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50 Pág., In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratifcation vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV, This work was supported in part through Laulima Govern ment Solutions, LLC prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272201800013C. J.H.K. performed this work as an employee of Tunnell Government Services (TGS), a subcontractor of Laulima Gov ernment Solutions, LLC under Contract No. HHSN272201800013C. This work was also funded in part by Contract No. HSHQDC 15-C-00064 awarded by DHS S&T for the management and operation of The National Biodefense Analysis and Countermeasures Center, a federally funded research and development center operated by the Battelle National Biodefense Institute (V.W.); and NIH contract HHSN272201000040I/HHSN27200004/D04 and grant R24AI120942 (N.V., R.B.T.). S.S. acknowledges support from the Mississippi Agri cultural and Forestry Experiment Station (MAFES), USDA-ARS pro ject 58-6066-9-033 and the National Institute of Food and Agriculture, US Department of Agriculture, Hatch Project, under Accession Num ber 1021494.

The family

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., La lista completa de autores que integran el documento puede consultarse en el archivo., Facultad de Ciencias Agrarias y Forestales, Centro de Investigaciones en Fitopatología, Facultad de Ciencias Exactas, Instituto de Biotecnologia y Biologia Molecular

Thrips palmi (Thysanoptera: Thripidae) is an important pest of vegetables, ornamentals, and legumes worldwide. Besides damage caused by feeding, it transmits several tospoviruses. Identification of T. palmi at an early stage is crucial in implementing appropriate pest management strategies. Morpho-taxonomic identification of T. palmi based on the adult stage is time-consuming and needs taxonomic expertise. Here, we report a rapid, on-site, field-based assay for identification of T. palmi based on recombinase polymerase amplification (RPA), its first application in insects. RPA primers designed based on 3′ polymorphisms of the Internal Transcribed Spacer 2 region efficiently discriminated T. palmi without any cross-reactivity to other predominant thrips species. RPA was performed with crude DNA, extracted from single T. palmi simply by crushing in sterile distilled water and could be completed within 20 min by holding the reaction tubes in the hand. The assay was further simplified by using fluorescent as well as colorimetric dyes thus eliminating the gel-electrophoresis steps. The presence of T. palmi was visualized by a change in color from dark blue to sky blue. The assay was validated with known thrips specimens and found to be effective in diagnosing the presence of T. palmi in natural vegetation. This on-site, rapid assay for diagnosis of T. palmi can be used by non-expert personnel in the field of quarantine and pest management.

Culturing insect cells in vitro has witnessed remarkable growth in the past decades. Over 600 cell lines of dipteran, hemipteran, and lepidopteran insects have been reported so far. Besides its wide adoption for the production of recombinant proteins, viral pesticides, and vaccines, insect cell culture is increasingly being used as a tool for basic research in genetics, molecular biology, biochemistry, and virology. Since its first application in plant virus studies in 1956, insect cell culture has become an indispensable tool to study the complex and intimate interactions of plant viruses and their insect vectors. The in vitro cell cultures of several insect vectors of plant viruses including leafhoppers, planthoppers, aphids, thrips, and whiteflies have been successfully employed to understand the functions of viral proteins and receptor-mediated endocytosis into vector cells. This review summarizes the development of novel media and insect cell culture systems and their applications in plant virus research.

MicroRNAs (miRNAs) play key regulatory roles in the plant’s response to biotic and abiotic stresses and have fundamental functions in plant–virus interactions. The study of changes in miRNAs in response to virus infection can provide molecular details for a better understanding of virus-host interactions. Maize Iranian mosaic virus (MIMV) infects maize and certain other poaceous plants but miRNA changes in response to MIMV infection are unknown. In the present study, we compared the miRNA profiles of MIMV-infected and uninfected maize and characterized their predicted roles in response to the virus. Small RNA sequencing of maize identified 257 conserved miRNAs of 26 conserved families in uninfected and MIMV-infected maize libraries. Among them, miR395, miR166 and miR156 family members were highly represented. Small RNA data were confirmed using RT-qPCR. In addition, 33 potential novel miRNAs were predicted. The data show that 13 miRNAs were up-regulated and 113 were down-regulated in response to MIMV infection. Several of those miRNAs are known to be important in the response to plant pathogens. To determine the potential roles of individual miRNAs in response to MIMV, miRNA targets, predicted interactions with circular RNAs and comparative transcriptome data were analyzed. The expression profiles of different miRNAs in response to MIMV provide novel insights into the roles of miRNAs in the interaction between MIMV and maize plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03134-1.

