Integration of Rubus yellow net virus in the raspberry genome: A story centuries in the making.

Rubus yellow net virus (RYNV) belongs to genus Badnavirus. Badnaviruses are found in plants as endogenous, inactive or activatable sequences, and/or in episomal (infectious and active) forms. To assess the state of RYNV in Rubus germplasm, we sequenced the genomes of various cultivars and mined eight raspberry whole genome datasets. Bioinformatics analysis revealed the presence of a diverse array of endogenous RYNV (endoRYNV) sequences that differ significantly in their structure; some lineages have nearly complete, yet non-functional genomes whereas others have rudimentary, short sequence fragments. We developed assays to genotype the main lineages as well as the only known episomal lineage present in the United States. This study discloses the widespread presence of endoRYNVs in commercial raspberries, likely because breeding efforts have focused on a limited pool of germplasm that harbored endoRYNVs.

Genomic analyses of a widespread blueberry virus in the United States.

Screening of blueberry accessions using high throughput sequencing revealed the presence of a new virus. Genomic structure and sequence are similar to that of nectarine stem pitting associated virus (NSPaV), a member of the genus Luteovirus, family Tombusviridae. The full genome of the new luteovirus, tentatively named blueberry virus L (BlVL), was characterized and analyzed. Similar to NSPaV, BlVL does not contain readily identifiable movement proteins in any of the seven isolates sequenced. More than 600 samples collected from five states were screened and 79% were found infected, making BlVL the most widespread blueberry virus in the United States.

Evidence of Rubus Yellow Net Virus Integration into the Red Raspberry Genome.

Rubus yellow net virus (RYNV) infects Rubus spp., causing a severe decline when present in mixed infections with other viruses. RYNV belongs to the family Caulimoviridae, also known as plant pararetroviruses, which can exist as episomal or integrated elements (endogenous). Most of integrated pararetroviruses are noninfectious; however, a few cases have been reported where they excised from the plant genome and formed infectious particles. Graft transmission onto indicator plants R. occidentalis "Munger" has been the standard test method for RYNV detection in certification programs. Previously, it was noticed that some RYNV PCR-positive plants did not induce symptoms on "Munger", suggesting an integration event. In this study, bio-indexing and different molecular techniques were employed to differentiate between integrated and episomal RYNV sequences. Reverse transcription-PCR using RYNV-specific oligonucleotides after DNase treatment generated positive results for the virus in graft transmissible isolates (episomal) only. To confirm these results, rolling circle amplification on DNA preparations from the same samples resulted in amplicons identified as RYNV only from plants with graft transmissible RYNV. High-throughput sequencing was used to identify the RYNV-like sequences present in the host DNA. These results indicate the integration of RYNV into the red raspberry genome and highlight the necessity to recognize this phenomenon (integration) in future Rubus quarantine and certification programs.

Microbial-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pest Management.

RNA interference (RNAi) is a convenient tool to identify and characterize biological functions in organisms. Recently, it has become an alternative to chemical insecticides as a biologically based control agent. This promising technology has the potential to avoid many problems associated with conventional chemical insecticides. In order for RNAi application to be practical for field use, a major hurdle is the development of a cost-effective system of double-stranded RNA (dsRNA) production for a large quantity of dsRNA. A handful of research reports has demonstrated microbial-based dsRNA production using L4440 vector and HT115 (DE3) Escherichia coli for application to vertebrate and invertebrate systems. However, the dsRNA yield, production efficiency, and biological purity from this in vitro system is still unclear. Thus, our study detailed biochemical and molecular tools for large-scale dsRNA production using the microbial system and investigated the production efficiency and yield of crude and purified dsRNAs. An unrelated insect gene, green fluorescent protein (GFP), and an insect neuropeptide gene, pyrokinin (PK) identified from Drosophila suzukii, were used to construct the recombinant L4440 to be expressed in the HT115 (DE3) cell. A considerable amount of dsRNA, 19.5 µg/mL of liquid culture, was isolated using ultrasonic disruption followed by phenol extraction. The sonication method was further evaluated to extract crude dsRNA without the additional phenol extraction and nuclease treatments and also to reduce potential bacterial viability. The results suggest that the ultrasonic method saved time and costs to isolate crude dsRNA directly from large volumes of cell culture without E coli contamination. We investigated whether the injection of PK dsRNA into flies resulted in increased adult mortality, but it was not statistically significant at 95% confidence level. In this study, the microbial-based dsRNA production has potential for applied RNAi technology to complement current insect pest management practices.

