Identification of a new nucleopolyhedrovirus isolated from the olive leaf moth, Palpita vitrealis, from two locations in Egypt.

The olive leaf moth (jasmine moth), Palpita vitrealis (Lepidoptera: Crambidae), is an important insect pest of olives in several Mediterranean countries. A new alphabaculovirus was isolated from diseased larvae of P. vitrealis in Egypt, first in Giza in spring 2005 and again in Marsa Matrouh in 2019.The larvae exhibited typical symptoms of a baculovirus infection. Light and scanning electron microscopy studies revealed polyhedral occlusion bodies. Transmission electron microscopy of ultrathin sections of purified OBs revealed virions with multiple embedded nucleocapsids. The identity of the two virus isolates was confirmed by sequencing the partial polyhedrin and lef-8 genes, and sequence comparison suggested a relationship to group I alphabaculoviruses. Therefore, this virus was termed Palpita vitrealis nucleopolyhedrovirus (PaviNPV). Whole genome sequencing of the PaviNPV isolate from Giza (Gz05) revealed a genome of 117,533 bp, 131 open reading frames (ORFs) and four homologous repeat (hr) regions. Phylogenetic reconstruction and genetic distance analyses using 38 core genes indicated that PaviNPV should be considered to belong to a novel species within the genus Alphabaculovirus. In bioassays, PaviNPV was highly virulent against second-instar larvae of P. vitrealis. The study reports a novel baculovirus that might have potential as a biological control agent of the olive leaf moth.

Biological and Genetic Characterization of Physostegia Chlorotic Mottle Virus in Europe Based on Host Range, Location, and Time.

Application of high throughput sequencing (HTS) technologies enabled the first identification of Physostegia chlorotic mottle virus (PhCMoV) in 2018 in Austria. Subsequently, PhCMoV was detected in Germany and Serbia on tomatoes showing severe fruit mottling and ripening anomalies. We report here how prepublication data-sharing resulted in an international collaboration across eight laboratories in five countries, enabling an in-depth characterization of PhCMoV. The independent studies converged toward its recent identification in eight additional European countries and confirmed its presence in samples collected 20 years ago (2002). The natural plant host range was expanded from two to nine species across seven families, and we confirmed the association of PhCMoV presence with severe fruit symptoms on economically important crops such as tomato, eggplant, and cucumber. Mechanical inoculations of selected isolates in the greenhouse established the causality of the symptoms on a new indexing host range. In addition, phylogenetic analysis showed a low genomic variation across the 29 near-complete genome sequences available. Furthermore, a strong selection pressure within a specific ecosystem was suggested by nearly identical sequences recovered from different host plants through time. Overall, this study describes the European distribution of PhCMoV on multiple plant hosts, including economically important crops on which the virus can cause severe fruit symptoms. This work demonstrates how to efficiently improve knowledge on an emergent pathogen by sharing HTS data and provides a solid knowledge foundation for further studies on plant rhabdoviruses.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

ICTV Virus Taxonomy Profile: Caulimoviridae.

Caulimoviridae is a family of non-enveloped reverse-transcribing plant viruses with non-covalently closed circular dsDNA genomes of 7.1-9.8 kbp in the order Ortervirales. They infect a wide range of monocots and dicots. Some viruses cause economically important diseases of tropical and subtropical crops. Transmission occurs through insect vectors (aphids, mealybugs, leafhoppers, lace bugs) and grafting. Activation of infectious endogenous viral elements occurs in Musa balbisiana, Petunia hybrida and Nicotiana edwardsonii. However, most endogenous caulimovirids are not infectious. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Caulimoviridae, which is available at ictv.global/report/caulimoviridae.

Complete genome sequence and construction of an infectious full-length cDNA clone of celery latent virus - an unusual member of a putative new genus within the Potyviridae.

