Necrotic Ringspot Virus Research Articles

Development of colorimetric one-step reverse transcription loop-mediated isothermal amplification for rapid and visualized detection of tomato necrotic ringspot virus

Development of colorimetric one-step reverse transcription loop-mediated isothermal amplification for rapid and visualized detection of tomato necrotic ringspot virus

Viral Diversity in Mixed Tree Fruit Production Systems Determined through Bee-Mediated Pollen Collection

Commercially cultivated Prunus species are commonly grown in adjacent or mixed orchards and can be infected with unique or commonly shared viruses. Apple (Malus domestica), another member of the Rosacea and distantly related to Prunus, can share the same growing regions and common pathogens. Pollen can be a major route for virus transmission, and analysis of the pollen virome in tree fruit orchards can provide insights into these virus pathogen complexes from mixed production sites. Commercial honey bee (Apis mellifera) pollination is essential for improved fruit sets and yields in tree fruit production systems. To better understand the pollen-associated virome in tree fruits, metagenomics-based detection of plant viruses was employed on bee and pollen samples collected at four time points during the peak bloom period of apricot, cherry, peach, and apple trees at one orchard site. Twenty-one unique viruses were detected in samples collected during tree fruit blooms, including prune dwarf virus (PDV) and prunus necrotic ringspot virus (PNRSV) (Genus Ilarvirus, family Bromoviridae), Secoviridae family members tomato necrotic ringspot virus (genus Nepovirus), tobacco necrotic ringspot virus (genus Nepovirus), prunus virus F (genus Fabavirus), and Betaflexiviridae family member cherry virus A (CVA; genus Capillovirus). Viruses were also identified in composite leaf and flower samples to compare the pollen virome with the virome associated with vegetative tissues. At all four time points, a greater diversity of viruses was detected in the bee and pollen samples. Finally, the nucleotide sequence diversity of the coat protein regions of CVA, PDV, and PNRSV was profiled from this site, demonstrating a wide range of sequence diversity in pollen samples from this site. These results demonstrate the benefits of area-wide monitoring through bee pollination activities and provide new insights into the diversity of viruses in tree fruit pollination ecosystems.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for In-Field Detection of American Plum Line Pattern Virus.

American plum line pattern virus (APLPV) is the most infrequently reported Ilarvirus infecting stone fruit trees and is of sufficient severity to be classified as an EPPO quarantine A1 pathogen. In late spring, yellow line pattern symptoms were observed on leaves in a few flowering cherries (Prunus serrulata Lindl.) grown in a public garden in Northwest Italy. RNA extracts from twenty flowering cherries were submitted to Ilarvirus multiplex and APLPV-specific RT-PCR assays already reported or developed in this study. One flowering cherry (T22) with mixed prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) infection also showed infection with APLPV. Blastn analysis of PCR products of the full coat protein (CP) and movement protein (MP) genes obtained from flowering cherry T22 showed 98.23% and 98.34% nucleotide identity with reference APLPV isolate NC_003453.1 from the USA. Then, a LAMP-specific assay was designed to facilitate the fast and low-cost identification of this virus either in the laboratory or directly in the field. The developed assay allowed not only the confirmation of APLPV (PSer22IT isolate) infection in the T22 flowering cherry but also the identification of APLPV in an asymptomatic flowering cherry tree (TL1). The LAMP assay successfully worked with crude flowering cherry extracts, obtained after manually shaking a single plant extract in the ELISA extraction buffer for 3-5 min. The developed rapid, specific and economic LAMP assay was able to detect APLPV using crude plant extracts rather that RNA preparation in less than 20 min, making it suitable for in-field detection. Moreover, the LAMP assay proved to be more sensitive in APLPV detection in flowering cherry compared to the specific one-step RT-PCR assay. The new LAMP assay will permit the estimation of APLPV geographic spread in the territory, paying particular attention to surrounding gardens and propagated flowering cherries in ornamental nurseries.

