Complete genome sequences of three Prunus-infecting nepoviruses: apricot latent ringspot virus, myrobalan latent ringspot virus, and a novel virus from smooth stone peach (Prunus mira Koehne).

The complete genome sequences of two poorly studied Prunus-infecting nepoviruses, apricot latent ringspot virus (ALRSV) and myrobalan latent ringspot virus (MLRSV) were determined, confirming that they are members of subgroup C. Serological, biological, and molecular data, in particular a low level (58.8%) of amino acid sequence identity in the coat protein, suggest that ALRSV and MLRSV should be considered taxonomically distinct. In addition, data mining of public RNASeq data from wild and ornamental Prunus identified two contigs representing the nearly complete genome of a new subgroup A nepovirus from a smooth stone peach (Prunus mira) dataset (SRR8369794) from the Himalayas, for which the name "Prunus mira virus A" is proposed.

Almond Can Be Infected by Plum Pox Virus-D Isolate Penn4 and Is a Transmission-Competent Host.

Although it is currently eradicated from the United States, Plum pox virus (PPV) poses an ongoing threat to U.S. stone fruit production. Although almond (Prunus dulcis) is known to be largely resistant to PPV, there is conflicting evidence about its potential to serve as an asymptomatic reservoir host for the virus and thus serve as a potential route of entry. Here, we demonstrate that both Tuono and Texas Mission cultivars can be infected by the U.S. isolate PPV Dideron (D) Penn4 and that Tuono is a transmission-competent host, capable of serving as a source of inoculum for aphid transmission of the virus. These findings have important implications for efforts to keep PPV out of the United States and highlight the need for additional research to test the susceptibility of almond to other PPV-D isolates.

Viral Reservoir Capacity of Wild Prunus Alternative Hosts of Plum Pox Virus Through Multiple Cycles of Transmission and Dormancy.

Plum pox virus (PPV) is a significant pathogen of Prunus worldwide and is known for having a broad experimental host range. Many of these hosts represent epidemiological risks as potential wild viral reservoirs. A comparative study of the PPV reservoir capacity of three commonly found native North American species, western choke cherry (Prunus virginiana var. demissa), black cherry (Prunus serotina), and American plum (Prunus americana) was conducted. Pennsylvania isolates of PPV-D were transmitted from the original host peach (Prunus persica cv. GF305) to all three species. Viral accumulation and transmission rates to alternative hosts and peach were monitored over the course of five vegetative growth and cold induced dormancy (CID) cycles. The three alternative host species demonstrated differences in their ability to maintain PPV-D and the likelihood of transmission to additional alternative hosts or back transmission to peach. Western choke cherry had low (5.8%) initial infection levels, PPV-D was not transmissible to additional western choke cherry, and transmission of PPV-D from western choke cherry to peach was only possible before the first CID cycle. Black cherry had intermediate initial infection levels (26.6%) but did not maintain high infection levels after repeated CID cycles. Conversely, American plum had a high level (50%) of initial infection that was not significantly different from initial infection in peach (72.2%) and maintained moderate levels (15 to 25%) of infection and PPV-D transmission to both American plum and peach through all five cycles of CID. Our results indicate that American plum has the greatest potential to act as a reservoir host for Pennsylvania isolates of PPV-D.

Molecular Plant-Plum Pox Virus Interactions.

Plum pox virus, the agent that causes sharka disease, is among the most important plant viral pathogens, affecting Prunus trees across the globe. The fabric of interactions that the virus is able to establish with the plant regulates its life cycle, including RNA uncoating, translation, replication, virion assembly, and movement. In addition, plant-virus interactions are strongly conditioned by host specificities, which determine infection outcomes, including resistance. This review attempts to summarize the latest knowledge regarding Plum pox virus-host interactions, giving a comprehensive overview of their relevance for viral infection and plant survival, including the latest advances in genetic engineering of resistant species.

Occurrence and detection of little cherry virus 1, little cherry virus 2, cherry green ring mottle virus, cherry necrotic rusty mottle virus, and cherry virus A in stone fruit trees in Poland.

To investigate the occurrence of little cherry virus 1 (LChV-1), little cherry virus 2 (LChV-2), cherry green ring mottle virus (CGRMV), cherry necrotic rusty mottle virus (CNRMV), and cherry virus A (CVA) in stone fruit trees in Poland, leaf samples were collected from sweet and sour cherry, peach, and apricot trees. Two sets of primers were used to increase the effectiveness of virus detection. The RT-PCR results indicated that the most frequently detected virus in all of the tested samples was CVA (60%), followed by CGRMV (13%), CNRMV (12%), LChV-1 (11%), and LChV-2 (4%). CVA and CNRMV were not detected in peaches. Mixed infections of these viruses were frequently detected. Keywords: little- cherry virus 1; little cherry virus 2; cherry green ring mottle virus; cherry necrotic rusty mottle virus; cherry virus A; RT-PCR.

