A Polyvalent Tool for Detecting Coguviruses in Multiple Hosts Allowed the Identification of a Novel Seed-Transmitted Coguvirus Infecting Brassicaceae.

The genus Coguvirus, a recently established genus in the family Phenuiviridae, includes several species whose members infect both woody and herbaceous hosts, suggesting a broader host range and wider distribution than previously. To gain insights into the epidemiology and biology of coguviruses, a polyvalent reverse transcription-PCR assay using degenerate primers was developed. The specificity of the assay for coguviruses was confirmed by testing citrus and apple plants infected by previously reported coguviruses and/or several unrelated viruses. The expected 236-bp amplicon was obtained from citrus, apple, pear, watermelon, and several species of the family Brassicaceae. Sequencing of the PCR amplicons allowed the identification, for the first time in Italy and/or Europe, of several coguviruses in multiple hosts, confirming the effectiveness of the assay. Moreover, a new virus, tentatively named Brassica oleracea Torzella virus 1 (BoTV1), was detected in several plants of Torzella cabbage. The complete +genome of BoTV1, determined by high-throughput sequencing and 5' rapid amplification of cDNA ends, revealed that it has the typical molecular features of coguviruses and fulfils the current criteria to be classified as a member of a new species, for which the tentative name Coguvirus torzellae is proposed. The same polyvalent assay was also used to investigate and confirm that BoTV1 is transmitted through seeds in black cabbage, thus providing the first evidence on the relevance of this natural transmission mode in the epidemiology of coguviruses. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

2020 taxonomic update for phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Rabenchromenone and Rabenzophenone, Phytotoxic Tetrasubstituted Chromenone and Hexasubstituted Benzophenone Constituents Produced by the Oak-Decline-Associated Fungus Fimetariella rabenhorstii.

A new phytotoxic tetrasubstituted chromen-4-one (1) and a new hexasubstituted benzophenone (2), named rabenchromenone and rabenzophenone, respectively, were isolated from the culture filtrates of Fimetariella rabenhorstii, an oak-decline-associated fungus in Iran. Rabenchromenone and rabenzophenone, isolated together with known moniliphenone (3) and coniochaetone A (4), were characterized as methyl 3-chloro-1,8-dihydroxy-6-methyl-9-oxo-1,9-dihydrocyclopenta[b]chromene-1-carboxylate and methyl 4-chloro-2-(2,6-dihydroxy-4-methylbenzoyl)-3-hydroxybenzoate, respectively, by spectroscopic methods (primarily nuclear magnetic resonance and high-resolution electrospray ionization mass spectrometry). The R absolute configuration at C-1 of rabenchromenone was determined by quantum chemical calculations and electronic circular dichroism experiments. All metabolites (1-4) were tested by leaf puncture on tomato and oak plants. All compounds were active in this assay by causing in both plants a necrosis diameter in the range of 0.2-0.7 cm. Specifically, rabenzophenone (2) was found to be the most phytotoxic compound in both plants.

Overview of Biotic Stresses in Pepper (Capsicum spp.): Sources of Genetic Resistance, Molecular Breeding and Genomics.

Pepper (Capsicum spp.) is one of the major vegetable crops grown worldwide largely appreciated for its economic importance and nutritional value. This crop belongs to the large Solanaceae family, which, among more than 90 genera and 2500 species of flowering plants, includes commercially important vegetables such as tomato and eggplant. The genus includes over 30 species, five of which (C. annuum, C. frutescens, C. chinense, C. baccatum, and C. pubescens) are domesticated and mainly grown for consumption as food and for non-food purposes (e.g., cosmetics). The main challenges for vegetable crop improvement are linked to the sustainable development of agriculture, food security, the growing consumers' demand for food. Furthermore, demographic trends and changes to climate require more efficient use of plant genetic resources in breeding programs. Increases in pepper consumption have been observed in the past 20 years, and for maintaining this trend, the development of new resistant and high yielding varieties is demanded. The range of pathogens afflicting peppers is very broad and includes fungi, viruses, bacteria, and insects. In this context, the large number of accessions of domesticated and wild species stored in the world seed banks represents a valuable resource for breeding in order to transfer traits related to resistance mechanisms to various biotic stresses. In the present review, we report comprehensive information on sources of resistance to a broad range of pathogens in pepper, revisiting the classical genetic studies and showing the contribution of genomics for the understanding of the molecular basis of resistance.

