Rayapati A. Naidu

Grapevine leafroll-associated virus 3

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.

A Leafhopper-Transmissible DNA Virus with Novel Evolutionary Lineage in the Family Geminiviridae Implicated in Grapevine Redleaf Disease by Next-Generation Sequencing

A graft-transmissible disease displaying red veins, red blotches and total reddening of leaves in red-berried wine grape (Vitis vinifera L.) cultivars was observed in commercial vineyards. Next-generation sequencing technology was used to identify etiological agent(s) associated with this emerging disease, designated as grapevine redleaf disease (GRD). High quality RNA extracted from leaves of grape cultivars Merlot and Cabernet Franc with and without GRD symptoms was used to prepare cDNA libraries. Assembly of highly informative sequence reads generated from Illumina sequencing of cDNA libraries, followed by bioinformatic analyses of sequence contigs resulted in specific identification of taxonomically disparate viruses and viroids in samples with and without GRD symptoms. A single-stranded DNA virus, tentatively named Grapevine redleaf-associated virus (GRLaV), and Grapevine fanleaf virus were detected only in grapevines showing GRD symptoms. In contrast, Grapevine rupestris stem pitting-associated virus, Hop stunt viroid, Grapevine yellow speckle viroid 1, Citrus exocortis viroid and Citrus exocortis Yucatan viroid were present in both symptomatic and non-symptomatic grapevines. GRLaV was transmitted by the Virginia creeper leafhopper (Erythroneura ziczac Walsh) from grapevine-to-grapevine under greenhouse conditions. Molecular and phylogenetic analyses indicated that GRLaV, almost identical to recently reported Grapevine Cabernet Franc-associated virus from New York and Grapevine red blotch-associated virus from California, represents an evolutionarily distinct lineage in the family Geminiviridae with genome characteristics distinct from other leafhopper-transmitted geminiviruses. GRD significantly reduced fruit yield and affected berry quality parameters demonstrating negative impacts of the disease. Higher quantities of carbohydrates were present in symptomatic leaves suggesting their possible role in the expression of redleaf symptoms.

Grapevine Leafroll: A Complex Viral Disease Affecting a High-Value Fruit Crop

Grapevine (Vitis spp.) is one of the most widely grown fruit crops in the world. It is a deciduous woody perennial vine for which the cultivation of domesticated species began approximately 6,000 to 8,000 years ago in the Near East. Grapevines are broadly classified into red- and white-berried cultivars based on their fruit skin color, although yellow, pink, crimson, dark blue, and black-berried cultivars also exist. Grapevines can be subject to attacks by many different pests and pathogens, including graft-transmissible agents such as viruses, viroids, and phytoplasmas. Among the virus and virus-like diseases, grapevine leafroll disease (GLD) is by far the most widespread and economically damaging viral disease of grapevines in many regions around the world. The global expansion of the grape and wine industry has seen a parallel increase in the incidence and economic impact of GLD. Despite the fact that GLD was recognized as a potential threat to grape production for several decades, our knowledge of the nature of the disease is still quite limited due to a variety of challenges related to the complexity of this virus disease, the association of several distinct GLD-associated viruses, and contrasting symptoms in red- and white-berried cultivars. In view of the growing significance of GLD to wine grape production worldwide, this feature article provides an overview of the state of knowledge on the biology and epidemiology of the disease and describes management strategies currently deployed in vineyards.

High-throughput sequence analysis of small RNAs in grapevine (Vitis vinifera L.) affected by grapevine leafroll disease

Grapevine leafroll disease (GLRD) is one of the most economically important virus diseases of grapevine (Vitis spp.) worldwide. In this study, we used high-throughput sequencing of cDNA libraries made from small RNAs (sRNAs) to compare profiles of sRNA populations recovered from own-rooted Merlot grapevines with and without GLRD symptoms. The data revealed the presence of sRNAs specific to Grapevine leafroll-associated virus 3, Hop stunt viroid (HpSVd), Grapevine yellow speckle viroid 1 (GYSVd-1) and Grapevine yellow speckle viroid 2 (GYSVd-2) in symptomatic grapevines and sRNAs specific only to HpSVd, GYSVd-1 and GYSVd-2 in nonsymptomatic grapevines. In addition to 135 previously identified conserved microRNAs in grapevine (Vvi-miRs), we identified 10 novel and several candidate Vvi-miRs in both symptomatic and nonsymptomatic grapevine leaves based on the cloning of miRNA star sequences. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) of selected conserved Vvi-miRs indicated that individual members of an miRNA family are differentially expressed in symptomatic and nonsymptomatic leaves. The high-resolution mapping of sRNAs specific to an ampelovirus and three viroids in mixed infections, the identification of novel Vvi-miRs and the modulation of certain conserved Vvi-miRs offers resources for the further elucidation of compatible host-pathogen interactions and for the provision of ecologically relevant information to better understand host-pathogen-environment interactions in a perennial fruit crop.