From the discovery of a novel arepavirus in diseased arecanut palms (Areca catechu L.) in India to the identification of known and novel arepaviruses in bee and plant transcriptomes through data-mining

Arecanut palm is a commercially important plantation crop valued for its nut. In this investigation, we report the discovery of a putative novel arepavirus, named areca palm necrotic ringspot virus 2 (ANRSV2), in necrotic ringspot diseased areca palms in Bantwal, Dakshina Kannada, Karnataka, India through RNA-sequencing and transmission electron microscopy. Further, the presence of ANRSV2 in the diseased samples was confirmed through reverse transcriptase-polymerase chain reaction assays. In addition, by mining public domain transcriptome data for arepaviral sequences, we identified a putative novel arepavirus in Psychotria rubra, a non-palm host. We recovered the genome sequences of the areca palm necrotic ringspot virus in honey bees, tomato, Onobrychis viciifolia, and Rhamnus heterophylla. These findings broaden our comprehension of arepaviral diversity and host range, and suggest an intriguing possibility of pollen-mediated arepaviral transmission that necessitates empirical validation. Further studies are needed to understand the biology of identified putative novel arepaviruses.

Comprehensive Metatranscriptomic Analysis of Plant Viruses in Imported Frozen Cherries and Blueberries.

The possibility of new viruses emerging in various regions worldwide has increased due to a combination of factors, including climate change and the expansion of international trading. Plant viruses spread through various transmission routes, encompassing well-known avenues such as pollen, seeds, and insects. However, research on potential transmission routes beyond these known mechanisms has remained limited. To address this gap, this study employed metatranscriptomic analysis to ascertain the presence of plant viruses in imported frozen fruits, specifically cherries and blueberries. This analysis aimed to identify pathways through which plant viruses may be introduced into countries. Virome analysis revealed the presence of six species of plant viruses in frozen cherries and blueberries: cherry virus A (CVA), prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), prunus virus F (PrVF), blueberry shock virus (BlShV), and blueberry latent virus (BlLV). Identifying these potential transmission routes is crucial for effectively managing and preventing the spread of plant viruses and crop protection. This study highlights the importance of robust quality control measures and monitoring systems for frozen fruits, emphasizing the need for proactive measures to mitigate the risk associated with the potential spread of plant viruses.

Effect of virus infection on the fruit quality of sour cherry cultivar Łutówka

A survey was carried out on a commercial sour cherry fruit orchard located in Lublin province in Poland to determine the influence of viruses on the fruit quality of sour cherry cv. Łutówka. Leaf samples from trees of sour cherry cv. Łutówka were tested for Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Little cherry virus 1 (LChV-1), Little cherry virus 2 (LChV-2), Cherry virus A (CVA), Cherry green ring mottle virus (CGRMV), Cherry necrotic rusty mottle virus (CNRMV), Cherry rasp leaf virus (CRLV) and Cherry mottle leaf virus (CMLV) using the RT-PCR technique. The results indicated that PNRSV and PNRSV+CVA infected the samples. PDV, LChV-1, LChV-2, CGRMV, CNRMV, CRLV, and CMLV were not detected in any of the tested sour cherry trees. The effect of virus infection on the chemical composition of sour cherry fruits was investigated. The anthocyanin, total phenolic and vitamin C contents, and antioxidant activity were evaluated. The total phenolic compound, vitamin C contents, and antioxidant activity were significantly higher in PNRSV- and PNRSV+CVA-infected than in virus-free sour cherry fruits. The total anthocyanin content in PNRSV- or PNRSV+CVA-infected fruits was lower than in control trees. To our knowledge, this is the first report in the world about the effect of PNRSV or PNRSV+CVA infection on the anthocyanin compounds, total polyphenolic compounds, vitamin C contents, and the antioxidant activity of sour cherry fruits.

Rubisco small subunit (RbCS) is co-opted by potyvirids as the scaffold protein in assembling a complex for viral intercellular movement.

