Exploring the viral landscape of saffron through metatranscriptomic analysis.

Saffron (Crocus sativus L.), a historically significant crop valued for its nutraceutical properties, has been poorly explored from a phytosanitary perspective. This study conducted a thorough examination of viruses affecting saffron samples from Spanish cultivars, using high-throughput sequencing alongside a systematic survey of transcriptomic datasets from Crocus sativus at the Sequence Read Archive. Our analysis unveiled a broad diversity and abundance, identifying 17 viruses across the 52 analyzed libraries, some of which were highly prevalent. This includes known saffron-infecting viruses and previously unreported ones. In addition, we discovered 7 novel viruses from the Alphaflexiviridae, Betaflexiviridae, Potyviridae, Solemoviridae, and Geminiviridae families, with some present in libraries from various locations. These findings indicate that the saffron-associated virome is more complex than previously reported, emphasizing the potential of phytosanitary analysis to enhance saffron productivity.

Real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for detection of cassava brown streak viruses.

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the most economically important viral disease of cassava. As cassava is a vegetatively propagated crop, the development of rapid and sensitive diagnostics would aid in the identification of virus-free planting material and development of effective management strategies. In this study, a rapid, specific and sensitive real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed for real-time detection of CBSV and UCBSV. The RT-RPA was able to detect as little as 2 pg/µl of purified RNA obtained from infected cassava leaves, a sensitivity equivalent to that obtained by quantitative real-time reverse transcription PCR (qRT-PCR), within 20 min at 37 °C. Further, the RT-RPA detected each target virus directly from crude leaf and stem extracts, avoiding the tedious and costly isolation of high-quality RNA. The developed RT-RPA assay provides a valuable diagnostic tool that can be adopted by cassava seed certification and virus resistance breeding programs to ensure distribution of virus-free cassava planting materials to farmers. This is the first report on the development and validation of crude sap-based RT-RPA assay for the detection of cassava brown streak viruses (UCBSV and CBSV) infection in cassava plants.

Arachis virus Y, a new potyvirid from Brazilian forage peanut (Arachis pintoi).

The complete nucleotide sequence of a new member of the family Potyviridae, which we propose to name "Arachis virus Y" (ArVY), is reported from forage peanut plants (Arachis pintoi) exhibiting virus-like symptoms. The ArVY positive-sense RNA genome is 9,213 nucleotides long and encodes a polyprotein with 2,947 amino acids that is predicted to be cleaved into 10 mature proteins. The complete single open reading frame (ORF) of ArVY shares 47% and 34% nucleotide and amino acid sequence identity, respectively, with the closest related virus, soybean yellow shoot virus. Electron microscopic analysis revealed elongated viral particles typical of those found in plant cells infected with potyviruses.

Complete genome sequence of a German isolate of spartina mottle virus supports its classification as a member of the proposed genus "Sparmovirus" within the family Potyviridae.

Spartina mottle virus (SpMV), an unassigned member of the family Potyviridae, has been known since 1980, when it was first described in England and Wales in symptomatic plants of the genus Spartina. In infected cells, flexuous particles and pinwheel inclusion bodies were found that resemble those of potyvirids. To date, the NCBI database contains only two partial sequences of a German (Nessmersiel) and an Italian (Assisi) isolate, suggesting that SpMV could be the first member of a new genus, called "Sparmovirus", in the family Potyviridae. In this study, the first complete genome sequence of the German SpMV isolate (SpMV Ger) was determined. The genome of SpMV is a single-stranded, monopartite, polyadenylated RNA consisting of 9376 nucleotides. Sequence analysis revealed a genome organization similar to that of classical potyviruses, including many conserved features. In phylogenetic analysis, SpMV could not be assigned to any of the known genera, but it showed the closest relationship to rymoviruses and common reed chlorotic stripe virus (CRCSV, unassigned). Sequence comparisons confirmed that a new genus should be established containing SpMV, CRCSV, and three Bermuda grass mosaic virus isolates, which are considered divergent strains of SpMV.

Bymovirus-induced yellow mosaic diseases in barley and wheat: viruses, genetic resistances and functional aspects.