Sequence diversity, population genetics and potential recombination events in grapevine rupestris stem pitting-associated virus in Pacific North-West vineyards

Grapevine rupestris stem pitting-associated virus (GRSPaV; genus Foveavirus, family Flexiviridae) is present in many grape-growing regions of the world. A total of 84 full-length coat protein (CP) sequences and 57 sequences representing the helicase-encoding region (HR) of the RNA-dependent RNA polymerase were obtained from wine grape cultivars grown in the Pacific North-West (PNW) of the United States and their molecular diversity was compared with corresponding sequences previously reported from other grape-growing regions. In pairwise comparisons, the CP sequences from PNW isolates showed identities between 80 and 100% at the nucleotide level and the HR sequences showed identities between 79 and 100%. A global phylogenetic analysis of the CP and HR sequences revealed segregation of GRSPaV isolates into four major lineages with isolates from PNW distributed in all four lineages, indicating a lack of clustering by geographical origin. Scion cultivars grafted onto rootstock were found to contain mixtures of more genetic variants belonging to different lineages than own-rooted cultivars. Assessment of population genetic parameters found that the CP was more variable than the HR region. The discordant gene phylogenies obtained for some CP and HR sequences and the identification of potential recombination events involving parents from different lineages provided strong evolutionary evidence for genetic diversity among GRSPaV isolates. These results underscore the highly variable nature of the virus with implications for grapevine health status and distribution of virus-tested planting materials. This study also contributes to an increased understanding of molecular population genetics of viruses infecting deciduous woody perennials.

Grapevine leafroll-associated virus 1 Occurs as Genetically Diverse Populations

The genetic diversity of 34 isolates of Grapevine leafroll-associated virus 1 (GLRaV-1) from different wine, table, and ornamental grape cultivars in California, New York, and Washington States in the United States was investigated. Segments of the heat-shock protein 70 homolog (HSP70h) gene, coat protein (CP) gene, coat protein duplicate 2 (CPd2) gene, and open reading frame 9 (p24) were amplified by reverse-transcription polymerase chain reaction, cloned, and sequenced. A pairwise comparison of nucleotide sequences revealed intra- and interisolate sequence diversity, with CPd2 and HSP70h being the most and the least divergent, respectively, among the four genomic regions studied. The normalized values for the ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site indicated different purifying selection pressures acting on each of the four genomic regions, with the CP and CPd2 being subjected to the strongest and weakest functional constraints, respectively. A global phylogenetic analysis of sequences from the four genomic regions revealed segregation of GLRaV-1 isolates into three major clades and a lack of clearly defined clustering by geographical origin. In contrast, only two lineages were apparent when the CP and CPd2 gene sequences were used in phylogenetic analyses. Putative recombination events were revealed among the HSP70h, CP, and p24 sequences. The genetic landscape of GLRaV-1 populations presented in this study provides a foundation for better understanding of the epidemiology of grapevine leafroll disease across grape-growing regions in the United States. In addition, this study will benefit grape clean plant programs across the country in improving the sanitary status of planting materials provided to nurseries and grape growers.