Plant viruses must move through plasmodesmata (PD) to complete their life cycles. For viruses in the Potyviridae family (potyvirids), three viral factors (P3N-PIPO, CI, and CP) and few host proteins are known to participate in this event. Nevertheless, not all the proteins engaging in the cell-to-cell movement of potyvirids have been discovered. Here, we found that HCPro2 encoded by areca palm necrotic ring spot virus (ANRSV) assists viral intercellular movement, which could be functionally complemented by its counterpart HCPro from a potyvirus. Affinity purification and mass spectrometry identified several viral factors (including CI and CP) and host proteins that are physically associated with HCPro2. We demonstrated that HCPro2 interacts with both CI and CP in planta in forming PD-localized complexes during viral infection. Further, we screened HCPro2-associating host proteins, and identified a common host protein in Nicotiana benthamiana-Rubisco small subunit (NbRbCS) that mediates the interactions of HCPro2 with CI or CP, and CI with CP. Knockdown of NbRbCS impairs these interactions, and significantly attenuates the intercellular and systemic movement of ANRSV and three other potyvirids (turnip mosaic virus, pepper veinal mottle virus, and telosma mosaic virus). This study indicates that a nucleus-encoded chloroplast-targeted protein is hijacked by potyvirids as the scaffold protein to assemble a complex to facilitate viral movement across cells.

The complete genome sequence of tomato necrotic ringspot virus in chilli in Thailand derived from next-generation sequencing.

Tomato necrotic ringspot virus (TNRV) was first reported in Thailand in 2011, where it continues to reduce the yield and quality of pepper and tomato crops. Here, we report the complete genome sequence of TNRV isolate chilli-CR derived from next-generation sequencing. The TNRV genome comprises 16,595 nucleotides (nt) on three RNA segments. The L RNA is 8,858 nt, the M RNA is 4,724 nt, and the S RNA is 3,013 nt in length. The genome structure and organization are typical of orthotospoviruses, encoding five proteins, named L, NSm, GNGC, NSs, and N. Pairwise comparison of each genomic RNA segment and its deduced amino acid (aa) sequence showed that TNRV chilli-CR shares 73.6-82.3% nt sequence identity and 81.1-91.9% aa sequence identity with pepper chlorotic spot virus (PCSV). Similar phylogenetic groupings were observed based on each genomic RNA or deduced aa sequence, and with concatenated genomic RNA sequences. The clustering of TNRV and PCSV in all phylogenetic analyses, and the 78.9% overall nt sequence identity observed using the concatenated genomic RNAs suggest that TNRV is a distinct orthotospovirus and that analysis of concatenated orthotospovirus genome sequences will be of value in future phylogenetic studies of this virus group.

Plant virus diversity in bee and pollen samples from apple (Malus domestica) and sweet cherry (Prunus avium) agroecosystems.

Honey bee (Apis mellifera) pollination is widely used in tree fruit production systems to improve fruit set and yield. Many plant viruses can be associated with pollen or transmitted through pollination, and can be detected through bee pollination activities. Honey bees visit multiple plants and flowers in one foraging trip, essentially sampling small amounts of pollen from a wide area. Here we report metagenomics-based area-wide monitoring of plant viruses in cherry (Prunus avium) and apple (Malus domestica) orchards in Creston Valley, British Columbia, Canada, through bee-mediated pollen sampling. Plant viruses were identified in total RNA extracted from bee and pollen samples, and compared with profiles from double stranded RNA extracted from leaf and flower tissues. CVA, PDV, PNRSV, and PVF coat protein nucleotide sequences were aligned and compared for phylogenetic analysis. A wide array of plant viruses were identified in both systems, with cherry virus A (CVA), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), and prunus virus F (PVF) most commonly detected. Citrus concave gum associated virus and apple stem grooving virus were only identified in samples collected during apple bloom, demonstrating changing viral profiles from the same site over time. Different profiles of viruses were identified in bee and pollen samples compared to leaf and flower samples reflective of pollen transmission affinity of individual viruses. Phylogenetic and pairwise analysis of the coat protein regions of the four most commonly detected viruses showed unique patterns of nucleotide sequence diversity, which could have implications in their evolution and management approaches. Coat protein sequences of CVA and PVF were broadly diverse with multiple distinct phylogroups identified, while PNRSV and PDV were more conserved. The pollen virome in fruit production systems is incredibly diverse, with CVA, PDV, PNRSV, and PVF widely prevalent in this region. Bee-mediated monitoring in agricultural systems is a powerful approach to study viral diversity and can be used to guide more targeted management approaches.