Silencing of one copy of the translation initiation factor eIFiso4G in Japanese plum (Prunus salicina) impacts susceptibility to Plum pox virus (PPV) and small RNA production.

BACKGROUND: In plants, host factors encoded by susceptibility (S) genes are indispensable for viral infection. Resistance is achieved through the impairment or the absence of those susceptibility factors. Many S genes have been cloned from model and crop species and a majority of them are coding for members of the eukaryotic translation initiation complex, mainly eIF4E, eIF4G and their isoforms. The aim of this study was to investigate the role of those translation initiation factors in susceptibility of stone fruit species to sharka, a viral disease due to Plum pox virus (PPV). RESULTS: For this purpose, hairpin-inducing silencing constructs based on Prunus persica orthologs were used to generate Prunus salicina (Japanese plum) 4E and 4G silenced plants by Agrobacterium tumefaciens-mediated transformation and challenged with PPV. While down-regulated eIFiso4E transgenic Japanese plums were not regenerated in our conditions, eIFiso4G11-, but not the eIFiso4G10-, silenced plants displayed durable and stable resistance to PPV. We also investigated the alteration of the si- and mi-RNA profiles in transgenic and wild-type Japanese plums upon PPV infection and confirmed that the newly generated small interfering (si) RNAs, which are derived from the engineered inverted repeat construct, are the major contributor of resistance to sharka. CONCLUSIONS: Our results indicate that S gene function of the translation initiation complex isoform is conserved in Prunus species. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of the different isoforms of proteins involved in this complex to breed for resistance to sharka in fruit trees.

Estimation of the dispersal distances of an aphid-borne virus in a patchy landscape.

Characterising the spatio-temporal dynamics of pathogens in natura is key to ensuring their efficient prevention and control. However, it is notoriously difficult to estimate dispersal parameters at scales that are relevant to real epidemics. Epidemiological surveys can provide informative data, but parameter estimation can be hampered when the timing of the epidemiological events is uncertain, and in the presence of interactions between disease spread, surveillance, and control. Further complications arise from imperfect detection of disease and from the huge number of data on individual hosts arising from landscape-level surveys. Here, we present a Bayesian framework that overcomes these barriers by integrating over associated uncertainties in a model explicitly combining the processes of disease dispersal, surveillance and control. Using a novel computationally efficient approach to account for patch geometry, we demonstrate that disease dispersal distances can be estimated accurately in a patchy (i.e. fragmented) landscape when disease control is ongoing. Applying this model to data for an aphid-borne virus (Plum pox virus) surveyed for 15 years in 605 orchards, we obtain the first estimate of the distribution of flight distances of infectious aphids at the landscape scale. About 50% of aphid flights terminate beyond 90 m, which implies that most infectious aphids leaving a tree land outside the bounds of a 1-ha orchard. Moreover, long-distance flights are not rare-10% of flights exceed 1 km. By their impact on our quantitative understanding of winged aphid dispersal, these results can inform the design of management strategies for plant viruses, which are mainly aphid-borne.

The complete nucleotide sequence and genome organization of a novel virus of the order Tymovirales isolated from Prunus davidiana (Carr.) Franch. in Liaoning, China.

In September 2017, a yellow spot leaf disease was noted on the leaves of Prunus davidiana (Carr.) Franch. plants in Liaoning, China, and spherical virions (approx. 30 nm in diameter) were later observed in preparations of symptomatic leaves. Subsequent deep sequencing of small RNA revealed the presence of a virus in these symptomatic leaves The complete genome of this viral isolate consists of 6,072 nucleotides, excluding the poly(A) tail. The virus showed the closest genetic relationship to grapevine-associated tymo-like virus, reported in Colmar, France (GaTLV, MH383239), which is the sole member of the newly proposed genus "Gratylivirus" within the order Tymovirales, which is currently unassigned to a particular family. The virus clustered closely with GaTLV in a phylogenetic tree constructed based on complete genomic sequences. On the basis of the nucleotide and amino acid sequences of the replicase and coat protein genes, this virus shares the highest (although still relatively low) sequence similarity with those of GaTLV (41.6%-60.8% identity), indicating that the virus is a distinct member of the order Tymovirales, for which the name "prunus yellow spot-associated virus" (PYSaV) is proposed. To our knowledge, this is the first report of a virus naturally infecting P. davidiana.