Alternate hosts of African cassava mosaic virus and East African cassava mosaic Cameroon virus in Nigeria

Cassava mosaic disease (CMD) caused by African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) is the major constraint to cassava production in Nigeria. Sequences of the DNA-A component of ACMV and EACMCV isolates from leguminous plant species (Senna occidentalis, Leucana leucocephala and Glycine max), castor oil plant (Ricinus communis), a weed host (Combretum confertum) and a wild species of cassava (Manihot glaziovii) were determined. All ACMV isolates from these hosts showed 96–98% nucleotide sequence identity with cassava isolates from West Africa. EACMCV was found only in four hosts (S. occidentalis, L. leucocephala, C. confertum, M. glaziovii), and sequences of these isolates showed 96–99% identity with cassava isolates from West Africa. These results provide definitive evidence for the natural occurrence of ACMV and EACMCV in plant species besides cassava.

Importance and Genetic Diversity of Vegetable-Infecting Tospoviruses in India

A survey for Peanut bud necrosis virus (PBNV), Watermelon bud necrosis virus (WBNV), Capsicum chlorosis virus (CaCV), and Iris yellow spot virus (IYSV) was conducted between 2002 and 2009 in the major vegetable-growing areas in India. PBNV was documented widely in tomato and chili peppers in 14 states representing southern, north-western, north-eastern, and central regions and WBNV was predominantly detected in watermelons and cucurbits in all except north-eastern regions. In addition, the expanded host range of PBNV to watermelons and other cucurbits and WBNV to tomato and chili peppers was observed leading to natural mixed infection of the two viruses. IYSV was found in onion in southern, central, and north-eastern regions and CaCV in tomato and chili peppers in northern and southern regions, respectively. Phylogenetic analysis of the nucleocapsid gene revealed segregation of field isolates of PBNV and WBNV into two distinct subclades, whereas isolates of CaCV and IYSV each clustered into a single clade. A proposal for establishing WBNV as a distinct tospovirus species is made based on the molecular characterization of small- (S) and medium- (M) RNA segments.

Grapevine leafroll disease and associated viruses: a unique pathosystem.

Grapevine leafroll is the most complex and intriguing viral disease of grapevine (Vitis spp.). Several monopartite closteroviruses (family Closteroviridae) from grapevines have been molecularly characterized, yet their role in disease etiology is not completely resolved. Hence, these viruses are currently designated under the umbrella term of Grapevine leafroll-associated viruses (GLRaVs). This review examines our current understanding of the genetically divergent GLRaVs and highlights the emerging picture of several unique aspects of the leafroll disease pathosystem. A systems biology approach using contemporary technologies in molecular biology, -omics, and cell biology aids in exploring the comparative molecular biology of GLRaVs and deciphering the complex network of host-virus-vector interactions to bridge the gap between genomics and phenomics of leafroll disease. In addition, grapevine-infecting closteroviruses have a great potential as designer viruses to pursue functional genomics and for the rational design of novel disease intervention strategies in this agriculturally important perennial fruit crop.

Pheromone-Based Monitoring of Pseudococcus maritimus (Hemiptera: Pseudococcidae) Populations in Concord Grape Vineyards

ABSTRACT The grape mealybug, Pseudococcus maritimus (Ehrhorn), is the dominant mealybug in Washingtons Concord grape vineyards (Vitis labrusca L.). It is a direct pest of fruit clusters and a vector of grapevine leafroll-associated viruses. Using traps baited with the sex pheromone of Ps. maritimus, we determined the optimal trap density for monitoring Ps. maritimus, with the goal of providing a more rapid monitoring method for Ps. maritimus than visual surveys. Varying densities of pheromone-baited traps (one, four, and eight traps per 12.14 ha) were deployed in Concord vineyards to monitor Ps. maritimus seasonal phenology in 2010 and 2011. In both years, flights of adult males were detected in early May and captures peaked twice per season in mid-June and mid-August, indicating two generations each year. Trap data were analyzed using Taylors Power Law, Iwaos patchiness regression, and the K parameter of the negative binomial model to determine optimal sample size. The formula using the K parameter provided the lowest required sample size, showing that four to eight traps per 12.14 ha were needed to provide 30% sampling precision efficiency throughout the entire season. Fewer traps were needed during flight peaks when trap capture numbers were great. Only one pheromone-baited trap per 12.14 ha was sufficient to provide Ps. maritimus flight phenology data to make informed management decisions. Species-specific pheromone-baited traps deployed for Pianococcus ficus (Signoret), Pseudococcus longispinus (Targioni Tozzetti), and Pseudococcus viburni (Signoret) did not detect any of these species in the vineyards sampled.