Monopartite Genome Research Articles

Phylogeographic analysis of Begomovirus coat and replication-associated proteins.

Begomoviruses are globally distributed plant pathogens that significantly limit crop production. These viruses are traditionally described according to phylogeographic distribution and categorized into two groups: begomoviruses from the Africa, Asia, Europe and Oceania (AAEO) region and begomoviruses from the Americas. Monopartite begomoviruses are more common in the AAEO region, while bipartite viruses predominate in the Americas, where the begomoviruses lack the V2/AV2 gene involved in inter-cellular movement and RNA silencing suppression found in AAEO begomoviruses. While these features are generally accepted as lineage-defining, the number of known species has doubled due to sequence-based discovery since 2010. To re-evaluate the geographic groupings after the rapid expansion of the genus, we conducted phylogenetic analyses for begomovirus species representatives of the two longest and most conserved begomovirus proteins: the coat and replication-associated proteins. Both proteins still largely support the broad AAEO and Americas begomovirus groupings, except for sweet potato-infecting begomoviruses that form an independent, well-supported clade for their coat protein regardless of the region they were isolated from. Our analyses do not support more fine-scaled phylogeographic groupings. Monopartite and bipartite genome organizations are broadly interchanged throughout the phylogenies, and the absence of the V2/AV2 gene is highly reflective of the split between Americas and AAEO begomoviruses. We observe significant evidence of recombination within the Americas and within the AAEO region but rarely between the regions. We speculate that increased globalization of agricultural trade, the invasion of polyphagous whitefly vector biotypes and recombination will blur begomovirus phylogeographic delineations in the future.

Engineering a Sinorhizobium meliloti Chassis with Monopartite, Single Replicon Genome Configuration.

Multipartite bacterial genomes pose challenges for genome engineering and the establishment of additional replicons. We simplified the tripartite genome structure (3.65 Mbp chromosome, 1.35 Mbp megaplasmid pSymA, 1.68 Mbp chromid pSymB) of the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Strains with bi- and monopartite genome configurations were generated by targeted replicon fusions. Our design preserved key genomic features such as replichore ratios, GC skew, KOPS, and coding sequence distribution. Under standard culture conditions, the growth rates of these strains and the wild type were nearly comparable, and the ability for symbiotic nitrogen fixation was maintained. Spatiotemporal replicon organization and segregation were maintained in the triple replicon fusion strain. Deletion of the replication initiator-encoding genes, including the oriVs of pSymA and pSymB from this strain, resulted in a monopartite genome with oriC as the sole origin of replication, a strongly unbalanced replichore ratio, slow growth, aberrant cellular localization of oriC, and deficiency in symbiosis. Suppressor mutation R436H in the cell cycle histidine kinase CckA and a 3.2 Mbp inversion, both individually, largely restored growth, but only the genomic rearrangement recovered the symbiotic capacity. These strains will facilitate the integration of secondary replicons in S. meliloti and thus be useful for genome engineering applications, such as generating hybrid genomes.

Molecular characterization and identification of Stellaria aquatica virus C, a novel putative member of the genus Closterovirus infecting Stellaria aquatica in South Korea.

A novel virus infecting Stellaria aquatica plants, tentatively named "Stellaria aquatica virus C" (StAVC), was identified in Gangwon-do Province, South Korea. Its monopartite genome consists of a single-stranded RNA of 15,024 nucleotides, and it shares 38.24 to 56.2% nucleotide sequence identity with known closterovirus genome sequences. Its genome contains nine hypothetical open reading frames. These encode the multifunctional protein RNA-dependent RNA polymerase (RdRp), hydrophobic protein (P7), heat shock protein 70 homolog (HSP70h), coat protein homolog (CPh), minor coat protein (CPm), and major coat protein (CP), along with proteins involved in suppressing RNA silencing. Phylogenetic analysis reveals that, based on its HSP70h amino acid sequence, StAVC is closely related to members of the genus Closterovirus within the family Closteroviridae. This is the first record of the full genome sequence of StAVC in South Korea.