First identification and molecular characterization of a novel cherry robigovirus.

A large contig with sequence similarities to members of the genus Robigovirus was identified by high-throughput sequencing analysis from a symptomless cherry accession. The complete genome sequence of this new virus is 8,384 nucleotides in length, excluding the 3' poly(A) tail. Its genome organization is very similar to those of four known robigoviruses, encoding a putative replicase, three 'triple gene block' proteins, a coat protein, and an unknown protein, 2a. Unlike the four cherry robigoviruses, the new virus does not contain a putative ORF5a. The full-length genome of the virus, which is provisionally named "cherry robigovirus 5" (CRV-5), is 52-57% identical to genome sequences of other robigoviruses. Phylogenetic analysis showed that CRV-5 and other robigoviruses group in a cluster, supporting its assignment to a new species in the genus Robigovirus.

Analyzing the Influence of Landscape Aggregation on Disease Spread to Improve Management Strategies.

Epidemiological models are increasingly used to predict epidemics and improve management strategies. However, they rarely consider landscape characteristics although such characteristics can influence the epidemic dynamics and, thus, the effectiveness of disease management strategies. Here, we present a generic in silico approach which assesses the influence of landscape aggregation on the costs associated with an epidemic and on improved management strategies. We apply this approach to sharka, one of the most damaging diseases of Prunus trees, for which a management strategy is already applied in France. Epidemic simulations were carried out with a spatiotemporal stochastic model under various management strategies in landscapes differing in patch aggregation. Using sensitivity analyses, we highlight the impact of management parameters on the economic output of the model. We also show that the sensitivity analysis can be exploited to identify several strategies that are, according to the model, more profitable than the current French strategy. Some of these strategies are specific to a given aggregation level, which shows that management strategies should generally be tailored to each specific landscape. However, we also identified a strategy that is efficient for all levels of landscape aggregation. This one-size-fits-all strategy has important practical implications because of its simple applicability at a large scale.

Improving Management Strategies of Plant Diseases Using Sequential Sensitivity Analyses.

Improvement of management strategies of epidemics is often hampered by constraints on experiments at large spatiotemporal scales. A promising approach consists of modeling the biological epidemic process and human interventions, which both impact disease spread. However, few methods enable the simultaneous optimization of the numerous parameters of sophisticated control strategies. To do so, we propose a heuristic approach (i.e., a practical improvement method approximating an optimal solution) based on sequential sensitivity analyses. In addition, we use an economic improvement criterion based on the net present value, accounting for both the cost of the different control measures and the benefit generated by disease suppression. This work is motivated by sharka (caused by Plum pox virus), a vector-borne disease of prunus trees (especially apricot, peach, and plum), the management of which in orchards is mainly based on surveillance and tree removal. We identified the key parameters of a spatiotemporal model simulating sharka spread and control and approximated optimal values for these parameters. The results indicate that the current French management of sharka efficiently controls the disease, but it can be economically improved using alternative strategies that are identified and discussed. The general approach should help policy makers to design sustainable and cost-effective strategies for disease management.

Development of a Real-Time RT-PCR for the Universal Detection of LChV1 and Study of the Seasonal Fluctuation of the Viral Titer in Sweet Cherry Cultivars.

Little cherry virus 1 (LChV1) is a sweet cherry pathogen which has lately been reported in other Prunus spp. LChV1 variability makes reliable detection a challenging undertaking. The objective of this work was to develop a rapid, sensitive, and reliable one-tube, real-time reverse-transcription polymerase chain reaction (RT-PCR) for the detection and quantification of LChV1. Primers and a TaqMan probe were designed, using conserved regions of the capsid protein gene. Detection range was evaluated using several divergent viral isolates. The amplification efficiency of the method was estimated at 96.7%, whereas the detection limit was about 100 RNA copies. The protocol was applied in the study of virus fluctuation within leaves and phloem tissue throughout the year and the best periods to test and plant tissues to sample were determined. Comparative analysis of this method with a previously published nested RT-PCR revealed the higher analytical and diagnostic sensitivity of the new test, making it a reliable tool that can be used in routine testing and certification programs.

Discovery of Negative-Sense RNA Viruses in Trees Infected with Apple Rubbery Wood Disease by Next-Generation Sequencing.