Nyamiviridae is a family of viruses in the order Mononegavirales, with unsegmented (except for members of the genus Tapwovirus), negative-sense RNA genomes of 10–13 kb. Nyamviruses have a genome organisation and content similar to that of other mononegaviruses. Nyamiviridae includes several genera that form monophyletic clades on phylogenetic analysis of the RNA polymerase. Nyamiviruses have been found associated with diverse invertebrates as well as land- and seabirds. Members of the genera Nyavirus and Socyvirus produce enveloped, spherical virions. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Nyamiviridae, which is available at ictv.global/report/nyamiviridae.

Maize Iranian mosaic virus (MIMV) infects several gramineous plants and is an economically important nucleorhabdovirus in Iran. Maize responds to MIMV infection at the transcriptional level. Alternative splicing (AS) is a mechanism that generates multiple mRNAs from a single pre-mRNA, often encoding protein isoforms with functional differences. We carried out genome-wide analysis of AS responses to MIMV in maize seedlings and identified genes involved in this molecular response. The AS events we investigated included skipped exons, alternative 3′ splice site, alternative 5′ splice site, mutually exclusive exons, and retained introns. In total 10,881 maize genes showed AS, of which 601 genes were involved in response to MIMV-infection and 186 were found only in uninfected maize. AS was identified in some of the genes that are involved in disease resistance or pathogenicity pathways. We demonstrated that in MIMV-infects maize, host genes that are involved in symptom development, virus multiplication, resistance to pathogens and host-pathogen interaction are affected by AS mechanism. Gene network analysis showed that ten genes represent the hubs for the protein network in maize and that they are involved in response to pathogen attack and include 26S proteasome, 14–3-3-like protein A, Rop family, mitogen-activated protein kinase, ubiquitin and serine/threonine-protein kinases. In conclusion, we showed that AS occurs as a transcriptional regulatory mechanism in maize response to MIMV infection and we identified genes that have the key roles in pathogenicity pathways that were differentially spliced in infected seedlings.

Melon thrips (Thrips palmi) is the principal insect pest of vegetable and ornamental plants worldwide. In addition to inflicting feeding injuries on host plants, they act as vectors of economically damaging tospoviruses. Application of insecticides and host plant resistance has proven largely ineffective in the management of T. palmi. However, increasingly, genetic knowledge is being successfully utilized to manage insect pests. A number of recent studies have enriched the genetic database of T. palmi. In this review, we report on genetics of T. palmi for species identification and to study its populations structure. For example, a DNA polymorphism analysis of global T. palmi populations has revealed the existence of 29 haplotypes. The T. palmi population can be divided into three lineages based on phylogenetic analysis. A high maximum intraspecific distance is indicative of cryptic species within T. palmi populations. Regarding climate adaptability, the upregulation of trehalose biosynthesis genes, and genes encoding several heat-shock proteins is expected to enable T. palmi thermal adaptation to both cold and hot conditions. Genetics of T. palmi has shown that resistance to conventional insecticides like cypermethrin appears to be linked to mutations in the sodium channel. On the other hand, resistance to imidacloprid appears to be a result of cytochrome P450-mediated detoxification. T. palmi genes potentially involved in tospovirus transmission are also reviewed and new molecular tools for genetic study presented. Molecular modeling and docking analyses of groundnut bud necrosis virus glycoprotein demonstrated protein–protein interactions with T. palmi vacuolar ATP synthase E subunit, cathepsin, clathrin, adaptor protein 2, and enolase. Overall, a better knowledge of T. palmi genes will assist elucidation of thrips identification, means of adaptation in diverse ecological niches, mechanisms of insecticide resistance, and transmission of plant viruses. This data will be an invaluable tool for integrated thrips management.