A circular single-stranded DNA mycovirus infects plants and confers broad-spectrum fungal resistance

Circular single-stranded DNA (ssDNA) viruses have been rarely found in fungi, and the evolutionary and ecological relationships among ssDNA viruses infecting fungi and other organisms remain unclear. In this study, a novel circular ssDNA virus, tentatively named Diaporthe sojae circular DNA virus 1 (DsCDV1), was identified in the phytopathogenic fungus Diaporthe sojae isolated from pear trees. DsCDV1 has a monopartite genome (3185 nt in size) encapsidated in isometric virions (21–26 nm in diameter). The genome comprises seven putative open reading frames encoding a discrete replicase (Rep) split by an intergenic region, a putative capsid protein (CP), several proteins of unknown function (P1–P4), and a long intergenic region. Notably, the two split parts of DsCDV1 Rep share high identities with the Reps of Geminiviridae and Genomoviridae, respectively, indicating an evolutionary linkage with both families. Phylogenetic analysis based on Rep or CP sequences placed DsCDV1 in a unique cluster, supporting the establishment of a new family, tentatively named Gegemycoviridae, intermediate to both families. DsCDV1 significantly attenuates fungal growth and nearly erases fungal virulence when transfected into the host fungus. Remarkably, DsCDV1 can systematically infect tobacco and pear seedlings, providing broad-spectrum resistance to fungal diseases. Subcellular localization analysis revealed that DsCDV1 P3 is systematically localized in the plasmodesmata, while its expression in trans-complementation experiments could restore systematic infection of a movement-deficient plant virus, suggesting that P3 is a movement protein. DsCDV1 exhibits unique molecular and biological traits not observed in other ssDNA viruses, serving as a link between fungal and plant ssDNA viruses and presenting an evolutionary connection between ssDNA viruses and fungi. These findings contribute to expanding our understanding of ssDNA virus diversity and evolution, offering potential biocontrol applications for managing crucial plant diseases.

Whole genome sequence analysis of shallot virus X from India reveals it to be a natural recombinant with positive selection pressure

BackgroundShallots are infected by various viruses like Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Shallot latent virus (SLV) and Shallot virus X (ShVX). In India, they have been found to be persistently infected by ShVX. ShVX also infects onion and garlic in combination with other carlaviruses and potyviruses. ShVX is a member of genus Allexivirus of family Alphaflexiviridae. ShVX has a monopartite genome, which is represented by positive sense single-stranded RNA. Globally, only six complete and 3 nearly complete genome sequences of ShV X are reported to date. This number is insufficient to measure a taxon’s true molecular diversity. Moreover, the complete genome sequence of ShVX from Asia has not been reported as yet. Therefore, this study was undertaken to generate a complete genome sequence of ShVX from India.ResultsShallot virus X (ShVX) is one of the significant threats to Allium crop production. In this study, we report the first complete genome sequence of the ShVX from India through Next-generation sequencing (NGS). The complete genome of the ShVX (Accession No. OK104171), from this study comprised 8911 nucleotides. In-silico analysis of the sequence revealed variability between this isolate and isolates from other countries. The dissimilarities are spread all over the genome specifically some non-coding intergenic regions. Statistical analysis of individual genes for site-specific selection indicates a positive selection in NABP region. The presence of a recombination event was detected in coat protein region. The sequence similarity percentage and phylogenetic analysis indicate ShVX Indian isolate is a distinctly different isolate. Recombination and site-specific selection may have a function in the evolution of this isolate. This is the first detailed study of the ShVX complete genome sequence from Southeast Asia.ConclusionThis study presents the first report of the entire genome sequence of an Indian isolate of ShVX along with an in-depth exploration of its evolutionary traits. The findings highlight the Indian variant as a naturally occurring recombinant, emphasizing the substantial role of recombination in the evolution of this viral species. This insight into the molecular diversity of strains within a specific geographical region holds immense significance for comprehending and forecasting potential epidemics. Consequently, the insights garnered from this research hold practical value for shaping ShVX management strategies and providing a foundation for forthcoming studies delving into its evolutionary trajectory.