Apple rubbery wood is a disease of apple found around the world, often associated with Apple flat limb disease, and regulated in many countries. Despite its long history in apple cultivation, the disease's causal agent has remained elusive. In this study, next-generation sequencing (NGS) was used to identify and characterize several related novel viral agents from apple rubbery wood-infected plants, which have been named Apple rubbery wood virus (ARWV) 1 and 2. Additional specimens with apple rubbery wood disease tested positive by polymerase chain reaction with primers designed to ARWV 1 and 2 genomic RNA segments. In an NGS-based screening of over 100 Malus and 100 Prunus specimens from a collection of virus-infected trees, only one Malus specimen was found to be infected with ARWV not known to be infected with the disease, which strongly suggests that ARWV is not commonly found in Malus spp. or other fruit trees. The two viruses are most closely related to members of the order Bunyavirales. Three RNA segments (large, medium, and small) were characterized and the viruses likely represent a new genus under the family Phenuiviridae, with a suggested name of Rubodvirus (Rubbery wood virus).

Prunus geminivirus A: A Novel Grablovirus Infecting Prunus spp.

Increased use of metagenomics for routine virus diagnosis has led to the characterization of several genus level geminiviruses from tree fruit long thought to exclusively host RNA viruses. In this study, the identification and molecular characterization of a novel geminivirus is reported for the first time in Prunus spp. The virus, provisionally named Prunus geminivirus A (PrGVA), was identified by Illumina sequencing from an asymptomatic plum tree. PrGVA was subsequently confirmed by rolling cycle amplification, cloning, and Sanger sequencing of its complete genome (3,174 to 3,176 nucleotides) from an additional 18 (9 apricot and 9 plum) field isolates. Apart from the nonanucleotide motif TAATATT↓AC present in its virion strand origin of replication, other conserved motifs of PrGVA support its geminiviral origin. PrGVA shared highest complete genome (73 to 74%), coat protein amino acid (83 to 85%) and rep-associated amino acid (74%) identities with Grapevine red blotch virus (GRBV). PrGVA was graft but not mechanically transmissible. Quantitative polymerase chain reaction screening of Prunus spp. in the National Clonal Germplasm Repository collection using newly designed primers and probes revealed 69.4% (apricot), 55.8% (plum), and 8.3% (cherry) incidences of PrGVA. PrGVA is proposed as a novel member of the genus Grablovirus based on its close genome and phylogenetic relationship with GRBV.

An efficient viral vector for functional genomic studies of Prunus fruit trees and its induced resistance to Plum pox virus via silencing of a host factor gene.

RNA silencing is a powerful technology for molecular characterization of gene functions in plants. A commonly used approach to the induction of RNA silencing is through genetic transformation. A potent alternative is to use a modified viral vector for virus-induced gene silencing (VIGS) to degrade RNA molecules sharing similar nucleotide sequence. Unfortunately, genomic studies in many allogamous woody perennials such as peach are severely hindered because they have a long juvenile period and are recalcitrant to genetic transformation. Here, we report the development of a viral vector derived from Prunus necrotic ringspot virus (PNRSV), a widespread fruit tree virus that is endemic in all Prunus fruit production countries and regions in the world. We show that the modified PNRSV vector, harbouring the sense-orientated target gene sequence of 100-200 bp in length in genomic RNA3, could efficiently trigger the silencing of a transgene or an endogenous gene in the model plant Nicotiana benthamiana. We further demonstrate that the PNRSV-based vector could be manipulated to silence endogenous genes in peach such as eukaryotic translation initiation factor 4E isoform (eIF(iso)4E), a host factor of many potyviruses including Plum pox virus (PPV). Moreover, the eIF(iso)4E-knocked down peach plants were resistant to PPV. This work opens a potential avenue for the control of virus diseases in perennial trees via viral vector-mediated silencing of host factors, and the PNRSV vector may serve as a powerful molecular tool for functional genomic studies of Prunus fruit trees.

Sugars and organic acids in plum fruit affected by Plum pox virus.

BACKGROUND: Plum pox virus (PPV) causes severe economic losses in stone fruit production, but little is known about its effect on plum fruit composition. In this study, the influence of PPV on sugars and organic acids was evaluated in a susceptible plum (Prunus domestica L.) cultivar. RESULTS: PPV infection significantly affected the content and composition of sugars and organic acids. The composition of necrotic tissue was modified the most. A short-time infected tree yielded fruit with similar sugar composition to fruit from a healthy tree, but the decline of organic acids was faster. Prematurely ripened symptomatic fruit had reduced fruit weight and low sugar content. CONCLUSION: Infected trees of the studied cultivar produce fruit of inferior quality. Fruits are not suitable for processing, especially when most of them exhibit visual symptoms of PPV infection. © 2016 Society of Chemical Industry.

Complete genome sequence of three tomato ringspot virus isolates: evidence for reassortment and recombination.