Taxonomy of the order Bunyavirales: update 2019.

In February 2019, following the annual taxon ratification vote, the order Bunyavirales was amended by creation of two new families, four new subfamilies, 11 new genera and 77 new species, merging of two species, and deletion of one species. This article presents the updated taxonomy of the order Bunyavirales now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Dicer-like and RNA-dependent RNA polymerase gene family identification and annotation in the cultivated Solanum tuberosum and its wild relative S. commersonii.

MAIN CONCLUSION: We provide advances in DCL and RDR gene diversity in Solanaceae. We also shed light on DCL and RDR gene expression in response to cold stress. DICER-like (DCL) and RNA-dependent RNA polymerase (RDR) genes form the core components to trigger small non-coding RNA (ncRNA) production. In spite of this, little is known about the two gene families in non-model plant species. As their genome sequences are now available, the cultivated potato (Solanum tuberosum) and its cold-tolerant wild relative Solanum commersonii offer a valuable opportunity to advance our understanding of the above genes. To determine the extent of diversification and evolution of DCLs and RDRs in these species, we performed a comparative analysis. Seven DCLs were identified in the two species, whereas seven and six RDR genes were found in S. tuberosum and S. commersonii, respectively. Based on phylogenetic analysis with DCLs and RDRs from several species, we provide evidence for an increase in their number in both potato species. We also disclosed that tandem duplications played a major role in the evolution of these gene families in Solanaceae. DCL and RDR expression was investigated in different tissues and under cold and virus stresses, with divergent profiles of the tandem duplicated genes being found in different tissues. DCL paralogs showed a contrasting expression in S. tuberosum and S. commersonii following cold stress and virus infection. By contrast, no change in RDR transcript activity was detected following both stresses. Overall, this study provides the first comparative genomic analysis of the core components of the RNAi machinery in Solanaceae and offers a scaffold for future functional analysis of these gene families.

Characterization by Deep Sequencing of Prunus virus T, a Novel Tepovirus Infecting Prunus Species.

Double-stranded RNAs purified from a cherry tree collected in Italy and a plum tree collected in Azerbaijan were submitted to deep sequencing. Contigs showing weak but significant identity with various members of the family Betaflexiviridae were reconstructed. Sequence comparisons led to the conclusion that the viral isolates identified in the analyzed Prunus plants belong to the same viral species. Their genome organization is similar to that of some members of the family Betaflexiviridae, with three overlapping open reading frames (RNA polymerase, movement protein, and capsid protein). Phylogenetic analyses of the deduced encoded proteins showed a clustering with the sole member of the genus Tepovirus, Potato virus T (PVT). Given these results, the name Prunus virus T (PrVT) is proposed for the new virus. It should be considered as a new member of the genus Tepovirus, even if the level of nucleotide identity with PVT is borderline with the genus demarcation criteria for the family Betaflexiviridae. A reverse-transcription polymerase chain reaction detection assay was developed and allowed the identification of two other PrVT isolates and an estimate of 1% prevalence in the large Prunus collection screened. Due to the mixed infection status of all hosts identified to date, it was not possible to correlate the presence of PrVT with specific symptoms.

A Novel Self-Cleaving Viroid-Like RNA Identified in RNA Preparations from a Citrus Tree Is Not Directly Associated with the Plant.

