielding both higher viral protein and virion concentrations in western blot and transmission electron microscopy observations, respectively. Immunogold labelling of thin sections through N. benthamiana petioles revealed filamentous particles in the phloem cells of GLRaV-3 positive plants. Comparison of assembled whole genomes from GLRaV-3 infected V. vinifera vs. N. benthamiana revealed substitutions in the 5' UTR. These results open new avenues and opportunities for GLRaV-3 research.

Grapevine leafroll disease is associated with several species of phloem-limited grapevine leafroll-associated viruses (GLRaV), some of which are transmitted by mealybugs and scale insects. The grape phylloxera, Daktulosphaira vitifoliae (Fitch) Biotype A (Hemiptera: Phylloxeridae), is a common vineyard pest that feeds on the phloem of vine roots. There is concern that these insects may transmit one or more GLRaV species, particularly GLRaV-2, a species in the genus Closterovirus. A field survey was performed in vineyards with a high incidence of grapevine leafroll disease and D. vitifoliae was assessed for acquisition of GLRaV. In greenhouse experiments, the ability of D. vitifoliae to transmit GLRaV from infected root sections or vines to co-planted virus-free recipient vines was tested. There were no GLRaV-positive D. vitifoliae in the field survey, nor did D. vitifoliae transmit GLRaV-1, â2, â3, or -4LV in greenhouse transmission experiments. Some insects tested positive for GLRaV after feeding on infected source vines in the greenhouse, however there was no evidence of virus transmission to healthy plants. These findings, in combination with the sedentary behaviour of the soil biotype of D. vitifoliae, make it unlikely that D. vitifoliae is a vector of any GLRaV.

Several mealybug species are vectors of grapevine leafroll-associated viruses (GLRaV), which cause the economically important grapevine leafroll disease in grape-producing regions worldwide. The mealybug Ferrisia gilli Gullan is a new pest of grapevines in El Dorado County, located in the Sierra Foothill wine-growing region of California. GLRaV species 1, 2, 3, and 4LV have been detected in vineyards with symptomatic vines in the Sierra Foothills. We conducted controlled virus acquisition and transmission experiments using source vine accessions infected with different combinations of GLRaV. We determined that F. gilli acquired GLRaV 1, 2, 3, and 4LV, and transmitted GLRaV-3 and GLRaV-4LV to uninfected recipient vines. Like numerous other mealybug species, in addition to causing direct damage to vines, F. gilli poses a threat to the grape industry as a vector of economically damaging viruses.

The roles of proteins encoded by members of the genus Ampelovirus, family Closteroviridae are largely inferred by sequence homology or analogy to similarly located ORFs in related viruses. This study employed yeast two-hybrid and bimolecular fluorescence complementation assays to investigate interactions between proteins of grapevine leafroll-associated virus 3 (GLRaV-3). The p5 movement protein, HSP70 homolog, coat protein, and p20B of GLRaV-3 were all found to self-interact, however, the mechanism by which p5 interacts remains unknown due to the absence of a cysteine residue crucial for the dimerisation of the closterovirus homolog of this protein. Although HSP70h forms part of the virion head of closteroviruses, in GLRaV-3, it interacts with the coat protein that makes up the body of the virion. Silencing suppressor p20B has been shown to interact with HSP70h, as well as the major coat protein and the minor coat protein. The results of this study suggest that the virion assembly of a member of the genus Ampelovirus occurs in a similar but not identical manner to those of other genera in the family Closteroviridae. Identification of interactions of p20B with virus structural proteins provides an avenue for future research to explore the mechanisms behind the suppression of host silencing and suggests possible involvement in other aspects of the viral replication cycle.