Citrus black spot (Phyllosticta citricarpa) is an economically important disease of citrus in Australia. A closely related endophyte, P. capitalensis, also occurs on citrus in Australia, and the two fungi are known to co-exist in orchards. The diversity of other species of Phyllosticta on Citrus in Australia is unknown. Citrus black spot is managed by fungicide treatment and options such as biological control may be better economic alternatives. We studied the diversity of Phyllosticta on Citrus in Australia with a phylogenetic species hypothesis. We report P. paracapitalensis for the first time and confirm the presence of P. capitalensis in Queensland. Many examined isolates of Phyllosticta previously identified as P. capitalensis were reidentified as P. paracapitalensis. We also provide evidence through Koch’s postulates that the two endophytic species are non-pathogenic on citrus fruit. In addition, disease incidence and severity of citrus black spot was significantly reduced when fruit were pre-inoculated with one of the endophytes 14 days prior to pathogen inoculation on the same fruit. Our results indicate these endophytes may have potential antagonistic effects against P. citricarpa under field conditions.

We identified a novel plant rhabdovirus infecting native joa (Solanum aculeatissimum) plants in Brazil. Infected plants showed yellow blotches on the leaves, and typical enveloped bacilliform rhabdovirus particles associated with the nucleus were seen in thin sections by electron microscopy. The virus could be graft-transmitted to healthy joa and tomato plants but was not mechanically transmissible. RT-PCR using degenerate plant rhabdovirus L gene primers yielded an amplicon from extracted total RNA, the sequence of which was similar to those of alphanucleorhabdoviruses. Based on close sequence matches, especially with the type member potato yellow dwarf virus (PYDV), we adopted a degenerate-primer-walking strategy towards both genome ends. The complete genome of joa yellow blotch-associated virus (JYBaV) is comprised of 12,965 nucleotides, is less than 75% identical to that of its closest relative PYDV, and clusters with PYDV and other alphanucleorhabdoviruses in L protein phylogenetic trees, suggesting that it should be taxonomically classified in a new species in the genus Alphanucleorhabdovirus, family Rhabdoviridae. The genome organization of JYBaV is typical of the 'PYDV-like' subgroup of alphanucleorhabdoviruses, with seven genes (N-X-P-Y-M-G-L) separated by conserved intergenic regions and flanked by partly complementary 3' leader and 5' trailer regions.

Unfortunately, the inclusion of original names (in non-Latin script) of the following authors caused problems with author name indexing in PubMed. Therefore, these original names were removed from XML data to correct the PubMed record. Mengji Cao, Yuya Chiaki, Hideki Ebihara, Jingjing Fu, George Fú Gāo, Tong Han, Jiang Hong, Ni Hong, Seiji Hongo, Masayuki Horie, Dàohóng Jiāng, Fujio Kadono, Hideki Kondō, Kenji Kubota, Shaorong Li, Longhui Li, Jiànróng Lǐ, Huazhen Liu, Tomohide Natsuaki, Sergey V. Netesov, Anna Papa, Sofia Paraskevopoulou, Liying Qi, Takahide Sasaya, Mang Shi, Xiǎohóng Shí, Zhènglì Shí, Yoshifumi Shimomoto, Jin‑Won Song, Ayato Takada, Shigeharu Takeuchi, Yasuhiro Tomitaka, Keizō Tomonaga, Shinya Tsuda, Changchun Tu, Tomio Usugi, Nikos Vasilakis, Jiro Wada, Lin‑Fa Wang, Guoping Wang, Yanxiang Wang, Yaqin Wang, Tàiyún Wèi, Shaohua Wen, Jiangxiang Wu, Lei Xu, Hironobu Yanagisawa, Caixia Yang, Zuokun Yang, Lifeng Zhai, Yong‑Zhen Zhang, Song Zhang, Jinguo Zhang, Zhe Zhang, Xueping Zhou. In addition, the publication call-out in the supplementary material was updated from issue 11 to issue 12. The original article has been corrected.