Bymovirus-induced yellow mosaic diseases seriously threaten global production of autumn-sown barley and wheat, which are two of the presently most important crops around the world. Under natural field conditions, the diseases are caused by infection of soil-borne plasmodiophorid Polymyxa graminis-transmitted bymoviruses of the genus Bymovirus of the family Potyviridae. Focusing on barley and wheat, this article summarizes the achievements on taxonomy, geography and host specificity of these disease-conferring viruses, as well as the genetics of resistance in barley, wheat and wild relatives. Moreover, based on recent progress of barley and wheat genomics, germplasm resources and large-scale sequencing, the exploration and isolation of corresponding resistant genes from wheat and barley as well as relatives, no matter what a large and complicated genome is present, are becoming feasible and are discussed. Furthermore, the foreseen advances on cloning of the resistance or susceptibility-encoding genes, which will provide the possibility to explore the functional interaction between host plants and soil-borne viral pathogens, are discussed as well as the benefits for marker-assisted resistance breeding in barley and wheat.

Bulked segregant RNA-sequencing (BSR-seq) identified a novel rare allele of eIF4E effective against multiple isolates of BaYMV/BaMMV.

KEY MESSAGE: A novel rare allele of the barley host factor gene eIF4E for BaMMV/BaYMV infection was identified in an Iranian landrace that showed broad resistance to barley yellow mosaic virus disease, and molecular markers facilitating efficient selection were developed. The soil-borne yellow mosaic virus disease caused by different strains of barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV) is a major threat to winter barley (Hordeum vulgare) production in Europe and East Asia. However, the exploration of resistant germplasm or casual genes for barley breeding is rather limited in relation to the rapid diversification of viral strains. Here, we identified an Iranian barley landrace 'HOR3298,' which represented complete resistance to BaYMV and BaMMV. In contrast to rym4 and rym5, which act as the predominant source in Europe and East Asia for breeding resistant cultivars over decades and which have been overcome by several virulent isolates, this landrace showed broad-spectrum resistance to multiple isolates of BaYMV/BaMMV in the fields of Germany and China. By employment of bulked segregant RNA sequencing, test for allelism, and haplotype analysis, a recessive resistance gene in 'HOR3298' was genetically mapped coincident with the host factor eukaryotic translation initiation factor 4E (eIF4E, causal gene of rym4 and rym5). The eIF4EHOR3298 allele encoded for a novel haplotype that contained an exclusive nucleotide mutation (G565A) in the coding sequence. The easily handled markers were developed based on the exclusively rare variation, providing precise selection of this allele. Thus, this work provided a novel reliable resistance source and the feasible marker-assisted selection assays that can be used in breeding for barley yellow mosaic virus disease resistance in cultivated barley.

Complete genome sequence of a novel member of the family Potyviridae isolated from Phellodendron amurense Rupr. in Liaoning, China.

In September 2017, Phellodendron amurense Rupr. plants showing yellow ringspots on leaves were observed in Liaoning, China. Flexuous filamentous particles (~1000 × 13 nm) were observed in the sap prepared from symptomatic leaves. A virus was detected in the symptomatic leaves by sequencing small RNAs and assembling the genome sequence. The complete genomic RNA was found to be 10,457 nucleotides in length excluding the poly(A) tail and to have the closest phylogenetic relationship to rose yellow mosaic virus (RoYMV), the sole member of newly established genus Roymovirus in the family Potyviridae. The coat protein gene (CP) of this virus shares 49.2% nucleotide and 55.1% amino acid sequence identity with that of RoYMV. These results suggest that this virus, which was named "phellodendron yellow ringspot-associated virus" (PYRaV) is a new member of the genus Roymovirus.

The first complete genomic sequence of cardamom mosaic virus, a member of the genus Macluravirus (family Potyviridae).

The complete genome sequence of the KS isolate of cardamom mosaic virus (CdMV) was determined using transcriptome sequencing data from CdMV-infected Elettaria cardamomum as well as from overlapping cDNA clones made from RNA extracted from viral particles. The viral genome consists of 8249 nucleotides (nt) and encodes a large polyprotein of 2636 amino acids (aa). The polyprotein of CdMV shared 48.9%-67.4% aa sequence identity with other reported macluraviruses. Similar to the other members of genus Macluravirus, the genome of CdMV lacks the P1 coding region and the N-terminus of the HC-Pro coding region. The putative small open reading frame, PIPO, embedded within the P3 cistron, is preceded by a C(A)6 motif instead of G(A)6. Phylogenetic analysis based on the complete genome sequence aided the grouping of CdMV along with all other macluraviruses and showed that it is closely related to alpinia oxyphylla mosaic virus (AloMV). Among CdMV isolates, the KS isolate is most similar to the Appangala isolate based on disease symptoms and phylogeny.