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most prevalent and economically damaging virus in grapevines and is found on nearly all continents, except Antarctica. Ten mealybugs act as vector insects transmitting the GLRaV-3. Understanding the potential distribution range of vector insects under climate change is crucial for preventing and managing vector insects and controlling and delaying the spread of GLRaV-3. This study investigated the potential geographical range of insect vectors of GLRaV-3 worldwide using MaxEnt (maximum entropy) based on occurrence data under environmental variables. The potential distributions of these insects were projected for the 2030s, 2050s, 2070s, and 2090s under the three climate change scenarios. The results showed that the potential distribution range of most vector insects is concentrated in Southeastern North America, Europe, Asia, and Southeast Australia. Most vector insects contract their potential distribution ranges under climate-change conditions. The stacked model suggested that potential distribution hotspots of vector insects were present in Southeastern North America, Europe, Southeast Asia, and Southeast Australia. The potential distribution range of hotspots would shrink with climate change. These results provide important information for governmental decision-makers and farmers in developing control and management strategies against vector insects of GLRaV-3. They can also serve as references for studies on other insect vectors., Competing Interests: Declaration of competing interest The authors declare that they have no known conflict of interests. The manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Coinfections are common, leading to pathogen interactions during transmission and establishment in a host. However, few studies have tested the relative strengths of pathogen interactions in vectors and hosts that determine the outcome of infection. We tested interactions between two genetically distinct variants of the mealybug-transmitted Grapevine leafroll-associated virus 3. The transmission efficiency of each variant in single variant inoculations by two vector species was determined. The effects of vector species, a coinfected source, and simultaneous inoculation from multiple hosts to one host on variant establishment were examined. Within-vector interactions could have a role in transmission from hosts containing mixed infections, but not when vectors were moved from separate singly infected source plants to a single recipient plant. The invasive Planococcus ficus (Signoret) was a more efficient vector than Pseudococcus viburni (Signoret). Transmission efficiency of the two variants did not differ in single variant inoculations. Overall infections were the same whether from singly or coinfected source plants. In mixed inoculations, establishment of one variant was reduced. Mixed inoculations from two singly infected source plants resulted in fewer mixed infections than expected by chance. Therefore, the observed outcome was determined subsequent to host inoculation rather than in the vector. The outcome may be due to resource competition between pathogens. Alternatively apparent competition may be responsible; the pathogens' differential ability to overcome host defenses and colonize the host may determine the final outcome of new infections. Detailed knowledge of interactions between pathogens during transmission and establishment could improve understanding and management of disease spread.

Some diseases manifest as one characteristic set of symptoms to the host, but can be caused by multiple pathogens. Control treatments based on plant symptoms can make it difficult to effectively manage such diseases, as the biology of the underlying pathogens can vary. Grapevine leafroll disease affects grapes worldwide, and is associated with several viral species in the family Closteroviridae. Whereas some of the viruses associated with this disease are transmitted by insect vectors, others are only graft-transmissible. In three regions of California, we surveyed vineyards containing diseased vines and screened symptomatic plants for all known viral species associated with grapevine leafroll disease. Relative incidence of each virus species differed among the three regions regions, particularly in relation to species with known vectors compared with those only known to be graft-transmitted. In one region, the pathogen population was dominated by species not known to have an insect vector. In contrast, populations in the other surveyed regions were dominated by virus species that are vector-transmissible. Our survey did not detect viruses associated with grapevine leafroll disease at some sites with characteristic disease symptoms. This could be explained either by undescribed genetic diversity among these viruses that prevented detection with available molecular tools at the time the survey was performed, or a misidentification of visual symptoms that may have had other underlying causes. Based on the differences in relative prevalence of each virus species among regions and among vineyards within regions, we expect that region and site-specific management strategies are needed for effective disease control.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses affecting global grape and wine production. GLRaV-3 is the chief agent associated with grapevine leafroll disease (GLRD), the most prevalent and economically destructive grapevine viral disease complex. Response of grapevine to GLRaV-3 infection at the gene expression level is poorly characterized, limiting the understanding of GLRaV-3 pathogenesis and viral-associated symptom development. In this research, we used RNA-Seq to profile the changes in global gene expression of Cabernet franc, a premium red wine grape, analyzing leaf and berry tissues at three key different developmental stages. We have identified 1457 differentially expressed genes (DEGs) in leaves and 1181 DEGs in berries. The expression profiles of a subset of DEGs were validated through RT-qPCR, including those involved in photosynthesis (VvPSBP1), carbohydrate partitioning (VvSUT2, VvHT5, VvGBSS1, and VvSUS), flavonoid biosynthesis (VvUFGT, VvLAR1, and VvFLS), defense response (VvPR-10.3, and VvPR-10.7), and mitochondrial activities (ETFB, TIM13, and NDUFA1). GLRaV-3 infection altered source–sink relationship between leaves and berries. Photosynthesis and photosynthate assimilation were inhibited in mature leaves while increased in young berries. The expression of genes involved in anthocyanin biosynthesis increased in GLRaV-3-infected leaves, correlating with interveinal tissue reddening, a hallmark of GLRD symptoms. Notably, we identified changes in gene expression that suggest a compromised sugar export and increased sugar retrieval in GLRaV-3-infected leaves. Genes associated with mitochondria were down-regulated in both leaves and berries of Cabernet franc infected with GLRaV-3. Results of the present study suggest that GLRaV-3 infection may disrupt mitochondrial function in grapevine leaves, leading to repressed sugar export and accumulation of sugar in mature leaf tissues. The excessive sugar accumulation in GLRaV-3-infected leaves may trigger downstream GLRD symptom development and negatively impact berry quality. We propose a working model to account for the molecular events underlying the pathogenesis of GLRaV-3 and symptom development.

