Detection and genetic variability of newly identified dasheen mosaic virus in Poland

Dasheen mosaic virus (DsMV) is one of the most important viral pathogens of aroids and can cause major economic losses for ornamental crops. Here, we present the detection and molecular characterisation of DsMV isolates originating from Monstera adansonii plants in Poland. Moreover, the genetic variability of DsMV isolates was analyzed based on the coat protein gene ( CP) of the Polish and other DsMV isolates described to date. The presence of DsMV was confirmed by transmission electron microscopy (TEM) and reverse transcription polymerase chain reaction (RT-PCR) with specific, diagnostic primers in three out of ten examined plants. To obtain full-length sequences of CP, two pairs of primers were designed and used in the RT-PCR. The specificity of obtained products was confirmed by Sanger sequencing. The obtained sequences of CP were compared with 44 other DsMV sequences retrieved from the GenBank. Analyses revealed that DsMV population is very diverse. The variability of DsMV isolates was confirmed by low sequence identity and pervasive recombination events. The phylogenetic analysis was performed based on 37 non-recombinant CP sequences. The maximum-likelihood reconstruction revealed that the Polish isolates are distinct and grouped separately from other DsMV isolates. Due to the high genetic diversity, detecting the virus could be difficult. Nonetheless disease management relies strongly on a fast and accurate identification of the causal agent. To our knowledge this is the first report of DsMV in Poland

Molecular evolution of tomato black ring virus and de novo generation of a new type of defective RNAs during long-term passaging in different hosts

Tomato black ring virus (TBRV) is a worldwide-distributed RNA virus infecting a wide range of different host plants, including crop species, trees, shrubs, and weeds. Here, we investigated the molecular evolution of TBRV and its adaptability to different plant species. The TBRV-Pi isolate was used to generate five independent evolution lineages serially passaged in either quinoa, tobacco, or tomato plants. After 15 passages, the genetic variability present in all the lineages was characterized for the movement (MP) and coat (CP) coding cistrons. We addressed two main questions: to what extent does the amount of genetic variability in the TBRV genome depend on the host species, and are there host species-specific adaptive mutations? Overall, 201 different nucleotide substitutions emerged during the evolution experiment, with some of them appearing multiple times in different lineages; two of them (one in CP and one in MP) were unique for a particular host plant. We have shown that the degree of genetic differentiation depends on the host species in which the virus evolved, and that positive selection is operating upon certain residues, particularly in CP. Moreover, we have characterized new types of defective RNAs that arose during the TBRV-Pi evolution in tobacco. Furthermore, this is the first report of a defective RNA from the RNA2 of TBRV.Work in Poznań was supported by project 2015/17/B/NZ8/02407 from the National Science Centre in Poland to B.H.J. Work in València was supported by grant SPID201900X03998IV1 from Spain Ministerio de Ciencia e Innovación – FEDER to S.F.E.Peer reviewe

Rapid evolutionary dynamics of the Pepino mosaic virus – status and future perspectives

Pepino mosaic virus (PepMV) has emerged as an important pathogen of greenhouse tomato crops and is currently distributed worldwide. Population genetic studies have revealed a shift in the dominant PepMV genotype from European (EU) to Chilean 2 (CH2) in North America and several European countries. New genetic variants are constantly being created by mutation and recombination events. Single nucleotide substitutions in different parts of the genome were found to affect on development of symptoms resulting in new pathotypes and accumulation of viral RNA. The variability of the PepMV population has a great impact on designing specific diagnostic tools and developing efficient and durable strategies of disease control. In this paper we review the current knowledge about the PepMV population, the evolutionary dynamics of this highly infective virus, methods for its detection and plant protection strategies

Defective RNA particles of Tomato black ring virus: origin, structure and biological effect