We identified a novel plant rhabdovirus infecting native joá (Solanum aculeatissimum) plants in Brazil. Infected plants showed yellow blotches on the leaves, and typical enveloped bacilliform rhabdovirus particles associated with the nucleus were seen in thin sections by electron microscopy. The virus could be graft-transmitted to healthy joá and tomato plants but was not mechanically transmissible. RT-PCR using degenerate plant rhabdovirus L gene primers yielded an amplicon from extracted total RNA, the sequence of which was similar to those of alphanucleorhabdoviruses. Based on close sequence matches, especially with the type member potato yellow dwarf virus (PYDV), we adopted a degenerate-primer-walking strategy towards both genome ends. The complete genome of joá yellow blotch-associated virus (JYBaV) is comprised of 12,965 nucleotides, is less than 75% identical to that of its closest relative PYDV, and clusters with PYDV and other alphanucleorhabdoviruses in L protein phylogenetic trees, suggesting that it should be taxonomically classified in a new species in the genus Alphanucleorhabdovirus, family Rhabdoviridae. The genome organization of JYBaV is typical of the 'PYDV-like' subgroup of alphanucleorhabdoviruses, with seven genes (N-X-P-Y-M-G-L) separated by conserved intergenic regions and flanked by partly complementary 3' leader and 5' trailer regions.

Thrips are important pests of agricultural, horticultural, and forest crops worldwide. In addition to direct damages caused by feeding, several thrips species can transmit diverse tospoviruses. The present understanding of thrips–tospovirus relationships is largely based on studies of tomato spotted wilt virus (TSWV) and Western flower thrips (Frankliniella occidentalis). Little is known about other predominant tospoviruses and their thrips vectors. In this study, we report the progression of watermelon bud necrosis virus (WBNV) infection in its vector, melon thrips (Thrips palmi). Virus infection was visualized in different life stages of thrips using WBNV-nucleocapsid protein antibodies detected with FITC-conjugated secondary antibodies. The anterior midgut was the first to be infected with WBNV in the first instar larvae. The midgut of T. palmi was connected to the principal salivary glands (PSG) via ligaments and the tubular salivary glands (TSG). The infection progressed to the PSG primarily through the connecting ligaments during early larval instars. The TSG may also have an ancillary role in disseminating WBNV from the midgut to PSG in older instars of T. palmi. Infection of WBNV was also spread to the Malpighian tubules, hindgut, and posterior portion of the foregut during the adult stage. Maximum virus-specific fluorescence in the anterior midgut and PSG indicated the primary sites for WBNV replication. These findings will help to better understand the thrips–tospovirus molecular relationships and identify novel potential targets for their management. To our knowledge, this is the first report of the WBNV dissemination path in its vector, T. palmi.

Seven isolates of a putative cytorhabdovirus (family Rhabdoviridae, order Mononegavirales) designated as citrus-associated rhabdovirus (CiaRV) were identified in citrus, passion fruit, and paper bush from the same geographical area in China. CiaRV, bean-associated cytorhabdovirus (Brazil), and papaya virus E (Ecuador) should be taxonomically classified in the species Papaya cytorhabdovirus. Due to natural mutations, the glycoprotein (G) and P4 genes were impaired in citrus-infecting isolates of CiaRV, resulting in an atypical rhabdovirus genome organization of 3′ leader-N-P-P3-M-L-5′ trailer. The P3 protein of CiaRV shared a common origin with begomoviral movement proteins (family Geminiviridae). Secondary structure analysis and trans-complementation of movement-deficient tomato mosaic virus and potato virus X mutants by CiaRV P3 supported its function in viral cell-to-cell trafficking. The wide geographical dispersal of CiaRV and related viruses suggests an efficient transmission mechanism, as well as an underlying risk to global agriculture. Both the natural phenomenon and experimental analyses demonstrated presence of the “degraded” type of CiaRV in citrus, in parallel to “undegraded” types in other host plant species. This case study shows a plant virus losing the function of an important but nonessential gene, likely due to host shift and adaption, which deepened our understanding of course of natural viral diversification.