An update on apple chlorotic leaf spot virus status of sweet cherry in Bulgaria

The sweet cherry (Prunus avium L.) is one of the most important stone fruit species in Bulgaria. The cherry is susceptible to many viruses. To gain a better insight into the phyto-virologic status of sweet cherry, a survey for the presence of apple chlorotic leaf spot virus (ACLSV), cherry leafroll virus (CLRVR) and raspberry ringspot virus (RpRSV), in addition to ilarviruses, was carried out in 32 commercial and 6 collection orchards in ten regions of Bulgaria between 2017 and 2022. A total of 1503 sweet cherry samples were collected from symptomatic and symptomless trees. These samples were tested by cocktail ELISA for ACLSV and DAS-ELISA for CLRVR, RpRSV ?nd additionally for prune dwarf virus (PDV) and prunus necrotic ringspot virus (PNRSV) to detect mixed infections. Of the three viruses studied, only ACLSV was detected in commercial and collection sweet cherry orchards in all regions with intensive cherry cultivation in Bulgaria, in addition to ilarviruses. The extent of ACLSV virus infection was 8.8%. Single ACLSV infection was the most common, affecting 79.6% of infected sweet cherry trees, followed by ACLSV and PDV (14.4%) and least frequently by ACLSV and PNRSV (6.0%). The presence of ACLSV in orchards of different ages, including ?young? orchards, indicates that virus-free propagation material must be used when establishing new orchards to prevent the spread of pathogens with the planting material.

First report of prunus necrotic ringspot virus infecting rose (Rosa spp.) in Lebanon

Summary Prunus necrotic ringspot virus (PNRSV) was detected in several rose plants showing symptoms of rose mosaic disease (RMD) in Beqaa valley, Lebanon. PNRSV was found in 29 plants by molecular and serological analyses, while other viruses associated with RMD were absent. Although PNRSV is known to have a wide host range, the present paper reports the first occurrence of PNRSV on rose plants in Lebanon.

Examining the Effects of Prunus Necrotic Ringspot Virus (PNRSV) and Prune Dwarf Virus (PDV) on Peach (Prunus persica) Pollen Health

Pollen health is a key determinant of successful plant reproduction and crop yield. Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) are pollen-borne viruses of stone fruits, including peaches, primarily transmitted through vegetative propagation (i.e., grafting) and pollen. Here, we investigated the effects of PNRSV and PDV infection on peach pollen morphology, viability, and germination. Our scanning electron microscopy on pollen morphology, as well as pollen viability and germination assays, showed that PNRSV has cultivar-specific effects on pollen health; compared with pollen from virus-negative trees, PNRSV infection resulted in distorted shape, reduced viability, and germination of pollen grains of peach cultivar Red Top, but no significant effects on viability or germination were detected on cultivar Zee Lady pollen grains. PDV infection affected pollen width and reduced pollen viability but did not affect pollen germination when compared between PDV-positive and PDV-negative pollen grains. We discuss our results in terms of potential evolutionary consequences and implications on virus epidemiology and disease management in orchards. Investigating the effects of viral infection on pollen health will ultimately enhance our understanding of the transmission of pollen-borne viruses. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

An Evolutionary Perspective of Codon Usage Pattern, Dinucleotide Composition and Codon Pair Bias in Prunus Necrotic Ringspot Virus.