Resumen del trabajo presentado a la Conferencia Power of Viruses, celebrada en Poreč (Croatia) del 16 al 18 de mayo de 2018.Tomato black ring virus (TBRV) infects a wide range of economically important plants, and is distributed worldwide. The genome of TBRV consists of two single stranded RNAs of positive polarity. Both RNAs contain a small protein VPg at their S' ends and have polyadenylated 3' ends. RNA1 is responsible for viral replication and polyproteins' processing and RNA2 for encapsidation and movement in plant. The TBRV infection can be accompanied by subviral particles such as satellite or defective RNAs. Defective RNA particles are deletion/and/or rearrangement variants of the viral genomes created during replication. The presence of subviral RNAs might have a great impact on viral replication, accumulation and symptoms observed on infected plants. D RNAs which are referred to interfere with multiplication of their helper viruses are called defective interfering RNAs (DI RNAs). TBRV is the only member of the Nepovirus genus that is known to form defective RNA particles (D RNAs) during replication. In this study, we analyzed the origin and structure of D RNAs generated de novo during prolonged passages of TBRV isolates in Chenopodium quinoa plants. Moreover, the effect of D RNAs on helper virus replication and symptoms observed on infected plants was estimated. Two TBRV isolates, originated from Solanum lycopersicum and Lactuca sativa, were serially passaged in C. quinoa and after 1S passages the purified viral preparations were obtained. Viral RNA was isolated using phenol-chloroform method. The analysis of RNA profile revealed the presence of short, D RNA molecules. D RNAs were amplified and sequenced, and obtained sequences were compared with the helper virvs genome. C. quinoa, S. lycopersicum and Nicotiana tabacum were infected.with TBRV+D RNA and TBRV-D RNA. The symptoms and viral accumulation were manito red 7, 14, 21, and 28 dpi. The accumulation level of TBRV in each host was measured by RT-qPCR and statistical analyses were performed. The analysis revealed that D RNAs derived by interna! deletion in the RNA1 molecule of TBRV and contained a portian of S' non-coding region, a fragment of polymerase gene and almost entire 3' non-coding region. Plants infected with TBRV+D RNA displayed milder symptoms in comparison to those infected with TBRV-D RNA. Statistical analyses confirmed that D RNAs interfere with TBRV replication and thus should be referred to as defective interfering particles.Peer reviewe

High-Throughput Sequencing Facilitates Discovery of New Plant Viruses in Poland

Viruses cause epidemics on all major crops of agronomic importance, and a timely and accurate identification is essential for control. High throughput sequencing (HTS) is a technology that allows the identification of all viruses without prior knowledge on the targeted pathogens. In this paper, we used HTS technique for the detection and identification of different viral species occurring in single and mixed infections in plants in Poland. We analysed various host plants representing different families. Within the 20 tested samples, we identified a total of 13 different virus species, including those whose presence has not been reported in Poland before: clover yellow mosaic virus (ClYMV) and melandrium yellow fleck virus (MYFV). Due to this new finding, the obtained sequences were compared with others retrieved from GenBank. In addition, cucurbit aphid-borne yellows virus (CABYV) was also detected, and due to the recent occurrence of this virus in Poland, a phylogenetic analysis of these new isolates was performed. The analysis revealed that CABYV population is highly diverse and the Polish isolates of CABYV belong to two different phylogenetic groups. Our results showed that HTS-based technology is a valuable diagnostic tool for the identification of different virus species originating from variable hosts, and can provide rapid information about the spectrum of plant viruses previously not detected in a region

Genetic diversity of tomato black ring virus satellite RNAs and their impact on virus replication

This article belongs to the Collection State-of-the-Art Molecular Microbiology in Poland.Viral satellite RNAs (satRNAs) are small subviral particles that are associated with the genomic RNA of a helper virus (HV). Their replication, encapsidation, and movement depend on the HV. In this paper, we performed a global analysis of the satRNAs associated with different isolates of tomato black ring virus (TBRV). We checked the presence of satRNAs in 42 samples infected with TBRV, performed recombination and genetic diversity analyses, and examined the selective pressure affecting the satRNAs population. We identified 18 satRNAs in total that differed in length and the presence of point mutations. Moreover, we observed a strong effect of selection operating upon the satRNA population. We also constructed infectious cDNA clones of satRNA and examined the viral load of different TBRV isolates in the presence and absence of satRNAs, as well as the accumulation of satRNA molecules on infected plants. Our data provide evidence that the presence of satRNAs significantly affects viral load; however, the magnitude of this effect differs among viral isolates and plant hosts. We also showed a positive correlation between the number of viral genomic RNAs (gRNAs) and satRNAs for two analysed TBRV isolates.This study was supported from the projects 2017/25/B/NZ9/01715 from National Science Centre of Poland to B.H.-J. and PID2019-103998GB-I00 from Spain Agencia Estatal de Investigación to S.F.E.Peer reviewe