Genome-Wide Identification of Cotton MicroRNAs Predicted for Targeting Cotton Leaf Curl Kokhran Virus-Lucknow

Cotton leaf curl Kokhran virus (CLCuKoV) (genus, Begomovirus; family, Geminiviridae) is one of several plant virus pathogens of cotton (Gossypium hirsutum L.) that cause cotton leaf curl disease in Pakistan. Begomoviruses are transmitted by the whitefly Bemisia tabaci cryptic species group and cause economic losses in cotton and other crops worldwide. The CLCuKoV strain, referred to as CLCuKoV-Bur, emerged in the vicinity of Burewala, Pakistan, and was the primary causal virus associated with the second CLCuD epidemic in Pakistan. The monopartite ssDNA genome of (2.7 Kb) contains six open reading frames that encode four predicted proteins. RNA interference (RNAi)-mediated antiviral immunity is a sequence-specific biological process in plants and animals that has evolved to combat virus infection. The objective of this study was to design cotton locus-derived microRNA (ghr-miRNA) molecules to target strains of CLCuKoV, with CLCuKoV-Lu, as a typical CLCuD-begomovirus genome, predicted by four algorithms, miRanda, RNA22, psRNATarget, and RNA hybrid. Mature ghr-miRNA sequences (n = 80) from upland cotton (2n = 4x = 52) were selected from miRBase and aligned with available CLCuKoV-Lu genome sequences. Among the 80 cotton locus-derived ghr-miRNAs analyzed, ghr-miR2950 was identified as the most optimal, effective ghr-miRNA for targeting the CLCuKoV-Lu genome (nucleotide 82 onward), respectively, based on stringent criteria. The miRNA targeting relies on the base pairing of miRNA–mRNA targets. Conservation and potential base pairing of binding sites with the ghr-miR2950 were validated by multiple sequence alignment with all available CLCuKoV sequences. A regulatory interaction network was constructed to evaluate potential miRNA–mRNA interactions with the predicted targets. The efficacy of miRNA targeting of CLCuKoV was evaluated in silico by RNAi-mediated mRNA cleavage. This predicted targets for the development of CLCuD-resistant cotton plants.

Pepper mild mottle virus: a formidable foe of capsicum production—a review

Viruses are one of the major restraining factors in pepper cultivation globally. Among different viruses, pepper mild mottle virus (PMMoV) is one of the most detrimental plant viruses infecting Capsicum spp. belonging to the genus Tobamovirus and Virgaviridae family. It has a monopartite positive-sense single-stranded RNA genome of 6.35 kb size. On an average, PMMoV results in 15%–40% losses in capsicum fruit yield. However, the incidence of PMMoV can reach as high as 95%, leading to substantial yield losses ranging from 75% to 95%. The virus is transmitted via contact, soil, and seeds rather than via insect vectors. PMMoV, because of its seed-borne nature, now occurs worldwide. PMMoV mainly infects Capsicum spp. under natural conditions; however, it can experimentally be transmitted to other plants species belonging to the families Solanaceae, Cucurbitaceae, Labiatae, Chenopodiaceae, and Plantaginaceae. The resistance to tobamoviruses in capsicum is conferred by L locus. Mutations in the coat protein of PMMoV are responsible for the emergence of L-mediated resistance-breaking pathotypes. The highly contagious nature of the virus, seed transmission behavior, and the emergence of virulence complicate its management through a single approach. Therefore, efforts are directed towards providing a more practical and efficient integrated management solution using the RNA interference approach; exploitation of the L gene for resistance breeding; and the inhibitory potential of natural products, systemic resistance-inducing antagonistic bacteria, and chemically synthesized silver nanoparticles. Markers linked to L alleles have been observed to accelerate capsicum breeding programs through marker-assisted selection. In this study, an attempt has been made to compile the recent developments in PMMoV biology, pathogenic variability, genomic organization, and management strategies.

Tomato golden net virus and tomato yellow net virus: two novel New World begomoviruses with monopartite genomes.

Two novel tomato-infecting begomoviruses were discovered via high-throughput sequencing in Brazil. Both viruses were also Sanger-sequenced and displayed DNA-A components phylogenetically related to New World bipartite begomoviruses. The names tomato golden net virus (ToGNV) and tomato yellow net virus (ToYNV) were proposed. The majority of the New World begomoviruses has bipartite genomes. However, extensive analyses revealed that ToGNV and ToYNV have monopartite genomes, because no cognate DNA-B components were detected. Hence, they may comprise a unique group of monopartite New World begomoviruses, which have enormous biological, molecular, and plant breeding interest.

Identification of a novel dicistro-like virus associated with the roots of tomato plants.