The genome sequence of tomato ringspot virus (ToRSV, a subgroup C nepovirus) is currently available for one raspberry isolate. In this study, we describe the complete genome sequence of three additional isolates from raspberry (Rasp1-2014), grapevine (GYV-2014) and prunus (13C280). The degree of nucleotide sequence identity shared between RNA1 and RNA2 in the 5'-terminal 900 nucleotides and 3' untranslated region varied from 98-99 % (13C280, GYV-2014) to 80 % (Rasp1-2014). Phylogenetic studies revealed distinct origins for Rasp1-2014 RNA1 and RNA2, suggesting reassortment. Two recombination events were also identified in the 3' UTR and 5'-terminal region of RNA1.

Assessing the Mismatch Between Incubation and Latent Periods for Vector-Borne Diseases: The Case of Sharka.

The relative durations of the incubation period (the time between inoculation and symptom expression) and of the latent period (the time between inoculation and infectiousness of the host) are poorly documented for plant diseases. However, the extent of asynchrony between the ends of these two periods (i.e., their mismatch) can be a key determinant of the epidemic dynamics for many diseases and consequently it is of primary interest in the design of disease management strategies. In order to assess this mismatch, an experimental approach was developed and applied using sharka, a severe disease caused by Plum pox virus (PPV, genus Potyvirus, family Potyviridae) affecting trees belonging to the genus Prunus. Leaves of infected young peach trees were used individually as viral sources in aphid-mediated transmission tests carried out at different time points postinoculation in order to bracket symptom onset. By fitting a nonlinear logistic model to the obtained transmission rates, we demonstrated that the first symptoms appear on leaves 1 day before they rapidly become infectious. In addition, among symptomatic leaves, symptom intensity and transmission rate are positively correlated. These results strengthen the conclusion that, under our experimental conditions, incubation and latent periods of PPV infection are almost synchronous.

Genomic analyses of cherry rusty mottle group and cherry twisted leaf-associated viruses reveal a possible new genus within the family betaflexiviridae.

It is demonstrated that closely related viruses within the family Betaflexiviridae are associated with a number of diseases that affect sweet cherry (Prunus avium) and other Prunus spp. Cherry rusty mottle-associated virus (CRMaV) is correlated with the appearance of cherry rusty mottle disease (CRMD), and Cherry twisted leaf-associated virus (CTLaV) is linked to cherry twisted leaf disease (CTLD) and apricot ringpox disease (ARPD). Comprehensive analysis of previously reported full genomic sequences plus those determined in this study representing isolates of CTLaV, CRMaV, Cherry green ring mottle virus, and Cherry necrotic rusty mottle virus revealed segregation of sequences into four clades corresponding to distinct virus species. High-throughput sequencing of RNA from representative source trees for CRMD, CTLD, and ARPD did not reveal additional unique virus sequences that might be associated with these diseases, thereby further substantiating the association of CRMaV and CTLaV with CRMD and CTLD or ARPD, respectively. Based on comparison of the nucleotide and amino acid sequence identity values, phylogenetic relationships with other triple-gene block-coding viruses within the family Betaflexiviridae, genome organization, and natural host range, a new genus (Robigovirus) is suggested.

Detection of a New Luteovirus in Imported Nectarine Trees: A Case Study to Propose Adoption of Metagenomics in Post-Entry Quarantine.

In spring 2013, 5-year-old nectarine (Prunus persica) trees, grafted on peach rootstock Nemaguard, were found stunted in a propagation block in California. These trees had been propagated from budwood of three nectarine cultivars imported from France and cleared through the post-entry quarantine procedure. Examination of the canopy failed to reveal any obvious symptoms. However, examination of the trunks, after stripping the bark, revealed extensive pitting on the woody cylinder. To investigate the etiological agent, double-stranded RNA was extracted from bark scrapings from the scion and rootstock portions, and a cDNA library was prepared and sequenced using the Illumina platform. BLAST analysis of the contigs generated by the de novo assembly of sequence reads indicated the presence of a novel luteovirus. Complete sequence of the viral genome was determined by sequencing of three overlapping cDNA clones generated by reverse transcription-polymerase chain reaction (RT-PCR) and by rapid amplification of the 5'- and 3'-termini. The virus genome was comprised of 4,991 nucleotides with a gene organization similar to members of the genus Luteovirus (family Luteoviridae). The presence of the virus, tentatively named Nectarine stem pitting-associated virus, was confirmed in symptomatic trees by RT-PCR. Discovery of a new virus in nectarine trees after post-entry quarantine indicates the importance of including (i) metagenomic analysis by next-generation sequencing approach as an essential tool to assess the plant health status, and (ii) examination of the woody cylinders as part of the indexing process.