Citrus black spot, caused by

Thrips palmi (Thysanoptera: Thripidae) is an important pest of vegetables, ornamentals, and legumes worldwide. Besides damage caused by feeding, it transmits several tospoviruses. Identification of T. palmi at an early stage is crucial in implementing appropriate pest management strategies. Morpho-taxonomic identification of T. palmi based on the adult stage is time-consuming and needs taxonomic expertise. Here, we report a rapid, on-site, field-based assay for identification of T. palmi based on recombinase polymerase amplification (RPA), its first application in insects. RPA primers designed based on 3′ polymorphisms of the Internal Transcribed Spacer 2 region efficiently discriminated T. palmi without any cross-reactivity to other predominant thrips species. RPA was performed with crude DNA, extracted from single T. palmi simply by crushing in sterile distilled water and could be completed within 20 min by holding the reaction tubes in the hand. The assay was further simplified by using fluorescent as well as colorimetric dyes thus eliminating the gel-electrophoresis steps. The presence of T. palmi was visualized by a change in color from dark blue to sky blue. The assay was validated with known thrips specimens and found to be effective in diagnosing the presence of T. palmi in natural vegetation. This on-site, rapid assay for diagnosis of T. palmi can be used by non-expert personnel in the field of quarantine and pest management.

The genome of a novel rhabdovirus was detected in yerba mate (Ilex paraguariensis St. Hil.). The newly identified virus, tentatively named "yerba mate virus A" (YmVA), has a genome of 14,961 nucleotides. Notably, eight open reading frames were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, including two novel accessory genes, in the order 3′-N-P-3-4-M-G-L-8-5′. Sequence comparisons of the encoded proteins as well as phylogeneticanalysis suggest that YmVA is a new member of the genus Cytorhabdovirus, family Rhabdoviridae. YmVA?s unique genomic organization and phylogenetic relationships indicate that this virus likely represents a distinct evolutionary lineage among the cytorhabdoviruses. Fil: Bejerman, Nicolas. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigaciones Agropecuarias. Unidad de Fitopatologia y Modelizacion Agricola. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Unidad de Fitopatologia y Modelizacion Agricola.; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina Fil: Acevedo, Raúl Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina Fil: de Breuil, Soledad. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigaciones Agropecuarias. Unidad de Fitopatologia y Modelizacion Agricola. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Unidad de Fitopatologia y Modelizacion Agricola.; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina Fil: Ruiz, Oscar Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Sansberro, Pedro Alfonso. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigaciones Agropecuarias. Unidad de Fitopatologia y Modelizacion Agricola. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Unidad de Fitopatologia y Modelizacion Agricola.; Argentina Fil: Dietzgen, Ralf G.. The University of Queensland; Australia Fil: Nome, Claudia. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigaciones Agropecuarias. Unidad de Fitopatologia y Modelizacion Agricola. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Unidad de Fitopatologia y Modelizacion Agricola.; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina Fil: Debat, Humberto Julio. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigaciones Agropecuarias. Unidad de Fitopatologia y Modelizacion Agricola. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Unidad de Fitopatologia y Modelizacion Agricola.; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina

Circular RNAs (circRNAs) are covalently closed non-coding RNAs that are usually derived from exonic regions of genes, but can also arise from intronic and intergenic regions. Studies of circRNAs in humans, animals and several plant species have shown an altered population of circRNAs in response to abiotic and biotic stress. Recently it was shown that circRNAs also occur in maize, but it is unknown if maize circRNAs are responsive to stress. Maize Iranian mosaic virus (MIMV, genus Nucleorhabdovirus, family Rhabdoviridae) causes an economically important disease in maize and other gramineous crops in Iran. In this study, we used data from RNA-Seq of MIMV-infected maize and uninfected controls to identify differentially expressed circRNAs. Such circRNAs were confirmed by two-dimensional polyacrylamide gel electrophoresis, northern blot, RT-qPCR and sequencing. A total of 1443 circRNAs were identified in MIMV-infected maize and 1165 circRNAs in uninfected maize. Two hundred and one circRNAs were in common between MIMV-infected and uninfected samples. Of these, 155 circRNAs were up-regulated and 5 down-regulated in MIMV infected plants, compared to the uninfected control. This study for the first time identified and profiled circRNA expression in maize in response to virus infection. Moreover, we predict that 33 circRNAs may bind 23 maize miRNAs, possibly affecting plant metabolism and development. Our data suggest a role for circRNAs in plant cell regulation and response to biotic stress such as virus infection, and give new insights into the complexity of plant-microbe interactions.