Celery latent virus (CeLV) is an incompletely described plant virus known to be sap and seed transmissible and to possess flexuous filamentous particles measuring about 900 nm in length, suggesting it as a possible member of the family Potyviridae. Here, an Italian isolate of CeLV was transmitted by sap to a number of host plants and shown to have a single-stranded and monopartite RNA genome being 11 519 nucleotides (nts) in size and possessing some unusual features. The RNA contains a large open reading frame (ORF) that is flanked by a short 5' untranslated region (UTR) of 13 nt and a 3' UTR consisting of 586 nt that is not polyadenylated. CeLV RNA shares nt sequence identity of only about 40 % with other members of the Potyviridae (potyvirids). The CeLV polyprotein is notable in that it starts with a signal peptide, has a putative P3N-PIPO ORF and shares low aa sequence identity (about 18 %) with other potyvirids. Although potential cleavage sites were not identified for the N-terminal two-thirds of the polyprotein, the latter possesses a number of sequence motifs, the identity and position of which are characteristic of other potyvirids. Attempts at constructing an infectious full-length cDNA clone of CeLV were successful following Rhizobium radiobacter infiltration of Nicotiana benthamiana and Apium graveolens. CeLV appears to have the largest genome of all known potyvirids and some unique genome features that may warrant the creation of a new genus, for which we propose the name 'celavirus'.

Characterisation of a novel nucleorhabdovirus infecting alfalfa (Medicago sativa).

BACKGROUND: Nucleorhabdoviruses possess bacilliform particles which contain a single-stranded negative-sense RNA genome. They replicate and mature in the nucleus of infected cells. Together with viruses of three other genera of the family Rhabdoviridae, they are known to infect plants and can be transmitted by arthropod vectors, during vegetative propagation, or by mechanical means. In 2010, an alfalfa (Medicago sativa) plant showing virus-like symptoms was collected from Stadl-Paura, Austria and sent to Julius Kühn Institute for analysis. METHODS: Electron microscopy (EM) of leaf extracts from infected plants revealed the presence of rhabdovirus-like particles and was further used for ultrastructural analyses of infected plant tissue. Partially-purified preparations of rhabdovirus nucleocapsids were used for raising an antiserum. To determine the virus genome sequence, high throughput sequencing (HTS) was performed. RT-PCR primers were designed to confirm virus infection and to be used as a diagnostic tool. RESULTS: EM revealed bacilliform virions resembling those of plant-infecting rhabdoviruses. HTS of ribosomal RNA-depleted total RNA extracts revealed a consensus sequence consisting of 13,875 nucleotides (nt) and containing seven open reading frames (ORFs). Homology and phylogenetic analyses suggest that this virus isolate represents a new species of the genus Nucleorhabdovirus (family Rhabdoviridae). Since the virus originated from an alfalfa plant in Austria, the name alfalfa-associated nucleorhabdovirus (AaNV) is proposed. Viroplasms (Vp) and budding virions were observed in the nuclei of infected cells by EM, thus confirming its taxonomic assignment based on sequence data. CONCLUSIONS: In this study, we identified and characterised a new nucleorhabdovirus from alfalfa. It shared only 39.8% nucleotide sequence identity with its closest known relative, black currant-associated rhabdovirus 1. The virus contains an additional open reading frame (accessory gene) with unknown function, located between the matrix protein and the glycoprotein genes. Serological and molecular diagnostic assays were designed for future screening of field samples. Further studies are needed to identify other natural hosts and potential vectors.

A divergent strain of melon chlorotic spot virus isolated from black medic (Medicago lupulina) in Austria.