Viroid and viroid-like satellite RNAs are infectious, circular, non-protein coding RNAs reported in plants only so far. Some viroids (family Avsunviroidae) and viroid-like satellite RNAs share self-cleaving activity mediated by hammerhead ribozymes (HHRzs) endowed in both RNA polarity strands. Using a homology-independent method based on the search for conserved structural motifs of HHRzs in reads and contigs from high-throughput sequenced RNAseq libraries, we identified a novel small (550 nt) viroid-like RNA in a library from a Citrus reticulata tree. Such a viroid-like RNA contains a HHRz in both polarity strands. Northern blot hybridization assays showed that circular forms of both polarity strands of this RNA (tentatively named citrus transiently-associated hammerhead viroid-like RNA1 (CtaHVd-LR1)) exist, supporting its replication through a symmetric pathway of the rolling circle mechanism. CtaHVd-LR1 adopts a rod-like conformation and has the typical features of quasispecies. Its HHRzs were shown to be active during transcription and in the absence of any protein. CtaHVd-LR1 was not graft-transmissible, and after its first identification, it was not found again in the original citrus source when repeatedly searched in the following years, suggesting that it was actually not directly associated with the plant. Therefore, the possibility that this novel self-cleaving viroid-like RNA is actually associated with another organism (e.g., a fungus), in turn, transiently associated with citrus plants, is proposed.

Identification and Characterization of Citrus Concave Gum-Associated Virus Infecting Citrus and Apple Trees by Serological, Molecular and High-Throughput Sequencing Approaches.

Citrus concave gum-associated virus (CCGaV) is a negative-stranded RNA virus, first reported a few years ago in citrus trees from Italy. It has been reported in apple trees in the USA and in Brazil, suggesting a wider host range and geographic distribution. Here, an anti-CCGaV polyclonal antiserum to specifically detect the virus has been developed and used in a standard double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) that has been validated as a sensitive and reliable method to detect this virus both in citrus and apple trees. In contrast, when the same antiserum was used in direct tissue-blot immunoassay, CCGaV was efficiently detected in citrus but not in apple. Using this antiserum, the first apple trees infected by CCGaV were identified in Italy and the presence of CCGaV in several apple cultivars in southern Italy was confirmed by field surveys. High-throughput sequencing (HTS) allowed for the assembling of the complete genome of one CCGaV Italian apple isolate (CE-c3). Phylogenetic analysis of Italian CCGaV isolates from apple and citrus and those available in the database showed close relationships between the isolates from the same genus (Citrus or Malus), regardless their geographical origin. This finding was further confirmed by the identification of amino acid signatures specific of isolates infecting citrus or apple hosts. Analysis of HTS reads also revealed that the CE-c3 Italian apple tree, besides CCGaV, was simultaneously infected by several viruses and one viroid, including apple rubbery wood virus 2 which is reported for the first time in Italy. The complete or almost complete genomic sequences of the coinfecting agents were determined.

Phytotoxic metabolites from Stilbocrea macrostoma, a fungal pathogen of Quercus brantii in Iran.

Two phytotoxic furan derivatives were isolated, together with the well-known fungal and plant phytotoxin tyrosol, from the culture filtrates of Stilbocrea macrostoma. This fungal pathogen isolated from Quercus brantii trees induced wood necrosis and decline symptoms on the host plant in Iran. The two furan derivatives, isolated for the first time from Stilbocrea macrostoma, were identified by spectroscopic methods (essentially 1 D and 2 D 1H and 13C NMR and ESIMS spectroscopy) as 5-hydroxymethylfuraldehyde and 2,5-dihydroxymethylfuran. The phytotoxic activity of the three metabolites was evaluated by leaf puncture assay on holm oak (Quercus ilex L.) and tomato (Lycopersicon esculentum L.) leaves. All compounds induced necrosis on holm oak leaves while very low toxicity was showed against tomato leaves. The two furan derivatives were more toxic than tyrosol and particularly 5-hydroxymethylfuraldehyde was the most phytotoxic compound.

Organic Amendments Modulate Soil Microbiota and Reduce Virus Disease Incidence in the TSWV-Tomato Pathosystem.