Occurrence, Genetic Variability of Tomato Yellow Ring Orthotospovirus Population and the Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Its Rapid Detection

Tomato-infecting viruses have been considered as a serious threat to tomato crops in Poland. Therefore, during 2014–2021, 234 tomato samples delivered directly by greenhouse tomato growers to Plant Disease Clinic of IPP-NRI were tested. Eight virus species: pepino mosaic virus (PepMV), tomato yellow ring orthotospovirus (TYRV), tomato spotted wilt orthotospovirus (TSWV), potato virus Y (PVY), cucumber mosaic virus (CMV), tomato black ring virus (TBRV) and tomato mosaic virus (ToMV) were detected in single or mixed infection in 89 samples. The presence of TYRV was established for the first time in Poland in 2014. Since then, its presence has been observed in single and mixed infection with TSWV and CMV. Here, we analysed the genetic variability of TYRV population based on complete nucleocapsid (N) protein gene sequence of 55 TYRV isolates. Maximum-likelihood reconstruction revealed the presence of three distinct, well-supported phylogroups. Moreover, the effect of host species on virus diversity was confirmed. Therefore, RT-LAMP assay was developed for the rapid and efficient detection of TYRV isolates that can be implemented in field and greenhouse conditions

Molecular evolution of tomato black ring virus and de novo generation of a new type of defective RNAs during long‐term passaging in different hosts

Tomato black ring virus (TBRV) is a worldwide-distributed RNA virus infecting a wide range of different host plants, including crop species, trees, shrubs, and weeds. Here, we investigated the molecular evolution of TBRV and its adaptability to different plant species. The TBRV-Pi isolate was used to generate five independent evolution lineages serially passaged in either quinoa, tobacco, or tomato plants. After 15 passages, the genetic variability present in all the lineages was characterized for the movement (MP) and coat (CP) coding cistrons. We addressed two main questions: to what extent does the amount of genetic variability in the TBRV genome depend on the host species, and are there host species-specific adaptive mutations? Overall, 201 different nucleotide substitutions emerged during the evolution experiment, with some of them appearing multiple times in different lineages; two of them (one in CP and one in MP) were unique for a particular host plant. We have shown that the degree of genetic differentiation depends on the host species in which the virus evolved, and that positive selection is operating upon certain residues, particularly in CP. Moreover, we have characterized new types of defective RNAs that arose during the TBRV-Pi evolution in tobacco. Furthermore, this is the first report of a defective RNA from the RNA2 of TBRV.Work in Poznań was supported by project 2015/17/B/NZ8/02407 from the National Science Centre in Poland to B.H.J. Work in València was supported by grant SPID201900X03998IV1 from Spain Ministerio de Ciencia e Innovación – FEDER to S.F.E.Peer reviewe

Rapid evolutionary dynamics of the Pepino mosaic virus – status and future perspectives

Pepino mosaic virus (PepMV) has emerged as an important pathogen of greenhouse tomato crops and is currently distributed worldwide. Population genetic studies have revealed a shift in the dominant PepMV genotype from European (EU) to Chilean 2 (CH2) in North America and several European countries. New genetic variants are constantly being created by mutation and recombination events. Single nucleotide substitutions in different parts of the genome were found to affect on development of symptoms resulting in new pathotypes and accumulation of viral RNA. The variability of the PepMV population has a great impact on designing specific diagnostic tools and developing efficient and durable strategies of disease control. In this paper we review the current knowledge about the PepMV population, the evolutionary dynamics of this highly infective virus, methods for its detection and plant protection strategies