Tomato chlorosis virus (ToCV) is an emergent plant pathogen that causes a yellow leaf disorder in tomato and other solanaceous crops. ToCV is a positive-sense, single stranded (ss)RNA bipartite virus with long and flexuous virions belonging to the genus Crininivirus (family Closteroviridae). ToCV is phloem-limited, transmissible by whiteflies, and causes symptoms of interveinal chlorosis, bronzing, and necrosis in the lower leaves of tomato accompanied by a decline in vigor and reduction in fruit yield. The availability of infectious virus clones is a valuable tool for reverse genetic studies that has been long been hampered in the case of closterovirids due to their genome size and complexity. Here, attempts were made to improve the infectivity of the available agroinfectious cDNA ToCV clones (isolate AT80/99-IC from Spain) by adding the hepatitis delta virus (HDV) ribozyme fused to the 3′ end of both genome components, RNA1 and RNA2. The inclusion of the ribozyme generated a viral progeny with RNA1 3′ ends more similar to that present in the clone used for agroinoculation. Nevertheless, the obtained clones were not able to infect tomato plants by direct agroinoculation, like the original clones. However, the infectivity of the clones carrying the HDV ribozyme in Nicotiana benthamiana plants increased, on average, by two-fold compared with the previously available clones.

Tomato chlorosis virus (ToCV) is an emergent plant pathogen that causes a yellow leaf disorder in tomato and other solanaceous crops. ToCV is a positive-sense, single stranded (ss)RNA bipartite virus with long and flexuous virions belonging to the genus Crininivirus (family Closteroviridae). ToCV is phloem-limited, transmissible by whiteflies, and causes symptoms of interveinal chlorosis, bronzing, and necrosis in the lower leaves of tomato accompanied by a decline in vigor and reduction in fruit yield. The availability of infectious virus clones is a valuable tool for reverse genetic studies that has been long been hampered in the case of closterovirids due to their genome size and complexity. Here, attempts were made to improve the infectivity of the available agroinfectious cDNA ToCV clones (isolate AT80/99-IC from Spain) by adding the hepatitis delta virus (HDV) ribozyme fused to the 3′ end of both genome components, RNA1 and RNA2. The inclusion of the ribozyme generated a viral progeny with RNA1 3′ ends more similar to that present in the clone used for agroinoculation. Nevertheless, the obtained clones were not able to infect tomato plants by direct agroinoculation, like the original clones. However, the infectivity of the clones carrying the HDV ribozyme in Nicotiana benthamiana plants increased, on average, by two-fold compared with the previously available clones. [ABSTRACT FROM AUTHOR]