Prunus necrotic ringspot virus (PNRSV) is a significant virus of ornamental plants and fruit trees. It is essential to study this virus due to its impact on the horticultural industry. Several studies on PNRSV diversity and phytosanitary detection technology were reported, but the content on the codon usage bias (CUB), dinucleotide preference and codon pair bias (CPB) of PNRSV is still uncertain. We performed comprehensive analyses on a dataset consisting of 359 coat protein (CP) gene sequences in PNRSV to examine the characteristics of CUB, dinucleotide composition, and CPB. The CUB analysis of PNRSV CP sequences showed that it was not only affected by natural selection, but also affected by mutations, and natural selection played a more significant role compared to mutations as the driving force. The dinucleotide composition analysis showed an over-expression of the CpC/GpA dinucleotides and an under-expression of the UpA/GpC dinucleotides. The dinucleotide composition of the PNRSV CP gene showed a weak association with the viral lineages and hosts, but a strong association with viral codon positions. Furthermore, the CPB of PNRSV CP gene is low and is related to dinucleotide preference and codon usage patterns. This research provides reference for future research on PNRSV genetic diversity and gene evolution mechanism.

Whole Genome Characterization of Prunus Necrotic Ringspot Virus and Prune Dwarf Virus Infecting Stone Fruits in Russia

We conducted a survey of the phytosanitary status of the Prunus germplasm collection in the Nikita Botanical Gardens, Yalta, Russia. The virome of plants displaying virus-like symptoms was studied using Illumina MiSeq high-throughput sequencing. Reads related to prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), and ourmia-like virus 1 (OuLV1) were generated in a number of samples. Near complete genomes of two divergent PNRSV isolates, PDV isolate, and a contig partly covered OuLV1 genome were assembled de novo using the metaSPAdes program. The structure of the genomic RNA1, RNA2, and RNA3 of the new ilarvirus isolates was shown to be typical of PNRSV and PDV. This is the first report and characterization of the PNRSV and PDV full-length genomes from Russia, expanding the information on their geographical distribution and genetic diversity. An open reading frames (ORF)-based phylogeny of all full-length PNRSV and PDV genomes available in GenBank divided each ORF into two or three main clusters. A number of isolates migrated from one cluster to another cluster, depending on the analyzed genome segment. The different branching order may indicate reassortment in the evolutionary history of some PDV and PNRSV isolates.

Characterization of Prunus Necrotic Ringspot Virus and Cherry Virus A Infecting Myrobalan Rootstock.

Prunus necrotic ringspot virus (PNRSV) and cherry virus A (CVA) are two viruses that mainly infect plants of the genus Prunus. Full-length sequences of these two viruses, collected in the Czech Republic from Prunus cerasifera plants, were obtained via HTS sequencing. Phylogenetic analyses based on the NJ method and Splitstree tools showed that the Czech PNRSV isolate (ON088600-ON088602) is a divergent isolate from other molecular groups, sharing less than 97% pairwise nucleotide identity with members of other groups. The Czech CVA isolate (ON088603) belonged to molecular subgroup III-2, clustered with isolates from non-cherry hosts, and shared the highest pairwise nucleotide identity (99.7%) with an isolate of Australian origin.

Insights on the directional movement of Prunus necrotic ringspot virus between peach and wild Prunus species

Insights on the directional movement of Prunus necrotic ringspot virus between peach and wild Prunus species

Bees and thrips carry virus-positive pollen in peach orchards in South Carolina, United States.

Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) are pollen-borne viruses of important stone fruit crops, including peaches, which can cause substantial yield loss. Although both horizontal and vertical (i.e., seed) transmission of both viruses occurs through pollen, the role of flower-visiting insects in their transmission is not well understood. Bees and thrips reportedly spread PNRSV and PDV in orchards and greenhouse studies; however, the field spread of PNRSV and PDV in peach orchards in the southeastern United States is not explored. We hypothesized that bees and thrips may facilitate virus spread by carrying virus-positive pollen. Our 2-yr survey results show that 75% of captured bees are carrying virus-positive pollen and moving across the orchard while a subsample of thrips were also found virus positive. Based on morphology, Bombus, Apis, Andrena, Eucera, and Habropoda are the predominant bee genera that were captured in peach orchards. Understanding the role of bees and thrips in the spread of PNRSV and PDV will enhance our understanding of pollen-borne virus ecology.