Application of organic amendments is considered an eco-friendly practice to promote soil fertility and suppressiveness against a wide range of soil-borne pathogens. However, limited information is available about the capabilities of organic amendments to control virus disease. In this study, the suppressiveness of different organic amendments (i.e., compost manure, biochar, alfalfa straw, and glucose) was determined against the Tomato spotted wilt virus (TSWV) on tomato plants in a 1-year-long mesocosm experiment. Organic treatments were compared to the ordinary soil management based on mineral fertilizers and fumigation. Tomato seedlings were inoculated with TSWV and the infection and symptoms were assessed three weeks later. The disease incidence was higher in soil treated with mineral fertilizers and fumigation (> 80%) compared to the application of organic amendments, with alfalfa straw and biochar recording the lowest incidence (< 40%). Moreover, soil microbiota structure and diversity were assessed by high-throughput sequencing of bacterial and eukaryotic rRNA gene markers. Several members belonging to the bacterial phyla of Acidobacteria, Actinobacteria, Bacteroidetes, and Proteobacteria, as well as members of the fungal genus Acremonium, were positively associated with plant health. This study showed that conventional practices, by shifting microbiome composition, may increase TSWV incidence and severity.

WRKY genes family study reveals tissue-specific and stress-responsive TFs in wild potato species.

Wild potatoes, as dynamic resource adapted to various environmental conditions, represent a powerful and informative reservoir of genes useful for breeding efforts. WRKY transcription factors (TFs) are encoded by one of the largest families in plants and are involved in several biological processes such as growth and development, signal transduction, and plant defence against stress. In this study, 79 and 84 genes encoding putative WRKY TFs have been identified in two wild potato relatives, Solanum commersonii and S. chacoense. Phylogenetic analysis of WRKY proteins divided ScWRKYs and SchWRKYs into three Groups and seven subGroups. Structural and phylogenetic comparative analyses suggested an interspecific variability of WRKYs. Analysis of gene expression profiles in different tissues and under various stresses allowed to select ScWRKY045 as a good candidate in wounding-response, ScWRKY055 as a bacterial infection triggered WRKY and ScWRKY023 as a multiple stress-responsive WRKY gene. Those WRKYs were further studied through interactome analysis allowing the identification of potential co-expression relationships between ScWRKYs/SchWRKYs and genes of various pathways. Overall, this study enabled the discrimination of WRKY genes that could be considered as potential candidates in both breeding programs and functional studies.

A Negative-Stranded RNA Virus Infecting Citrus Trees: The Second Member of a New Genus Within the Order Bunyavirales.

A new RNA virus has been identified from a sweet orange tree in southern Italy. This virus, tentatively named citrus virus A (CiVA), has a bipartite genome composed of (i) a negative-stranded (ns) RNA1, encoding the viral RNA-dependent RNA polymerase (RdRp), and (ii) an ambisense RNA2, coding for the putative movement protein (MP) and nucleocapsid protein (NP), with the two open reading frames separated by a long AU-rich intergenic region (IR) adopting a hairpin conformation. CiVA genomic RNAs and the encoded proteins resemble those of the recently discovered citrus concave gum-associated virus (CCGaV). This CCGaV, a nsRNA virus associated with the ancient citrus concave gum disease, has been proposed as the representative member of a new genus tentatively named Coguvirus. Molecular and phylogenetic analyses presented here support the classification of CiVA, and likely of other two recently described nsRNA viruses infecting plants, in this new genus. By showing that the evolutionary origin of the MP of all the putative coguviruses likely differs from that of their respective RdRp and NP, this study also provides evidence of a likely modular genome evolution for these viruses. Moreover, phylogenetic data support the proposal that, during the evolutionary history of nsRNA viruses, the plant-infecting viruses most likely emerged from an invertebrate-infecting ancestor several times as independent events. CiVA was identified in a field sweet orange tree not showing any obvious symptom and was graft-transmitted to sweet orange, grapefruit, rough lemon and Dweet tangor indicator plants that did not developed symptoms. The capacity of infecting citrus hosts of several species was also confirmed by a preliminary survey that identified orange, mandarin, clementine and lemon trees as natural hosts of CiVA in several fields of southern Italy, again without any obvious association with specific symptoms.