The viromic profile of Polyscias balfouriana cv. Marginata, a perennial woody and ornamental plant, was determined using ribosomal RNA-depleted total RNA (rRNA-depleted totRNA) sequencing. Five viruses (i.e., polyscias mosaic virus, PoMV; one potential novel rhabdovirus; and three novel viruses of Betaflexiviridae and Closteroviridae) were detected and prevalence-surveyed in Hainan province, China. The genomes of polyscias capillovirus 1 (PCaV-1) and polyscias citrivirus 1 (PCiV-1) of family Betaflexiviridae were completed, and the genomes of polyscias crinivirus 1 (PCrV-1) of Closteroviridae were nearly completed lacking the 5′ and 3′ termini. PCaV-1 shares 68% genome nucleotide (nt) identity and 66% replicase (Rep) amino acid (aa) identity with homologues in apple stem grooving virus (ASGV). PCiV-1 shares 65% genome nt identity and 64% Rep aa identity with homologs in citrus leaf blotch virus (CLBV). Meeting the species demarcation criteria, PCaV-1 and PCiV-1 were considered to be new species in genera Capillovirus and Citrivirus, respectively. PCrV-1 shares high genome nt identity (62%), heat shock protein 70-like protein (HSP70h) and RNA-dependent RNA polymerase (RdRp) aa identity (78–80%) with homologues in tomato chlorosis virus (ToCV). We tentatively consider PCrV-1 to be an unclassified member of the Crinivirus genus. PoMV, PCaV-1, PCiV-1, and PCrV-1 are the prevalent viruses with >73% occurrence in the Xinglong Tropical Botanical Garden, Hainan, China.

This article reports on the first identification of Pineapple mealybug wilt-associated virus 1 (PMWaV-1) and 3 (PMWaV-3) in pineapple plants in Malaysia. Mealybug wilt of pineapple (MWP) is a disease that affects pineapple production globally. The study collected leaf samples from farms in Melaka and Johor and used PCR assays to confirm the presence of PMWaV-1, PMWaV-2, and PMWaV-3 in the samples. The findings suggest that PMWaVs could spread through virus-infected pineapple suckers, highlighting the importance of educating and training pineapple farmers in disease prevention and control. [Extracted from the article]

Background: the most widely distributed and virulent Grapevine leafroll-associated viruses (GLRaV) that affect grapevine are GLRaV-1 and GLRaV-3, transmitted semi-persistently by different mealybugs and soft scales, mainly causing downward rolling of the leaf margins and interveinal reddening. Methods: the main objectives of this study were to investigate the genetic structure and molecular diversity of GLRaV-1 and GLRaV-3 in 617 samples from 11 autochthonous Sicilian grapevine cultivars, ascertaining their presence and spread. The detection was implemented by serological and molecular analyses and subsequently phylogenetic analyses on selected Sicilian isolates were conducted. Results: in total, 33 and 138 samples resulted positive to GLRaV-1 and GLRaV-3, with an incidence of 5.34% and 22.36%, respectively; 9 out of the 11 cultivars resulted positive, while the presence of both viruses was not found in ‘Grillo’ and ‘Moscato’ cultivars. Conclusions: phylogenetic analyses of the coat protein (CP) gene of 12 GLRaV-1 selected sequences showed a close relationship with European isolates; the discrete nucleotide differentiation and positive selection could demonstrate a current increase in population fitness. The phylogenetic analyses of the CP gene of 31 GLRaV-3 Sicilian CP sequences demonstrates a close relationship between Sicilian and different countries isolates; a certain stability of GLRaV-3 in the different cultivars analyzed is suggested by the discrete differentiation nucleotide and negative selection of the Sicilian isolates.

Grapevine leafroll disease (GLD) is caused by a complex of vector-borne virus species in the family Closteroviridae. GLD is present in all grape-growing regions of the world, primarily affecting wine grape varieties. The disease has emerged in the last two decades as one of the major factors affecting grape fruit quality, leading to research efforts aimed at reducing its economic impact. Most research has focused on the pathogens themselves, such as improved detection protocols, with limited work directed toward disease ecology and the development of management practices. Here we discuss the ecology and management of GLD, focusing primarily on Grapevine leafroll-associated virus 3, the most important virus species within the complex. We contextualize research done on this system within an ecological framework that forms the backbone of the discussion regarding current and potential GLD management strategies. To reach this goal, we introduce various aspects of GLD biology and ecology, followed by disease management case studies from four different countries and continents (South Africa, New Zealand, California-USA, and France). We review ongoing regional efforts that serve as models for improved strategies to control this economically important and worldwide disease, highlighting scientific gaps that must be filled for the development of knowledge-based sustainable GLD management practices.

Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management.