First molecular characterization of plum pox virus strains in stone fruits of Tuscany (Central Italy)

Plum pox virus (PPV) a potyvirus in the family Potyviridae, is the etiologic agent of sharka, the worldwide most important and detrimental viral disease of stone fruits (genus Prunus). Although PPV was identified in Italy already in the 1970s and it has been largely reported in almost all Italian regions, a broad investigation and genetic characterization of PPV isolates was lacking in most of them, including Tuscany (Central Italy). To address this knowledge gap, in 2020 and 2021, field surveys were carried out in 80 commercial Tuscany orchards and a total of 690 leaf samples were collected from different Prunus species. PPV was identified in 25 and 12 peach and plum samples, respectively (more than 5% of tested samples), whereas no positive samples were reported in apricot. Eighteen of the 37 PPV positive samples showed mixed infections with other viruses and viroids, mostly Prunus necrotic ringspot virus in plum and peach latent mosaic viroid in peach. Molecular typing of PPV infected samples generated NIb/CP amplicons corresponding to PPV-Marcus (M) or PPV-Recombinant (Rec) strains. Furthermore, starting from the identification of eight PPV nucleotide sequences (among which five and two new PPV-M and PPV-Rec isolates, respectively), this study firstly identified the PPV-Mb subgroup in Italy, which was even prevalent than PPV-Ma. Finally, PPV-Rec isolates resulted phylogenetically close to Italian and Turkish isolates previously detected. Overall, the results here presented represent an important step to fill knowledge gaps about PPV in Tuscany, and we believe it may encourage other similar research to achieve more accurate data on PPV populations at both national and international levels.

The Virome of Babaco (Vasconcellea × heilbornii) Expands to Include New Members of the Rhabdoviridae and Bromoviridae.

Babaco (Vasconcellea × heilbornii) is a subtropical species in the Caricaceae family. The plant is native to Ecuador and represents an important crop for hundreds of families. The objective of this study was to characterize, at the genomic level, two new babaco viruses identified by high-throughput sequencing. The viruses, an ilarvirus and a nucleorhabdovirus, were found in a symptomatic babaco plant from a commercial nursery in the Azuay province of Ecuador. The tripartite genome of the new ilarvirus, provisionally named babaco ilarvirus 1 (BabIV-1), is related to subgroup 3 ilarviruses, including apple mosaic virus, apple necrotic mosaic virus, and prunus necrotic ringspot virus as the closest relatives. The genome of the nucleorhabdovirus, provisionally named babaco nucleorhabdovirus 1 (BabRV-1), showed the closest relation with joa yellow blotch-associated virus and potato yellow dwarf nucleorhabdovirus. Molecular-based detection methods found BabIV-1 and BabRV-1 in 21% and 36%, respectively, of plants surveyed in a commercial babaco nursery, highlighting the importance of enforcing virus testing and nursery certification programs for babaco.

Development of one step colorimetric RT-LAMP assays for rapid detection of Apple mosaic virus and Prunus necrotic ringspot virus

Apple mosaic virus (ApMV) and Prunus necrotic ringspot virus (PNRSV), belonging to genus Ilarvirus, cause significant losses to rose and other plants of the family Rosaceae. They are easily transmitted through mechanical or vegetative means. In our previous study, the occurrence of ApMV and PNRSV in rose plants was reported. In this study, as a first step towards the development of a colorimetric Reverse Transcriptase – Loop Mediated Isothermal Amplification (RT-LAMP) assay, two primer sets were designed, each containing six primers (F3, B3, FIP, BIP, LF and LB) targeting the coat protein genes of ApMV and PNRSV. After incubation of RT-LAMP reaction mix at an isothermal temperature (65 °C/30 min), the amplified products were visually confirmed with the nucleic acid intercalation dye SYBR Green I and the indicator dye Hydroxy-Naphthol Blue. The developed assays were virus specific and showed no cross amplification. Their sensitivity was 103 times higher than that of the corresponding RT-PCRs. The LAMP assays developed in this study are inexpensive, rapid and reliable for the early detection of ApMV and PNRSV, and could therefore be used in plant quarantine to control the risk of their spread.