Citrus black spot, caused by Phyllosticta citricarpa, is one of the most important fungal diseases in many citrus-growing regions with hot and humid summers. Ascospores and conidia are known to contribute to epidemic development of the disease. However, pathogenicity testing has never been done for pure ascospores produced from fully characterized P. citricarpa isolates, due to the inability to induce the sexual state in vitro. Recently, an in vitro mating technique was developed to readily produce pure P. citricarpa ascospores for use in host inoculation studies. To test the pathogenicity of P. citricarpa ascospores, we inoculated Troyer citrange leaves and Murcott tangor fruit with ascospores produced in vitro from characterized P. citricarpa isolates. Typical symptoms of citrus black spot occurred. Recovery of P. citricarpa isolates from symptomatic lesions and their characterization using genetic markers enabled us to identify recombinant genotypes among the isolates recovered from ascospore inoculations and, as such, fulfill Koch’s postulates for ascospores. We have also identified Troyer citrange seedlings as a potential model system for citrus black spot inoculation studies, because it allows typical symptoms of citrus black spot to be expressed with a much shorter latent period than on fruit. This will facilitate future studies of epidemiological aspects of P. citricarpa ascospores relative to conidia and improve our understanding of the citrus black spot pathosystem. The susceptibility of Troyer citrange seedlings will also facilitate experimenting with disease management methods, aimed at reducing the impact of citrus black spot.

In 2010, a novel cytorhabdovirus named alfalfa dwarf virus (ADV) was detected for the first time in lucerne crops in Argentina showing dwarfism, in mixed infections with several other viruses. ADV appears to be endemic to Argentina and has not been reported elsewhere. In this study, we have investigated the genetic variability of ADV based on the complete nucleoprotein (N) gene of 13 isolates from different lucerne-growing regions in Argentina. Phylogenetic and sequence identity analyses showed that all ADV isolates are closely related and have not diverged more than 1% in the N gene despite geographical separation. These data provide further evidence that ADV is new to science and emerged and spread very recently. A total of 43 single-nucleotide polymorphisms were identified between the ADV isolates studied. Analysis of N gene ORF sequence revealed a mutational bias, with more transitions than transversions. In all cases, the ratio of non-synonymous/synonymous nucleotide changes was

In 2010, a severe disease of lucerne (Medicago sativa L.) named alfalfa dwarf disease (ADD) was reported in Argentina. High throughput sequencing of diseased plants indicated the presence of five viruses, the (−) ssRNA virus, alfalfa dwarf virus (ADV), the (+) RNA viruses, alfalfa mosaic virus (AMV), bean leafroll virus (BLRV) and alfalfa enamovirus 1 (AEV-1) and the ssDNA virus, alfalfa leaf curl virus (ALCV). In this study, we determined which ADD-associated viruses are present in Australian lucerne that showed diverse virus-like symptoms. A duplex RT-PCR was developed for simultaneous detection of ADV and AMV using a cloned non-infectious ADV RNA fragment as positive control. Similarly, the presence of BLRV and AEV-1 was determined by duplex RT-PCR, and ALCV by PCR. Only AMV and BLRV that are endemic to Australia were detected. None of the novel exotic viruses ADV, AEV-1 and ALCV were detected in lucerne samples collected between 2015 and 2017. However, AMV and BLRV were detected in 78% and 70% of tested samples, respectively. Based on analysis of coat protein (CP) nucleotide sequences, Australian BLRV isolates are closely related to each other and to the Argentine Manfredi isolate. Phylogenetic analyses based on CP gene nucleotide sequences confirmed separation of AMV isolates into two subgroups. The majority of AMV isolates, including all those from Argentina and Australia, clustered in subgroup I with isolates from various hosts and geographic origins. All Australian AMV isolates were closely related to AMV isolated from ADD-affected lucerne in Argentina. Fil: Samarfard, S.. The University Of Queensland; Australia Fil: Bejerman, Nicolas. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet. Córdoba. Unidad de Fitopatología y Modernización Agrícola (UFYMA); Argentina Fil: Sharman, M.. The University Of Queensland; Australia Fil: Trucco, Verónica Milagros. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina Fil: Giolitti, Fabián José. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina Fil: Dietzgen, R. G.. The University Of Queensland; Australia