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

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

Strategies for the Construction of Cassava Brown Streak Disease Viral Infectious Clones.

Cassava brown streak disease (CBSD) has major impacts on yield and quality of the tuberous roots of cassava in Eastern and Central Arica. At least two Potyviridae species cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cloned viral genome sequences known as infectious clones (ICs) have been important in the study of other viruses, both as a means of standardising infectious material and characterising viral gene function. IC construction is often technically challenging for Potyviridae due to sequence instability in E. coli. Here, we evaluate three methods for the construction of infectious clones for CBSD. Whilst a simple IC for in vitro transcription was made for UCBSV isolate 'Kikombe', such an approach failed to deliver full-length clones for CBSV isolates 'Nampula' or 'Tanza', necessitating more complex approaches for their construction. The ICs successfully generated symptomatic infection in the model host N. benthamiana and in the natural host cassava. This shows that whilst generating ICs for CBSV is still a technical challenge, a structured approach, evaluating both in vitro and in planta transcription systems should successfully deliver ICs, allowing further study into the symptomology and virulence factors in this important disease complex.

Whitefly-transmitted viruses threatening cassava production in Africa.

Emerging plant viruses are one of the greatest problems facing crop production worldwide, and have severe consequences in the developing world where subsistence farming is a major source of food production, and knowledge and resources for management are limited. In Africa, evolution of two viral disease complexes, cassava mosaic begomoviruses (CMBs) (Geminiviridae) and cassava brown streak viruses (CBSVs) (Potyviridae), have resulted in severe pandemics that continue to spread and threaten cassava production. Identification of genetically diverse and rapidly evolving CMBs and CBSVs, extensive genetic variation in the vector, Bemisia tabaci (Hemiptera: Aleyrodidae), and numerous secondary endosymbiont profiles that influence vector phenotypes suggest that complex local and regional vector-virus-plant-environment interactions may be driving the evolution and epidemiology of these viruses.

Barley RNA viromes in six different geographical regions in Korea.

Barley is a kind of cereal grass belonging to the family Poaceae. To examine viruses infecting winter barley in Korea, we carried out a comprehensive study of barley RNA viromes using next-generation sequencing (NGS). A total of 110 barley leaf samples from 17 geographical locations were collected. NGS followed by extensive bioinformatics analyses revealed six different barley viromes: Barley yellow mosaic virus (BaYMV), Barley mild mosaic virus (BaMMV), Barley yellow dwarf virus (BYDV), Hordeum vulgare endornavirus (HvEV), and Barley virus G (BVG). BaYMV and HvEV were identified in all libraries, while other viruses were identified in some specific library. Based on the number of virus-associated reads, BaYMV was a dominant virus infecting winter barley in Korea causing yellow disease symptoms. We obtained nearly complete genomes of six BaYMV isolates and two BaMMV isolates. Phylogenetic analyses indicate that BaYMV and BaMMV were largely grouped based on geographical regions such as Asia and Europe. Single nucleotide polymorphisms analyses suggested that most BaYMV and BaMMV showed strong genetic variations; however, BaYMV isolate Jeonju and BaMMV isolate Gunsan exhibited a few and no SNPs, respectively, suggesting low level of genetic variation. Taken together, this is the first study of barley RNA viromes in Korea.

Complete genome sequence of a novel virus, classifiable within the Potyviridae family, which infects passion fruit (Passiflora edulis).

We report the complete nucleotide sequence of a new member of the Potyviridae family isolated from passion fruit plants grown in Israel, called Passiflora edulis symptomless virus (PeSV). The PeSV genome is 9,928 nucleotides long and encodes a 3,173 amino acids polyprotein that is predicted to be proteolytically cleaved into 10 mature peptides. Our phylogenetic analysis shows that PeSV represents a new species, and is most closely related to rose yellow mosaic virus (RoYMV). According to currently accepted criteria for genus demarcation, both viruses should be assigned as representative isolates of new species in the recently approved genus, Roymovirus, in the Potyviridae family.