A tenuivirus, referred to here as JKI 29327, was isolated from a black medic (Medicago lupulina) plant collected in Austria. The virus was mechanically transmitted to Nicotiana benthamiana, M. lupulina, M. sativa, Pisum sativum and Vicia faba. The complete genome was determined by high throughput sequencing. The genome of JKI 29327 consists of eight RNA segments closely related to those of melon chlorotic spot virus (MeCSV) isolate E11-018 from France. Since segments RNA 7 and 8 of JKI 29327 are shorter, its genome is slightly smaller (by 247 nts) than that of E11-018. Pairwise comparisons between the predicted virus proteins of JKI 29327 and their homologues in E11-018 showed aa identities ranging from 80.6 to 97.2%. Plants infected with E11-081 gave intermediate DAS-ELISA reactions with polyclonal antibodies to JKI 29327. Since JKI 29327 and E11-018 appear to be closely related both serologically and genetically, we propose to regard JKI 29327 as the black medic strain of MeCSV. To our knowledge, JKI 29327 represents the second tenuivirus identified from a dicotyledonous plant. Serological and molecular diagnostic methods were developed for future detection.

Caraway yellows virus, a novel nepovirus from Carum carvi.

A novel nepovirus was identified and characterised from caraway, and tentatively named caraway yellows virus (CawYV). Tubular structures with isomeric virus particles typical for nepoviruses were observed in infected tissues by electron microscopy. The whole genome of CawYV was identified by high throughput sequencing (HTS). It consists of two segments with 8026 nt for RNA1 and 6405 nt for RNA2, excluding the poly(A) tails. CawYV-RNA1 shared closest nt identity to peach rosette mosaic virus (PRMV) with 63%, while RNA2 shared 41.5% with blueberry latent spherical virus (BLSV). The amino acid sequences of the CawYV protease-polymerase (Pro-Pol) and capsid protein (CP) regions share the highest identities with those of the subgroup C nepoviruses. The Pro-Pol region shared highest aa identity with PRMV (80.1%), while the CP region shared 39.6% to soybean latent spherical virus. Phylogenetic analysis of the CawYV-Pro-Pol and -CP aa sequences provided additional evidence of their association with nepoviruses subgroup C. Based on particle morphology, genomic organization and phylogenetic analyses, we propose CawYV as a novel species within the genus Nepovirus subgroup C.

Mycoviruses Increase the Attractiveness of Fusarium graminearum for Fungivores and Suppress Production of the Mycotoxin Deoxynivalenol.

RNA viruses of the genera Ambivirus, Mitovirus, Sclerotimonavirus, and Partitivirus were found in a single isolate of Fusarium graminearum. The genomes of the mitovirus, sclerotimonavirus, and partitivirus were assigned to previously described viruses, whereas the ambivirus genome putatively represents a new species, named Fusarium graminearum ambivirus 1 (FgAV1). To investigate the effect of mycoviruses on the fungal phenotype, the spontaneous loss of mycoviruses during meiosis and the transmission of mycoviruses into a new strain via anastomosis were used to obtain isogenic F. graminearum strains both with and without mycoviruses. Notable effects observed in mycovirus-harboring strains were (i) the suppression of the synthesis of trichothecene mycotoxins and their precursor trichodiene, (ii) the suppression of the synthesis of the defense compound aurofusarin, (iii) the stimulation of the emission of 2-methyl-1-butanol and 3-methyl-1-butanol, and (iv) the increased attractiveness of fungal mycelia for fungivorous collembolans. The increased attractiveness of mycovirus-infected filamentous fungi to animal predators opens new perspectives on the ecological implications of the infection of fungi with viruses.

The nature and organization of satellite DNAs in Petunia hybrida, related, and ancestral genomes.