A Novel Genetic Variant of Grapevine leafroll-associated virus-3 (GLRaV-3) from Idaho Grapevines.

Grapevine leafroll-associated virus-3 (GLRaV-3) is a major constraint on profitable grapevine cultivation. The virus is transmitted efficiently by mealybugs and soft scale insects, or through vegetative propagation by cuttings, and is present worldwide, wherever grapevines are grown. GLRaV-3 exists as a complex of genetic variants currently classified in several phylogenetic groups that can differ from each other by as much as 30% in nucleotide sequence of the whole genome. In the course of the GLRaV-3 testing of wine grapes in southern Idaho, plants of two grapevine cultivars were found to harbor a novel genetic variant of GLRaV-3, named ID45, which exhibited ≤80% nucleotide sequence identity level to the known GLRaV-3 isolates in its most conserved HSP70h gene. The ID45 variant caused no foliar symptoms in 'Cabernet Sauvignon' in the fall, and was demonstrated to have poor reactivity to commercial virus-specific antibodies. The entire 18,478-nt genome sequence of the GLRaV-3-ID45 was determined using a combination of high-throughput and conventional Sanger sequencing, and demonstrated to have typical organization for the genus Ampelovirus (family Closteroviridae), with only 70 to 77% identity level to the GLRaV-3 genomes from other established phylogroups. We concluded that ID45 represented a new phylogenetic group IX of GLRaV-3. Database search using ID45 nucleotide sequence as a query suggested that this novel ID45 variant is present in at least one other grape-growing state in the U.S., California, and in Brazil. An RT-PCR based test was developed to distinguish ID45 from the predominant GLRaV-3 phylogroup I found in Idaho in single and mixed infections.

A Virus in American Blackcurrant (Ribes americanum) with Distinct Genome Features Reshapes Classification in the Tymovirales.

A novel virus with distinct genome features was discovered by high throughput sequencing in a symptomatic blackcurrant plant. The virus, tentatively named Ribes americanum virus A (RAVA), has distinct genome organization and molecular features bridging genera in the order Tymovirales. The genome consists of 7106 nucleotides excluding the poly(A) tail. Five open reading frames were identified, with the first encoding a putative viral replicase with methyl transferase (MTR), AlkB, helicase, and RNA dependent RNA polymerase (RdRp) domains. The genome organization downstream of the replicase resembles that of members of the order Tymovirales with an unconventional triple gene block (TGB) movement protein arrangement with none of the other four putative proteins exhibiting significant homology to viral proteins. Phylogenetic analysis using replicase conserved motifs loosely placed RAVA within the Betaflexiviridae. Data strongly suggest that RAVA is a novel virus that should be classified as a species in a new genus in the Betaflexiviridae or a new family within the order Tymovirales.

High Risk Blueberry Viruses by Region in North America; Implications for Certification, Nurseries, and Fruit Production.

There is limited information on the distribution of blueberry viruses in the U.S. or around the world other than where the viruses were first discovered and characterized. A survey for blueberry viruses was carried out in the U.S. in 2015⁻2017. Most blueberry viruses have been characterized to the point that sensitive diagnostic assays have been developed. These assays are based on ELISA or variations of PCR, which were employed here to determine the presence of blueberry viruses in major blueberry production and nursery areas of the U.S. The viruses included in this study were: blueberry fruit drop (BFDaV), blueberry latent (BlLV), blueberry leaf mottle (BLMoV), blueberry mosaic (BlMaV), blueberry red ringspot (BRRV), blueberry scorch (BlScV), blueberry shock (BlShV), blueberry shoestring (BlSSV), blueberry virus A (BVA), peach rosette mosaic (PRMV), tobacco ringspot (TRSV), and tomato ringspot (ToRSV). In the Pacific Northwest BlShV was the most widespread virus, with BlScV and ToRSV detected in a limited number of fields in Oregon and Washington, but BlScV was widespread in British Columbia. In the upper midwest, the nematode-borne (ToRSV, TRSV), aphid-transmitted (BlSSV and BVA) and pollen-borne (BLMoV) viruses were most widespread. In the northeast, TRSV, ToRSV, and BlScV, were detected most frequently. In the southeast, BRRV and BNRBV were the most widespread viruses. BlLV, a cryptic virus with no known symptoms or effect on plant growth or yield was present in all regions. There are other viruses present at low levels in each of the areas, but with the lower incidence they pose minimal threat to nursery systems or fruit production. These results indicate that there are hotspots for individual virus groups that normally coincide with the presence of the vectors. The information presented highlights the high risk viruses for nursery and fruit production each pose a different challenge for control.