Maize Iranian mosaic virus (MIMV) is a negative-sense single-stranded RNA virus that is classified in the genus Nucleorhabdovirus, family Rhabdoviridae. The MIMV genome contains six open reading frames (ORFs) that encode in 3΄ to 5΄ order the nucleocapsid protein (N), phosphoprotein (P), putative movement protein (P3), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). In this study, we determined the first complete genome sequence of MIMV using Illumina RNA-Seq and 3'/5' RACE. MIMV genome ('Fars' isolate) is 12,426 nucleotides in length. Unexpectedly, the predicted N gene ORF of this isolate and of four other Iranian isolates is 143 nucleotides shorter than that of the MIMV coding-complete reference isolate 'Shiraz 1' (Genbank NC_011542), possibly due to a minor error in the previous sequence. Genetic variability among the N, P, P3 and G ORFs of Iranian MIMV isolates was limited, but highest in the G gene ORF. Phylogenetic analysis of complete nucleorhabdovirus genomes demonstrated a close evolutionary relationship between MIMV, maize mosaic virus and taro vein chlorosis virus.

Citrus black spot (Phyllosticta citricarpa) causes fruit blemishes and premature fruit drop, resulting in significant economic losses in citrus growing areas with summer rainfall across the globe. The mating type locus of P. citricarpa has recently been characterized, revealing the heterothallic nature of this pathogen. However, insight into the occurrence of mating and the impact of completing the sexual cycle of P. citricarpa was lacking. To investigate the occurrence and impact of sexual reproduction, we developed a method to reliably, and for the first time, produce ascospores of P. citricarpa on culture media. To demonstrate meiosis during the mating process, we identified recombinant genotypes through multilocus genotyping of single ascospores. Because the process of fertilization was not well understood, we experimentally determined that fertilization of P. citricarpa occurs via spermatization. Our results demonstrate that P. citricarpa is heterothallic and requires isolates of different MAT idiomorphs to be in direct physical contact, or for spermatia to fulfill their role as male elements to fertilize the receptive organs, in order to initiate the mating process. The impact of mating on the epidemiology of citrus black spot in the field is discussed.

RNA silencing in plants can be triggered by the introduction of an exogenous gene. Green fluorescent protein (GFP) has been widely used as a visual reporter to study RNA silencing and viral-mediated suppression of RNA silencing in the model plant Nicotiana benthamiana. In transgenic N. benthamiana plants expressing an endoplasmic reticulum targeted GFP variant (16c) known as mGFP5, RNA silencing can be induced by ectopic over-expression of mGFP5. However, other GFP variants can also be used to induce GFP silencing in these plants. We compared the efficiency to induce local and systemic silencing of two commonly used GFP variants: enhanced GFP (eGFP) and mGFP5. Using lettuce necrotic yellows virus (LNYV) P protein to suppress GFP silencing, we demonstrate that eGFP gene, which is 76% identical at the nucleotide level to the endogenously expressed mGFP5 in 16c plants, triggers silencing more slowly and concurrently prolongs detectable silencing suppressor activity of the weak LNYV P suppressor, compared to the homologous mGFP5 gene. The use of eGFP as RNA silencing inducer in wild type or 16c plants appears to be a useful tool in identifying and analysing weak viral RNA silencing suppressor proteins whose activity might otherwise have been masked when challenged by a stronger RNA silencing response. We also show that reducing the dosage of strong dsRNA silencing inducers in conjunction with their homologous GFP targets facilitates the discovery and analysis of "weaker" RNA silencing suppressor activities.

In October 2018, the order Mononegavirales was amended by the establishment of three new families and three new genera, abolishment of two genera, and creation of 28 novel species. This article presents the updated taxonomy of the order Mon-onegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).