Introduction: The garden petunia, Petunia hybrida (Solanaceae) is a fertile, diploid, annual hybrid species (2n=14) originating from P. axillaris and P. inflata 200 years ago. To understand the recent evolution of the P. hybrida genome, we examined tandemly repeated or satellite sequences using bioinformatic and molecular cytogenetic analysis. Methods: Raw reads from available genomic assemblies and survey sequences of P. axillaris N (PaxiN), P. inflata S6, (PinfS6), P. hybrida (PhybR27) and the here sequenced P. parodii S7 (PparS7) were used for graph and k-mer based cluster analysis of TAREAN and RepeatExplorer. Analysis of repeat specific monomer lengths and sequence heterogeneity of the major tandem repeat families with more than 0.01% genome proportion were complemented by fluorescent in situ hybridization (FISH) using consensus sequences as probes to chromosomes of all four species. Results: Seven repeat families, PSAT1, PSAT3, PSAT4, PSAT5 PSAT6, PSAT7 and PSAT8, shared high consensus sequence similarity and organisation between the four genomes. Additionally, many degenerate copies were present. FISH in P. hybrida and in the three wild petunias confirmed the bioinformatics data and gave corresponding signals on all or some chromosomes. PSAT1 is located at the ends of all chromosomes except the 45S rDNA bearing short arms of chromosomes II and III, and we classify it as a telomere associated sequence (TAS). It is the most abundant satellite repeat with over 300,000 copies, 0.2% of the genomes. PSAT3 and the variant PSAT7 are located adjacent to the centromere or mid-arm of one to three chromosome pairs. PSAT5 has a strong signal at the end of the short arm of chromosome III in P. axillaris and P.inflata, while in P. hybrida additional interstitial sites were present. PSAT6 is located at the centromeres of chromosomes II and III. PSAT4 and PSAT8 were found with only short arrays. Discussion: These results demonstrate that (i) repeat families occupy distinct niches within chromosomes, (ii) they differ in the copy number, cluster organization and homogenization events, and that (iii) the recent genome hybridization in breeding P. hybrida preserved the chromosomal position of repeats but affected the copy number of repetitive DNA.

Host range and molecular and ultrastructural analyses of Asparagus virus 1 pathotypes isolated from garden asparagus Asparagus officinalis L.

Asparagus samples were examined from growing areas of Germany and selected European as well as North, Central and South American countries. Overall, 474 samples were analyzed for Asparagus virus 1 (AV1) using DAS-ELISA. In our survey, 19 AV1 isolates were further characterized. Experimental transmission to 11 species belonging to Aizoaceae, Amarantaceae, Asparagaceae, and Solanaceae succeeded. The ultrastructure of AV1 infection in asparagus has been revealed and has been compared with the one in indicator plants. The cylindrical inclusion (CI) protein, a core factor in viral replication, localized within the cytoplasm and in systemic infections adjacent to the plasmodesmata. The majority of isolates referred to pathotype I (PI). These triggered a hypersensitive resistance in inoculated leaves of Chenopodium spp. and were incapable of infecting Nicotiana spp. Only pathotype II (PII) and pathotype III (PIII) infected Nicotiana benthamiana systemically but differed in their virulence when transmitted to Chenopodium spp. The newly identified PIII generated amorphous inclusion bodies and degraded chloroplasts during systemic infection but not in local lesions of infected Chenopodium spp. PIII probably evolved via recombination in asparagus carrying a mixed infection by PI and PII. Phylogeny of the coat protein region recognized two clusters, which did not overlap with the CI-associated grouping of pathotypes. These results provide evidence for ongoing modular evolution of AV1.

To Be Seen or Not to Be Seen: Latent Infection by Tobamoviruses.

Tobamoviruses are among the most well-studied plant viruses and yet there is still a lot to uncover about them. On one side of the spectrum, there are damage-causing members of this genus: such as the tobacco mosaic virus (TMV), tomato brown rugose fruit virus (ToBRFV) and cucumber green mottle mosaic virus (CGMMV), on the other side, there are members which cause latent infection in host plants. New technologies, such as high-throughput sequencing (HTS), have enabled us to discover viruses from asymptomatic plants, viruses in mixed infections where the disease etiology cannot be attributed to a single entity and more and more researchers a looking at non-crop plants to identify alternative virus reservoirs, leading to new virus discoveries. However, the diversity of these interactions in the virosphere and the involvement of multiple viruses in a single host is still relatively unclear. For such host-virus interactions in wild plants, symptoms are not always linked with the virus titer. In this review, we refer to latent infection as asymptomatic infection where plants do not suffer despite systemic infection. Molecular mechanisms related to latent behavior of tobamoviruses are unknown. We will review different studies which support different theories behind latency.