The first phlebo-like virus infecting plants: a case study on the adaptation of negative-stranded RNA viruses to new hosts.

A novel negative-stranded (ns) RNA virus associated with a severe citrus disease reported more than 80 years ago has been identified. Transmission electron microscopy showed that this novel virus, tentatively named citrus concave gum-associated virus, is flexuous and non-enveloped. Notwithstanding, its two genomic RNAs share structural features with members of the genus Phlebovirus, which are enveloped arthropod-transmitted viruses infecting mammals, and with a group of still unclassified phlebo-like viruses mainly infecting arthropods. CCGaV genomic RNAs code for an RNA-dependent RNA polymerase, a nucleocapsid protein and a putative movement protein showing structural and phylogenetic relationships with phlebo-like viruses, phleboviruses and the unrelated ophioviruses, respectively, thus providing intriguing evidence of a modular genome evolution. Phylogenetic reconstructions identified an invertebrate-restricted virus as the most likely ancestor of this virus, revealing that its adaptation to plants was independent from and possibly predated that of the other nsRNA plant viruses. These data are consistent with an evolutionary scenario in which trans-kingdom adaptation occurred several times during the history of nsRNA viruses and followed different evolutionary pathways, in which genomic RNA segments were gained or lost. The need to create a new genus for this bipartite nsRNA virus and the impact of the rapid and specific detection methods developed here on citrus sanitation and certification are also discussed.

Characterization of a putative novel nepovirus from Aeonium sp.

A virus was isolated from potted plants of an unidentified species of Aeonium, a succulent ornamental very common in Southern Italy, showing chlorotic spots and rings on both leaf surfaces. It was successfully transmitted by sap inoculation to a limited range of hosts, including Nicotiana benthamiana which was used for ultrastructural observations and virus purification. Virus particles are isometric, ca. 30nm in diameter, have a single type of coat protein (CP) subunits 54kDa in size, that encapsidate single-stranded positive-sense RNA species of 7549 (RNA1) and 4010 (RNA2) nucleotides. A third RNA molecule 3472 nts in size entirely derived from RNA2 was also found. The structural organization of both genomic RNAs and the cytopathological features were comparable to those of nepoviruses. In addition, amino acid sequence comparisons of CP and the Pro-Pol region (a sequence containing parts of the proteinase and polymerase) with those of other nepoviruses showed that the Aeonium virus belongs to the subgroup A of the genus Nepovirus and is phylogenetically close to, but serologically distinct from tobacco ringspot virus (TRSV). Based on the species demarcation criteria for the family Secoviridae, the virus under study appears to be a novel member of the genus Nepovirus for which the name of Aeonium ringspot virus (AeRSV) is proposed.

Genetic variation of populations of Citrus psorosis virus.

Citrus psorosis virus (CPsV), the type species of genus Ophiovirus, has a segmented, negative-stranded RNA genome. We examined the population structure and genetic variation of CPsV in three coding regions located in RNAs 1, 2 and 3, analysing 22 isolates from Argentina, California, Florida, Italy and Spain. Most isolates contained a predominant sequence and some minor variants. Estimations of the genetic diversity and phylogenetic clustering of isolates disclosed two populations, one comprising isolates from Spain, Italy, Florida and California and the other including the Argentinean isolates. Isolate CPV-4 (from Texas) included for comparison was distant from both groups, suggesting that it belongs to a third group. The low ratio between non-synonymous and synonymous nucleotide substitutions indicated strong selection for amino acid sequence conservation, particularly in the coat protein gene. Incongruent phylogenetic relationships in different genomic regions suggested that exchange of genomic segments may have contributed to CPsV evolution.