Viruses belonging to the family Dicistroviridae have a monopartite positive-sense single-stranded RNA genome and infect a variety of arthropods. Using high-throughput sequencing, we detected a novel dicistro-like virus, tentatively named "tomato root-associated dicistro-like virus" (TRaDLV), in the roots of tomato plants showing yellow mosaic symptoms on the leaves. The diseased tomato plants were coinfected with multiple plant viruses, and TRaDLV was present in the roots but not in the leaves. The genome of TRaDLV is 8726 nucleotides in length, excluding the poly(A) tail, and contains two open reading frames (ORFs) separated by an intergenic region (IGR). The TRaDLV genome showed characteristics similar to those of dicistroviruses, including the presence of a 3C-like protease domain, repeated amino acid sequences representing multiple copies of viral genome-linked protein (VPg)-like sequences in the ORF1 polyprotein, and a series of stem-loop structures resembling an internal ribosome entry site in the IGR. Phylogenetic analysis revealed that TRaDLV clustered with unclassified dicistro-like viruses from invertebrates or identified in samples of plant-derived material. These findings indicate the existence of a novel dicistro-like virus that may associate with plant roots or a root-inhabiting organism.

Characteristics of the Complete Genome of Ageratum Yellow Vein China Virus Infecting Sonchus oleraceus.

Sonchus (Sonchus oleraceus) originated from Europe and is now cultivated worldwide. The wild resources of sonchus are very abundant, and it has rich nutritional and medicinal value. In this study, 15 sonchus samples with typical symptoms showing leaf curling, vein thickening, and enations were collected from Guigang and Baise City of Guangxi, China. Diseased sonchus were identified by PCR detection, whole genome sequence amplification, and phylogenetic and recombination analysis. The results showed that all the samples were confirmed infected by begomoviruses, and three full-length viral genomes were obtained from 15 sonchus, named GG7-13, GG8-6, and BS63-5. The full genome lengths were 2,584, 2,735, and 2,746 nt, respectively. The nucleotide identities among the three isolates ranged from 92.67 to 99.93%. All of them shared the highest identities (greater than 91.69%) with other isolates of ageratum yellow vein China virus (AYVCNV) (available on GenBank). According to the guidelines of classification of begomoviruses, the virus isolates obtained in this study are different isolates of AYVCNV; a phylogenetic tree analysis showed that these isolates formed a large branch with three other Guangxi isolates of AYVCNV, indicating their close evolution. The genome structures of GG8-6 and BS63-5 are consistent with the monopartite genome virus of the begomoviruses, and both have six open reading frames (ORFs), while GG7-13 has a 151-nt deletion between C2 and C3, resulting in a mutant strain of only five ORFs. This study is the first report on S. oleraceus infected by ageratum yellow vein China virus.

Bipartite Genomes in Enterobacterales: Independent Origins of Chromids, Elevated Openness and Donors of Horizontally Transferred Genes

Multipartite bacteria have one chromosome and one or more chromid. Chromids are believed to have properties that enhance genomic flexibility, making them a favored integration site for new genes. However, the mechanism by which chromosomes and chromids jointly contribute to this flexibility is not clear. To shed light on this, we analyzed the openness of chromosomes and chromids of the two bacteria, Vibrio and Pseudoalteromonas, both which belong to the Enterobacterales order of Gammaproteobacteria, and compared the genomic openness with that of monopartite genomes in the same order. We applied pangenome analysis, codon usage analysis and the HGTector software to detect horizontally transferred genes. Our findings suggest that the chromids of Vibrio and Pseudoalteromonas originated from two separate plasmid acquisition events. Bipartite genomes were found to be more open compared to monopartite. We found that the shell and cloud pangene categories drive the openness of bipartite genomes in Vibrio and Pseudoalteromonas. Based on this and our two recent studies, we propose a hypothesis that explains how chromids and the chromosome terminus region contribute to the genomic plasticity of bipartite genomes.

First report of Tomato leaf curl Karnataka virus associated with leaf curl disease of Zinnia elegans in India