Participation of Multifunctional RNA in Replication, Recombination and Regulation of Endogenous Plant Pararetroviruses (EPRVs).

Pararetroviruses, taxon Caulimoviridae, are typical of retroelements with reverse transcriptase and share a common origin with retroviruses and LTR retrotransposons, presumably dating back 1.6 billion years and illustrating the transition from an RNA to a DNA world. After transcription of the viral genome in the host nucleus, viral DNA synthesis occurs in the cytoplasm on the generated terminally redundant RNA including inter- and intra-molecule recombination steps rather than relying on nuclear DNA replication. RNA recombination events between an ancestral genomic retroelement with exogenous RNA viruses were seminal in pararetrovirus evolution resulting in horizontal transmission and episomal replication. Instead of active integration, pararetroviruses use the host DNA repair machinery to prevail in genomes of angiosperms, gymnosperms and ferns. Pararetrovirus integration - leading to Endogenous ParaRetroViruses, EPRVs - by illegitimate recombination can happen if their sequences instead of homologous host genomic sequences on the sister chromatid (during mitosis) or homologous chromosome (during meiosis) are used as template. Multiple layers of RNA interference exist regulating episomal and chromosomal forms of the pararetrovirus. Pararetroviruses have evolved suppressors against this plant defense in the arms race during co-evolution which can result in deregulation of plant genes. Small RNAs serve as signaling molecules for Transcriptional and Post-Transcriptional Gene Silencing (TGS, PTGS) pathways. Different populations of small RNAs comprising 21-24 nt and 18-30 nt in length have been reported for Citrus, Fritillaria, Musa, Petunia, Solanum and Beta. Recombination and RNA interference are driving forces for evolution and regulation of EPRVs.

Bacteria isolated from the cuticle of plant-parasitic nematodes attached to and antagonized the root-knot nematode Meloidogyne hapla.

Plant-parasitic nematodes are associated with specifically attached soil bacteria. To investigate these bacteria, we employed culture-dependent methods to isolate a representative set of strains from the cuticle of the infective stage (J2) of the root-knot nematode Meloidogyne hapla in different soils. The bacteria with the highest affinity to attach to J2 belonged to the genera Microbacterium, Sphingopyxis, Brevundimonas, Acinetobacter, and Micrococcus as revealed by 16S rRNA gene sequencing. Dynamics of the attachment of two strains showed fast adhesion in less than two hours, and interspecific competition for attachment sites. Isolates from the cuticle of M. hapla J2 attached to the lesion nematode Pratylenchus penetrans, and vice versa, suggesting similar attachment sites on both species. Removal of the surface coat by treatment of J2 with the cationic detergent CTAB reduced bacterial attachment, but did not prevent it. Some of the best attaching bacteria impaired M. hapla performance in vitro by significantly affecting J2 mortality, J2 motility and egg hatch. Most of the tested bacterial attachers significantly reduced the invasion of J2 into tomato roots, suggesting their beneficial role in soil suppressiveness against M. hapla.

Phage tail-like particles are versatile bacterial nanomachines - A mini-review.