Grapevine leafroll-associated virus-2 (GLRaV-2) and GLRaV-3 are both (+) ssRNA viruses of the family Closteroviridae and are involved in grapevine leafroll, the most destructive viral disease affecting the global grape/wine industry. Outbreaks of the disease were recently reported in Canada, causing serious concerns to the grape/wine industry. Reliable, sensitive and timely detection is key to the control of the disease. However, information on their seasonal dynamics and tissue distribution under cool climate conditions has been rather limited. We conducted a two-year comprehensive study to elucidate the temporal variation and spatial distribution of both viruses through symptom monitoring, Western blotting and RT-qPCR. Sampling was done monthly from commercial Cabernet Franc and Chardonnay vines from May to October in 2015 and 2016. Both viral RNA and capsid protein levels in leaves remained low during May and June, steadily increased from late July, and peaked in September or October. Interestingly, young berries collected in June contained high levels of viral RNA for both viruses. As expected, the viral RNA levels of GLRaV-2 detected by RT-qPCR using primers targeting the CP region were much higher than those by using primers targeting the genomic RNA. Surprisingly, virtually the same levels of viral RNA were detected for GLRaV-3 regardless of the targeted genomic regions. To ensure accurate detection of both viruses, we recommend using young berries early in the season and leaves and cambial scrapings from late July to harvest. To our knowledge, this is the first report on the seasonal dynamics and tissue distribution of two major grapevine viruses in Canada. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most prevalent and destructive virus species that contributes to grapevine leafroll disease, an economically damaging disease that affects vineyards globally. Grapevine virus A (GVA) is a virus species in the rugose wood complex and is associated with several vineyard diseases. Both virus species are transmitted by several mealybug species. Transmission efficiency is a major facet of pathogen spread and may be influenced by virus species interactions in the vector or host. We tested transmission efficiency of GLRaV-3 and GVA from nine field-collected source vine samples of Vitis vinifera cv Chardonnay by first instars of Planococcus ficus. Transmission of GLRaV-3 was 22% greater than transmission of GVA. Establishment of new mixed GLRaV-3/GVA infections did not differ significantly from single GLRaV-3 infections following inoculation by P. ficus. These results suggest that GVA may have a higher likelihood of establishing new infections in concert with GLRaV-3 than in single infections. [ABSTRACT FROM AUTHOR]

RNA silencing is a major antiviral mechanism in plants, which is counteracted by virus-encoded proteins with silencing suppression activity. ORFs encoding putative silencing suppressor proteins that share no structural or sequence homology have been identified in the genomes of four criniviruses. In this study, we investigated the RNA silencing suppression activity of several proteins encoded by the RNA1 (RdRp, p22) and RNA2 (CP, CPm and p26) of cucurbit chlorotic yellows virus (CCYV) using co-agroinfiltration assays on Nicotiana benthamiana plants. Our results indicate that p22 is a suppressor of local RNA silencing that does not interfere with cell-to-cell movement of the RNA silencing signal or with systemic silencing. Furthermore, comparisons of the suppression activity of CCYV p22 with that of two other well-known crinivirus suppressors (CYSDV p25 and ToCV p22) revealed that CCYV p22 is a weaker suppressor of local RNA silencing than the other two proteins. Finally, a comparative sequence analysis of the p22 genes of seven Greek CCYV isolates was performed, revealing a high level of conservation. Taken together, our research advances our knowledge about plant-virus interactions of criniviruses, an emergent group of pathogens that threatens global agriculture. [ABSTRACT FROM AUTHOR]

The cottony grape scale Pulvinaria vitis is a scale insect colonizing grapevine; however, its capacity as a vector of grapevine viruses is poorly known in comparison to other scale species that are vectors of viral species in the genera Ampelovirus and Vitivirus. The ability of P. vitis to transmit the ampeloviruses Grapevine leafroll-associated viruses [GLRaV]−1, −3, and −4, and the vitivirus Grapevine virus A (GVA), to healthy vine cuttings was assessed. The scale insects used originated from commercial vine plots located in Alsace, Eastern France. When nymphs sampled from leafroll-infected vineyard plants were transferred onto healthy cuttings, only one event of transmission was obtained. However, when laboratory-reared, non-viruliferous nymphs were allowed to acquire viruses under controlled conditions, both first and second instar nymphs derived from two vineyards were able to transmit GLRaV−1 and GVA. This is the first report of GLRaV−1 and GVA transmission from grapevine to grapevine by this species.