Tomato leaf curl Karnataka virus (ToLCKV) is a species of begomovirus which has a circular, ssDNA monopartite genome and is transmitted by the whitefly (Bemisia tabaci). Zinnia elegans is a popular ornamental bedding plant which is grown worldwide for its beautiful flowers. A few begomovirus species have been reported to infect Z. elegans, including Ageratum yellow vein China virus in Vietnam (Li et al., 2013), and Ageratum enation virus and Zinnia leaf curl virus in India (Kumar et al., 2010; Pandey & Tiwari, 2012). In March 2019 symptoms of excessive leaf curling with mild mosaic were observed on Z. elegans plants growing in a nursery in Bhopal, central India (Fig. 1). To investigate the aetiology of the disease total DNA was extracted from leaf samples from two diseased and one asymptomatic Z. elegans plant using a plant genomic DNA purification kit (Promega, USA), following the manufacturer's instructions. A begomovirus was detected from both diseased samples (but not the asymptomatic sample) by PCR using begomovirus-specific primers: CPIT-I/CPIT-T (AM180920/AM180921) (Singh, 2005), PALIv 1978/ PARIc 496 and PALIv 722/ PALIc 1960 (Rojas et al., 1993). The expected amplicons (~800 bp, 1.2 Kb and 1.2 Kb, respectively) were purified using a PCR Clean-up System (Promega, USA) and Sanger sequenced in both directions by Bioinnovations Connecting Technologies to Life (Mumbai, India). The consensus sequence from both samples was identical and the complete viral DNA-A genome of 2761 nucleotides was deposited in GenBank (Accession No. MK965196). Sequence analysis was done by BLASTn and showed that the DNA-A genome had the highest identity (96-97%) with ToLCKV (KX219744, HM007094, LN878123, U38239); Sunflower leaf curl virus (JX678965) and 94–95% with Tomato leaf curl virus (JX524172, KX831454). Phylogenetic analysis using neighbour-joining in MEGA v.7.0 showed a close relationship with isolates of ToLCKV and relationships with other begomoviruses (Fig. 2). The disease was successfully transmitted by B. tabaci from naturally infected Z. elegans to healthy seedlings of Nicotiana tabacum, Solanum lycopersicum and Z. elegans (each 3/3), symptoms being observed 30 days post inoculation. The test plants all tested positive by PCR using the begomovirus-specific primer pairs above. Positive amplicons were sequenced and the nucleotide sequences were identical to each other and the original isolate (MK965196). The present study demonstrates that Z. elegans is a host of ToLCKV. ToLCKV is a serious threat to the production of several economically important crops in India and has been reported at an incidence of >80% on soybean, sunflower and tomato in India (Malathi et al., 2017). Recently, ToLCKV and satellite molecules have been found associated with enation leaf curl disease of another ornamental plant, Pseuderanthemum reticulatum, in India (Ashwathappa et al., 2021). The whitefly transmission study provides additional evidence that ToLCKV causes the observed disease in Z. elegans and it is possible that ornamental plants may act as a reservoir of the virus. To the best of our knowledge, this is the first report of the association of ToLCKV with leaf curl disease of Z. elegans. The authors are thankful to the Vice Chancellor of Barkatullah University, Bhopal, India, for use of the facilities and the UGC, New Delhi, India for financial support under the UGC-BSR Research Start-Up grant.

A novel fungal negative-stranded RNA virus related to mymonaviruses in Auricularia heimuer.

Here, we report the characterization of a novel (-)ssRNA mycovirus isolated from Auricularia heimuer CCMJ1222, using a combination of RNA-seq, reverse transcription polymerase chain reaction, 5' and 3' rapid amplification of cDNA ends, and Sanger sequencing. Based on database searches, sequence alignment, and phylogenetic analysis, we designated the virus as "Auricularia heimuer negative-stranded RNA virus 1" (AhNsRV1). This virus has a monopartite RNA genome related to mymonaviruses (order Mononegavirales). The AhNsRV1 genome consists of 11,441 nucleotides and contains six open reading frames (ORFs). The largest ORF encodes a putative RNA-dependent RNA polymerase; the other ORFs encode hypothetical proteins with no conserved domains or known function. AhNsRV1 is the first (-)ssRNA virus and the third virus known to infect A. heimuer.

Three new mycoviruses identified in the apple replant disease (ARD)-associated fungus Rugonectria rugulosa