Late-summer Disease Symptoms in Western Washington Red Raspberry Fields Associated with Co-Occurrence of Phytophthora rubi, Verticillium dahliae, and Pratylenchus penetrans, but not Raspberry bushy dwarf virus.

Sixty percent of the $109 million processed red raspberry industry of the United States occurs in northern Washington State. In 2012, late-summer symptoms of vascular wilt and root disease were observed in many raspberry plantings. These symptoms were initially attributed to Verticillium dahliae. However, diagnostic tests for the pathogen were often contradictory and other soilborne pathogens (Phytophthora rubi and Pratylenchus penetrans) or Raspberry bushy dwarf virus (RBDV) might also have been involved. Therefore, a survey was conducted in 2013 and 2014 to (i) establish the incidence and soil population levels of V. dahliae in red raspberry production fields, (ii) compare among diagnostic methods and laboratories for detecting and quantifying V. dahliae from raspberry field soil, and (iii) assess which pathogens are associated with late-summer disease symptoms of raspberry. Plant and soil samples were collected from 51 disease sites and 20 healthy sites located in 24 production fields. Samples were analyzed for the presence and quantity of each pathogen using traditional plating and extraction methods (V. dahliae, P. rubi, and P. penetrans), quantitative polymerase chain reaction (qPCR) (V. dahliae and P. rubi), and enzyme-linked immunosorbent assay (RBDV). Results showed that V. dahliae was present in 88% of the production fields and that detection of the pathogen differed by method and by laboratory: qPCR detected V. dahliae in the soil from approximately three times as many sites (51 of 71 total sites) as by plating on NP10 semi-selective medium (15 of 71 total sites). Soil populations of V. dahliae were slightly greater at disease sites, but the pathogen was detected with similar frequency from healthy sites and it was rarely isolated from diseased plants (4%). P. rubi, P. penetrans, and RBDV were also common in production fields (79, 91, and 53% of fields, respectively). Both P. rubi (soil and root samples) and P. penetrans (root populations only), but not RBDV, were more frequently found at disease sites than healthy sites, and the amount of P. rubi detected by qPCR was greater from disease sites than healthy sites. In addition, P. rubi was isolated from 27% of the symptomatic plants located at disease sites. Regardless of detection method, V. dahliae, P. rubi, and P. penetrans, either with or without RBDV, were more likely to co-occur at disease sites (73%) than healthy sites (35%), suggesting that a soilborne disease complex is present in raspberry production fields. Results indicate that P. rubi is the primary pathogen most strongly associated with late-summer symptoms of disease, but root populations of P. penetrans and higher soil populations of V. dahliae may also be of concern. Therefore, disease control methods should focus on all three soilborne pathogens.

A new, widespread emaravirus discovered in blackberry.

A new virus was identified in blackberry plants exhibiting leaf mottling, chlorotic ringspots and curved midribs, symptoms associated with blackberry yellow vein disease (BYVD). The genome of the new virus, provisionally named blackberry leaf mottle-associated virus (BLMaV), was characterized and phylogenetic analysis revealed its close relationship to recognized members of the genus Emaravirus. BLMaV was transmitted by a yet to be described eriophyid mite species, further reinforcing its placement in the genus. Detection protocols were developed and used to determine the presence of the virus in plants collected from several areas in the U.S.A. The incidence of BLMaV was greater than 40% in BYVD-affected material.

Quarantine Regulations and the Impact of Modern Detection Methods.

Producers worldwide need access to the best plant varieties and cultivars available to be competitive in global markets. This often means moving plants across international borders as soon as they are available. At the same time, quarantine agencies are tasked with minimizing the risk of introducing exotic pests and pathogens along with imported plant material, with the goal to protect domestic agriculture and native fauna and flora. These two drivers, the movement of more plant material and reduced risk of pathogen introduction, are at odds. Improvements in large-scale or next-generation sequencing (NGS) and bioinformatics for data analysis have resulted in improved speed and accuracy of pathogen detection that could facilitate plant trade with reduced risk of pathogen movement. There are concerns to be addressed before NGS can replace existing tools used for pathogen detection in plant quarantine and certification programs. Here, we discuss the advantages and possible pitfalls of this technology for meeting the needs of plant quarantine and certification.

A novel emaravirus is associated with redbud yellow ringspot disease.