Type VI secretion systems and tailocins, two bacterial phage tail-like particles, have been reported to foster interbacterial competition. Both nanostructures enable their producer to kill other bacteria competing for the same ecological niche. Previously, type VI secretion systems and particularly R-type tailocins were considered highly specific, attacking a rather small range of competitors. Their specificity is conferred by cell surface receptors of the target bacterium and receptor-binding proteins on tailocin tail fibers and tail fiber-like appendages of T6SS. Since many R-type tailocin gene clusters contain only one tail fiber gene it was appropriate to expect small R-type tailocin target ranges. However, recently up to three tail fiber genes and broader target ranges have been reported for one plant-associated Pseudomonas strain. Here, we show that having three tail fiber genes per R-type tailocin gene cluster is a common feature of several strains of Gram-negative (often plant-associated) bacteria of the genus Kosakonia. Knowledge about the specificity of type VI secretion systems binding to target bacteria is even lower than in R-type tailocins. Although the mode of operation implicated specific binding, it was only published recently that type VI secretion systems develop tail fiber-like appendages. Here again Kosakonia, exhibiting up to three different type VI secretion systems, may provide valuable insights into the antagonistic potential of plant-associated bacteria. Current understanding of the diversity and potential of phage tail-like particles is fragmentary due to various synonyms and misleading terminology. Consistency in technical terms is a precondition for concerted and purposeful research, which precedes a comprehensive understanding of the specific interaction between bacteria producing phage tail-like particles and their targets. This knowledge is fundamental for selecting and applying tailored, and possibly engineered, producer bacteria for antagonizing plant pathogenic microorganisms.

Endogenous pararetroviruses: two-faced travelers in the plant genome.

Endogenous plant pararetroviruses (EPRVs) were identified as integrated counterparts of most members of the plant virus family Caulimoviridae and represent repetitive elements that are ubiquitous in the plant kingdom. They are often located in pericentromeric regions of plant chromosomes in the vicinity of retrotransposon sequences. Depending on their structure and sequence integrity, some EPRVs are able to replicate and to initiate viral infection. However, conservation of integrated sequences in plant genomes might indicate benefits for the host during evolution. Understanding EPRV activation and control by the host could have important implications for plant breeding strategies to prevent viral disease caused by EPRVs in newly generated cultivars.

Electron Microscopy Methods for Virus Diagnosis and High Resolution Analysis of Viruses.

The term "virosphere" describes both the space where viruses are found and the space they influence, and can extend to their impact on the environment, highlighting the complexity of the interactions involved. Studying the biology of viruses and the etiology of virus disease is crucial to the prevention of viral disease, efficient and reliable virus diagnosis, and virus control. Electron microscopy (EM) is an essential tool in the detection and analysis of virus replication. New EM methods and ongoing technical improvements offer a broad spectrum of applications, allowing in-depth investigation of viral impact on not only the host but also the environment. Indeed, using the most up-to-date electron cryomicroscopy methods, such investigations are now close to atomic resolution. In combination with bioinformatics, the transition from 2D imaging to 3D remodeling allows structural and functional analyses that extend and augment our knowledge of the astonishing diversity in virus structure and lifestyle. In combination with confocal laser scanning microscopy, EM enables live imaging of cells and tissues with high-resolution analysis. Here, we describe the pivotal role played by EM in the study of viruses, from structural analysis to the biological relevance of the viral metagenome (virome).

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida.

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.

Detection of petunia vein-clearing virus: model for the detection of DNA viruses in plants with homologous endogenous pararetrovirus sequences.

A number of cases of plant virus sequence integration into host plant genome have been reported. In at least two cases, endogenous pararetrovirus sequences are correlated strongly with subsequent episomal virus infection and there is circumstantial evidence that this also occurs for Petunia vein-clearing virus (PVCV). The detection of viruses is a critical component of plant health and therefore, it is important to have diagnostic procedures that differentiate between the detection of encapsidated viral DNA and homologous sequences in the host genome. PCR-based detection methods targeted at PVCV DNA have been tested and particular attention was paid to design controls that would indicate the existence of host DNA in the reaction. The use of ion-exchange chromatography for the partial purification of plant viruses from other cellular components, including chromosomal DNA, is described. The methods tested for PVCV detection are used to illustrate general principles for the specific detection of virus infections in host plants that carry homologous virus sequences in their genomes.