In this study, three new mycoviruses were identified co-infecting the apple replant disease (ARD)-associated root endophyte Rugonectria rugulosa. After dsRNA extraction, six viral fragments were visualized. Four fragments belong to a quadrivirus, which has a genome size of 17,166 bp. Each of the fragments of this quadrivirus has a single ORF encoding a protein. Two of these proteins are coat protein subunits, one ORF encodes the RdRp, and one protein has an unknown function. This virus was tentatively named rugonectria rugulosa quadrivirus 1 (RrQV1) as a member of the proposed new species Quadrivirus rugonectria. Another fragment represents the dsRNA intermediate form of a + ssRNA mitovirus with a genome size of 2410 nt. This virus encodes an RdRp and is tentatively called rugonectria rugulosa mitovirus 1 (RrMV1). RrMV1 is suggested as a member of a new species with the proposed name Mitovirus rugonectria. The sixth fragment belongs to the genome of an unclassified dsRNA virus tentatively called rugonectria rugulosa dsRNA virus 1 (RrV1). The monopartite dsRNA genome of RrV1 has a length of 8964 bp and contains two ORFs encoding a structure/gag protein and an RdRp. Full genomic sequences were determined and the genome structure as well as molecular properties are presented. After phylogenetic studies and sequence identity analyses, all three isolates are proposed as new mycoviruses. The results help to improve the understanding of the complexity of the factors involved in ARD and support the interest in mycoviral research. Subsequent analyses need to focus on the impact of mycoviruses on the biology and pathogenicity of ARD-associated fungi. The results of such studies could contribute to the development of mitigation strategies against the disease.

Identification and Characterization of Two Novel Geminiviruses Associated with Paper Mulberry (Broussonetia papyrifera) Leaf Curl Disease.

Paper mulberry (Broussonetia papyrifera) is a perennial woody plant used as source material for Cai Lun paper making, in traditional Chinese medicine, and as livestock feed. To identify the presence of viruses in paper mulberry plants affected by a disease with leaf curl symptoms, high-throughput sequencing of total RNA was performed. Analysis of transcriptome libraries allowed the reconstruction of two geminivirus-like genomes. Rolling-circle amplification and PCR with back-to-back primers confirmed the presence of two geminiviruses with monopartite genomes in these plants, with the names paper mulberry leaf curl virus 1 and 2 (PMLCV-1 and PMLCV-2) proposed. The genomes of PMLCV-1 (3,056 nt) and PMLCV-2 (3,757 to 3,763 nt) encode six proteins, with the V4 protein of PMLCV-1 and the V3 proteins of both viruses having low similarities to any known protein in databases. Alternative splicing of an intron, akin to that of mastre-, becurto-, capula-, and grabloviruses, was identified by small RNA (sRNA)-seq and RNA-seq reads mapping to PMLCV-1 and PMLCV-2 antisense transcripts. Phylogenetic analyses and pairwise comparisons showed that PMLCV-1 and PMLCV-2 are most closely related to, but distinct from, two unassigned geminiviruses, citrus chlorotic dwarf associated virus and mulberry mosaic dwarf associated virus, suggesting that they are two new members of the family Geminiviridae. Field investigation confirmed the close association of the two viruses with leaf curl symptoms in paper mulberry plants and that coinfection can aggravate the symptoms.

Description of a Novel Mycovirus in the Phytopathogen Fusarium culmorum and a Related EVE in the Yeast Lipomyces starkeyi.

A new mycovirus was found in the Fusarium culmorum strain A104-1 originally sampled on wheat in Belgium. This novel virus, for which the name Fusarium culmorum virus 1 (FcV1) is suggested, is phylogenetically related to members of the previously proposed family ‘’Unirnaviridae’’. FcV1 has a monopartite dsRNA genome of 2898 bp that harbors two large non-overlapping ORFs. A typical -1 slippery motif is found at the end of ORF1, advocating that ORF2 is translated by programmed ribosomal frameshifting. While ORF2 exhibits a conserved replicase domain, ORF1 encodes for an undetermined protein. Interestingly, a hypothetically transcribed gene similar to unirnaviruses ORF1 was found in the genome of Lipomyces starkeyi, presumably resulting from a viral endogenization in this yeast. Conidial isolation and chemical treatment were unsuccessful to obtain a virus-free isogenic line of the fungal host, highlighting a high retention rate for FcV1 but hindering its biological characterization. In parallel, attempt to horizontally transfer FcV1 to another strain of F. culmorum by dual culture failed. Eventually, a screening of other strains of the same fungal species suggests the presence of FcV1 in two other strains from Europe.