Yellow ringspot is the only virus-like disease reported in redbud (Cercis spp.) with symptoms including vein clearing, chlorotic ringspots and oak-leaf pattern. A putative new emaravirus was present in all trees displaying typical yellow ringspot symptoms and the name redbud yellow ringspot associated virus is proposed. The virus genome is composed of at least five RNA segments. Two coding regions were studied to determine isolate diversity with results pointing to a homogeneous virus population. Host range was evaluated using graft transmission and by testing species found in close proximity to infected trees. Mite transmission with Aculops cercidis, the predominant species found in redbud trees in the epicenter of the disease, was evaluated but was not found to be a vector of the virus. Based on this study and the accumulated knowledge on emaravirus evolution we propose that speciation is allopatric, with vectors being a major component of the process.

Identification of Extrachromosomal Circular DNA in Hop via Rolling Circle Amplification.

During a survey for new viruses affecting hop plants, a circular DNA molecule was identified via rolling circle amplification (RCA) and later characterized. A small region of the 5.7-kb long molecule aligned with a microsatellite region in the Humulus lupulus genome, and no coding sequence was identified. Sequence analysis and literature review suggest that the small DNA molecule is an extranuclear DNA element, specifically, an extrachromosomal circular DNA (eccDNA), and its presence was confirmed by electron microscopy. This work is the first report of eccDNAs in the family Cannabaceae. Additionally, this work highlights the advantages of using RCA to study extrachromosomal DNA in higher plants.

Evidence of sympatric speciation of elderberry carlaviruses.

Five new carlaviruses infecting elderberry were characterized and tentatively named as elderberry virus A-E (ElVA-ElVE). Their genome organization is similar to that of other carlaviruses with size ranging from 8540 to 8628 nucleotides, excluding the polyadenylated tails. ElVA, ElVB and ElVD share a common ancestor as do ElVC and ElVE, indicating that speciation may be sympatric with all viruses having emerged in elderberry. Analyses of the carlavirus conserved domains indicate that the 2-oxoglutarate and Fe(II)-dependent oxygenase motifs are reliable indicators of virus phylogenetic classification with recombination playing a significant role in the evolution of the genus. A universal RT-PCR assay that detects all the elderberry carlaviruses and potentially other members of the genus has been developed. This tool can be used for research and regulatory purposes as elderberry cultivation is rapidly expanding to new areas where the viruses may be absent.

Blueberry fruit drop-associated virus: A New Member of the Family Caulimoviridae Isolated From Blueberry Exhibiting Fruit-Drop Symptoms.

This study describes the nucleotide sequence and genome organization of a new DNA virus isolated from 'Bluecrop' blueberry plants exhibiting fruit-drop symptoms and named Blueberry fruit drop-associated virus (BFDaV). Blueberry fruit drop disease was first detected in blueberry plants in British Columbia, Canada in the late 1990s, and in a single field in northern Washington state in the United States in 2012. Infected bushes abort nearly 100% of their fruit about three weeks prior to harvest, when the berries are about 3 to 5 mm in diameter. At harvest, the affected plants appear taller than healthy ones as there is no fruit weighing down the branches. The virus was amplified from diseased material using rolling circle amplification, followed by enzyme digestion, cloning, and sequencing. The full genome of BFDaV is 9,850 bp in length and contains a single open reading frame, encoding for a polyprotein, and a large noncoding region. Based on the genome size and organization and phylogenetics, BFDaV is proposed as a new and the largest member of family Caulimoviridae. Finally, in mapping part of a field with fruit-drop symptoms, there was a nearly perfect correlation between the presence of the virus and fruit-drop symptoms.

Next-generation sequencing of elite berry germplasm and data analysis using a bioinformatics pipeline for virus detection and discovery.

Berry crops (members of the genera Fragaria, Ribes, Rubus, Sambucus, and Vaccinium) are known hosts for more than 70 viruses and new ones are identified continually. In modern berry cultivars, viruses tend to be asymptomatic in single infections and symptoms only develop after plants accumulate multiple viruses. Most certification programs are based on visual observations. Infected, asymptomatic material may be propagated in the nursery system and shipped to farms where plants acquire additional viruses and develop symptoms. This practice may result in disease epidemics with great impact to producers and the natural ecosystem alike. In this chapter we present work that allows for the detection of known and discovery of new viruses in elite germplasm, having the potential to greatly reduce virus dispersal associated with movement of propagation material.

Population structure of blueberry mosaic associated virus: Evidence of reassortment in geographically distinct isolates.