Genome organization and interaction with capsid protein in a multipartite RNA virus

We report the asymmetric reconstruction of the single-stranded RNA (ssRNA) content in one of the three otherwise identical virions of a multipartite RNA virus, brome mosaic virus (BMV). We exploit a sample consisting exclusively of particles with the same RNA content-specifically, RNAs 3 and 4-assembled in planta by agrobacterium-mediated transient expression. We find that the interior of the particle is nearly empty, with most of the RNA genome situated at the capsid shell. However, this density is disordered in the sense that the RNA is not associated with any particular structure but rather, with an ensemble of secondary/tertiary structures that interact with the capsid protein. Our results illustrate a fundamental difference between the ssRNA organization in the multipartite BMV viral capsid and the monopartite bacteriophages MS2 and Qβ for which a dominant RNA conformation is found inside the assembled viral capsids, with RNA density conserved even at the center of the particle. This can be understood in the context of the differing demands on their respective lifecycles: BMV must package separately each of several different RNA molecules and has been shown to replicate and package them in isolated, membrane-bound, cytoplasmic complexes, whereas the bacteriophages exploit sequence-specific "packaging signals" throughout the viral RNA to package their monopartite genomes.

Characterization of a New Monopartite Begomovirus with a Betasatellite Associated with Leaf Curl, Yellow Vein, and Vein Enation inHibiscus rosa-sinensis

A new begomovirus, tentatively named hibiscus yellow vein leaf curl virus (HYVLCV), was identified in Hibiscus rosa-sinensis plants showing symptoms of leaf curl, yellow vein, and vein enation on the undersides of the leaf in Taiwan. Sequence analysis of the full-length HYVLCV genome from the rolling cycle amplicon revealed a genome of 2,740 nucleotides that contains six open reading frames and a conserved sequence (5'-TAATATTAC-3') commonly found in geminiviral genomes. HYVLCV shares the highest nucleotide identity (88.8%) with cotton leaf curl Multan virus (CLCuMuV) genome, which is lower than the criteria (91%) set for species demarcation in the genus Begomovirus. No begomoviral DNA-B was detected; however, a begomovirus-associated DNA betasatellite (DNA-β) was detected. The DNA-β (1,355 nucleotides) shares the highest nucleotide identity (78.6%) with malvastrum yellow vein betasatellite (MaYVB). Because the identity is slightly higher than the criteria (78%) set for the species demarcation threshold for a distinct DNA-β species, the DNA-β of HYVLCV reported in this study is considered the same species of MaYVB and tentatively named MaYVB-Hib. An expected 1,498-bp fragment was amplified with two HYVLCV-specific primers from 10 of 11 field-collected samples. Four independent amplicons were sequenced, revealing 100% nucleotide identity with the HYVLCV genome. Agroinoculation of a dimer of the infectious monopartite genome alone to Nicotiana benthamiana resulted in mild symptoms at 28 days postinoculation (dpi); coagroinoculation with the DNA-β satellite resulted in severe symptoms at 12 dpi. HYVLCV could be transmitted to healthy H. rosa-sinensis by grafting, resulting in yellow vein symptoms at 30 dpi.

Reconstruction and Characterization of Full-Length Begomovirus and Alphasatellite Genomes Infecting Pepper through Metagenomics

In northwestern Argentina (NWA), pepper crops are threatened by the emergence of begomoviruses due to the spread of its vector, Bemisia tabaci (Gennadius). The genus Begomovirus includes pathogens that can have a monopartite or bipartite genome and are occasionally associated with sub-viral particles called satellites. This study characterized the diversity of begomovirus and alphasatellite species infecting pepper in NWA using a metagenomic approach. Using RCA-NGS (rolling circle amplification-next generation sequencing), 19 full-length begomovirus genomes (DNA-A and DNA-B) and one alphasatellite were assembled. This ecogenomic approach revealed six begomoviruses in single infections: soybean blistering mosaic virus (SbBMV), tomato yellow spot virus (ToYSV), tomato yellow vein streak virus (ToYVSV), tomato dwarf leaf virus (ToDfLV), sida golden mosaic Brazil virus (SiGMBRV), and a new proposed species, named pepper blistering leaf virus (PepBLV). SbBMV was the most frequently detected species, followed by ToYSV. Moreover, a new alphasatellite associated with ToYSV, named tomato yellow spot alphasatellite 2 (ToYSA-2), was reported for the first time in Argentina. For the Americas, this was the first report of an alphasatellite found in a crop (pepper) and in a weed (Leonurus japonicus). We also detected intra-species and inter-species recombination.