The population structure of blueberry mosaic associated virus (BlMaV), a putative member of the family Ophioviridae, was examined using 61 isolates collected from North America and Slovenia. The studied isolates displayed low diversity in the movement and nucleocapsid proteins and low ratios of non-synonymous to synonymous nucleotide substitutions, indicative of strong purifying selection. Phylogenetic analyses revealed grouping primarily based on geography with some isolates deviating from this rule. Phylogenetic incongruence in the two regions, coupled with detection of reassortment events, indicated the possible role of genetic exchange in the evolution of BlMaV.

Control of virus diseases of berry crops.

Virus control in berry crops starts with the development of plants free of targeted pathogens, usually viruses, viroids, phytoplasmas, and systemic bacteria, through a combination of testing and therapy. These then become the top-tier plants in certification programs and are the source from which all certified plants are produced, usually after multiple cycles of propagation. In certification schemes, efforts are made to produce plants free of the targeted pathogens to provide plants of high health status to berry growers. This is achieved using a systems approach to manage virus vectors. Once planted in fruit production fields, virus control shifts to disease control where efforts are focused on controlling viruses or virus complexes that result in disease. In fruiting fields, infection with a virus that does not cause disease is of little concern to growers. Virus control is based on the use of resistance and tolerance, vector management, and isolation.

A variant of Rubus yellow net virus with altered genomic organization.

Rubus yellow net virus (RYNV) is a member of the genus Badnavirus (family: Caulimoviridae). RYNV infects Rubus species causing chlorosis of the tissue along the leaf veins, giving an unevenly distributed netted symptom in some cultivars of red and black raspberry. Recently, a strain of RYNV was sequenced from a Rubus idaeus plant in Alberta, Canada, exhibiting such symptoms. The viral genome contained seven open reading frames (ORFs) with five of them in the sense-strand, including a large polyprotein. Here we describe a graft-transmissible strain of RYNV from Europe infecting cultivar 'Baumforth's Seedling A' (named RYNV-BS), which was sequenced using rolling circle amplification, enzymatic digestion, cloning and primer walking, and it was resequenced at a 5X coverage. This sequence was then compared with the RYNV-Ca genome and significant differences were observed. Genomic analysis identified differences in the arrangement of coding regions, promoter elements, and presence of motifs. The genomic organization of RYNV-BS consisted of five ORFs (four ORFs in the sense-strand and one ORF in the antisense-strand). ORFs 1, 2, and 3 showed a high degree of homology to RYNV-Ca, while ORFs 4 and 6 of RYNV-BS were quite distinct. Also, the predicted ORFs 5 and 7 in the RYNV-Ca were absent in the RYNV-BS sequence. These differences may account for the lack of aphid transmissibility of RYNV-BS.

Safeguarding Fruit Crops in the Age of Agricultural Globalization.

The expansion of fruit production and markets into new geographic areas provides novel opportunities and challenges for the agricultural and marketing industries. Evidence that fruit consumption helps prevent nutrient deficiencies and reduces the risk of cardiovascular disease and cancer has assisted in the expansion of all aspects of the fruit industry. In today's competitive global market environment, producers need access to the best plant material available in terms of genetics and health if they are to maintain a competitive advantage in the market. An ever-increasing amount of plant material in the form of produce, nursery plants, and breeding stock moves vast distances, and this has resulted in an increased risk of pest and disease introductions into new areas. One of the primary concerns of the global fruit industry is a group of systemic pathogens for which there are no effective remedies once plants are infected. These pathogens and diseases require expensive management and control procedures at nurseries and by producers locally and nationally. Here, we review (i) the characteristics of some of these pathogens, (ii) the history and economic consequences of some notable disease epidemics caused by these pathogens, (iii) the changes in agricultural trade that have exacerbated the risk of pathogen introduction, (iv) the path to production of healthy plants through the U.S. National Clean Plant Network and state certification programs, (v) the economic value of clean stock to nurseries and fruit growers in the United States, and (vi) current efforts to develop and harmonize effective nursery certification programs within the United States as well as with global trading partners.

A new ophiovirus is associated with blueberry mosaic disease.

Blueberry mosaic disease (BMD) was first described more than 60 years ago and is caused by a yet unidentified graft transmissible agent. A combination of traditional methods and next generation sequencing disclosed the presence of a new ophiovirus in symptomatic plants. The virus was detected in all BMD samples collected from several production areas of North America and was thus named blueberry mosaic associated virus. Phylogenetic analysis, supported by high bootstrap values, places the virus within the family Ophioviridae. The genome organization resembles that of citrus psorosis virus, the type member of the genus Ophiovirus. The implications of this discovery in BMD control and blueberry virus certification schemes are also discussed.