First Report of Cotton leaf curl Gezira virus and Its Associated Alphasatellite and Betasatellite from Disease Affected Okra Plants in the United States

Okra (Abelmoschus esculentus [L.] Moench) is an important fresh market vegetable crop in southern Texas, U.S.A. In fall 2018, an investigation was conducted to identify the agent(s) of a virus-like disease outbreak on a 4.9-ha okra (cv. Clemson Spineless) farm in Hidalgo Co., Texas. By mid-growing season, disease incidence was 100%, which led to total crop loss. Symptoms—foliar yellow mosaic, bleaching, and vein clearing—were reminiscent of those caused by begomoviruses (Begomovirus, Geminiviridae). Leaves collected from 10 randomly distributed symptomatic plants were subjected to total DNA isolation (Dellaporta et al. 1983) and rolling cycle amplification (RCA) (TempliPhi 100 Amplification Kit, GE Healthcare Biosciences). The RCA product was used as a template for PCR amplification of a diagnostic fragment of the DNA-A (Wyatt and Brown 1996) and DNA-B (Rojas et al. 1993) components and full-length alphasatellites (Bull et al. 2003) and betasatellites (Briddon et al. 2002). The expected-size amplicon corresponding to each of the four targets was obtained from all 10 samples, except the betasatellite, for which half of the expected ∼1,350-bp product resulted. Amplicons were cloned into the pJET1.2 plasmid vector, and inserts of the expected sizes were Sanger sequenced (n ≥ 3 per event). BLASTn searches of GenBank revealed an unexpectedly complex begomovirome, consisting of the monopartite cotton leaf curl Gezira virus (CLCuGV), cotton leaf curl Gezira alphasatellite (CLCuGA; Colecusatellite, Alphasatellitidae), cotton leaf curl Gezira betasatellite (CLCuGB; Betasatellite, Tolecusatellitidae), and the bipartite okra yellow mosaic Mexico virus (OkYMMV). Viral genome-, component-, and satellite-specific abutting primers were designed and used to PCR amplify the corresponding molecule from two RCA products in two different labs, followed by cloning and bidirectional Sanger sequencing of ≥3 clones per event. The complete genome sequences were analyzed comparatively with corresponding GenBank sequences of each genomic DNA segment using SDT1.2 (Muhire et al. 2014). The OkYMMV DNA-A of 2,613 nt (GenBank MN027195 to 96) and DNA-B of 2,594 nt (MN027197 to 98), each shared 99.5 and 99.7% maximum identity, respectively, with the DNA-A (HM035059) and -B (HM035060) of OkYMMV-[US:TX:09] from okra plants in Texas (Hernandez-Zepeda et al. 2010). The complete monopartite genome sequence of the CLCuGV okra isolates (MN027199 to 202) was 2,764 nt and shared 99.5 to 99.6% maximum identity with an isolate of CLCuGV from hollyhock plants in Jordan (CLCuGV-[JO:Hol:09]: GU945265). The closest relative of the CLCuGA okra isolate, of 1,354 nt (MN027203 to 05), was CLCuGA-[IL:IsSq-C171:11] (KT099176) from whiteflies collected from squash plants in Israel, at 99% identity. Several approaches by two independent laboratories to clone and sequence a full-length CLCuGB, involving restriction digestion from total DNA or RCA (n = 2) and/or PCR amplification with abutting primers designed based on the full-length CLCuGB sequences (AF397217, KT099178), yielded only “defective-sized” CLCuGB molecules of 676 to 730 nt (MN027206 to 220). The betasatellite half-mers lacked the βC1 coding region while sharing 96 to 99% identity with CLCuGB-[EG:OK03:02] (AF397217) from okra plants in Egypt and CLCuGB-[IL:IsSq-C9:11] (KT099178) from whiteflies. This is the first U.S. report of CLCuGV and its associated alphasatellite and betasatellite, which are endemic to Africa (Idris and Brown 2000). More recently, members of the CLCuGV-satellite complex have been reported in the African Sahel, Arabian Peninsula and Middle East, and Pakistan (Idris et al. 2014; Tahir et al. 2011; Tiendrebeogo et al. 2010). The source of this Old World CLCuGV-satellite complex into New World okra plants is unknown, but its introduction via infected plants or viruliferous whiteflies transported into southern Texas, with its intensive cotton and vegetable production, is plausible. Spread of this complex poses a serious threat because U.S. cotton and vegetable varieties harbor no resistance to CLCuGV.