Analysis of the complete genomic sequence of a novel virus, areca palm necrotic spindle-spot virus, reveals the existence of a new genus in the family Potyviridae.

A novel virus, tentatively named "areca palm necrotic spindle-spot virus" (ANSSV), was identified in Areca catechu L. in Hainan, China, and its complete genomic sequence was determined. Its positive-sense single-stranded RNA genome is comprised of 9,437 nucleotides (nt), excluding the poly (A) tail, and contains one large open reading frame encoding a polyprotein of 3,019 amino acids (aa). A Blastp search showed that the polyprotein of ANSSV shared a maximum of 31%-32% aa sequence identity (with 86%-95% coverage) with all seven known macluraviruses. Nucleotide sequence comparison of the ORF of ANSSV to those of macluraviruses revealed identities ranging from 41.0% to 44.6%, which is less than the inter-genus identity values for the family Potyviridae. Phylogenetic analysis based on either the aa or nt sequence of the polyprotein did not cluster ANSSV into any established or unassigned genus of the family Potyviridae. Therefore, we suggest that ANSSV is the first member of a previously unrecognized genus of the family Potyviridae.

Wuhan poty-like virus 1 is a possible member of the genus Macluravirus.

We have analysed the genome sequence of Wuhan poty-like virus 1 (WuPLV1), reported as an unclassified RNA virus in GenBank (Accession no: KX884573.1). Based on the polyprotein sequence identity (ranging from 55.2 to 71.1%), with classifiable members of the Macluravirus genus of the plant virus family Potyviridae, we suggest that WuPLV1 represents a possible new species of Macluravirus, although the virus was isolated from the Chinese land snail Mastigeulota kiangsinensis, which is not known to be a host or vector of macluraviruses.

Analysis of the complete genome sequence of a potyvirus from passion fruit suggests its taxonomic classification as a member of a new species.

The complete genomic sequence of a telosma mosaic virus (TeMV) isolate (named PasFru), identified in passion fruit in China, was determined. The entire RNA genome of PasFru comprises 10,049 nucleotides (nt) excluding the poly(A) tail and encodes a polyprotein of 3,173 amino acids (aa), flanked by 5' and 3' untranslated regions (UTR) of 276 and 251 nt, respectively. Compared with the previous TeMV isolate Hanoi from Telosma cordata, the only documented isolate with the entire genome sequence annotated, PasFru had an extra 87 nt and 89 aa residues at the 3'-end of 5'UTR and the N-terminus of the P1 protein, respectively, which contributed to the genome size difference between PasFru and Hanoi (10,049 nt versus 9,689 nt). Pairwise sequence comparisons showed that PasFru shares 73.6% nt and 80.9% aa sequence identity with the Hanoi isolate at the whole-genome and polyprotein level, respectively, and these values are below the corresponding threshold values for species demarcation in the family Potyviridae. These data suggest that TeMV-PasFru should be classified as a new member of the genus Potyvirus.

Metagenomic analysis of viruses associated with maize lethal necrosis in Kenya.

BACKGROUND: Maize lethal necrosis is caused by a synergistic co-infection of Maize chlorotic mottle virus (MCMV) and a specific member of the Potyviridae, such as Sugarcane mosaic virus (SCMV), Wheat streak mosaic virus (WSMV) or Johnson grass mosaic virus (JGMV). Typical maize lethal necrosis symptoms include severe yellowing and leaf drying from the edges. In Kenya, we detected plants showing typical and atypical symptoms. Both groups of plants often tested negative for SCMV by ELISA. METHODS: We used next-generation sequencing to identify viruses associated to maize lethal necrosis in Kenya through a metagenomics analysis. Symptomatic and asymptomatic leaf samples were collected from maize and sorghum representing sixteen counties. RESULTS: Complete and partial genomes were assembled for MCMV, SCMV, Maize streak virus (MSV) and Maize yellow dwarf virus-RMV (MYDV-RMV). These four viruses (MCMV, SCMV, MSV and MYDV-RMV) were found together in 30 of 68 samples. A geographic analysis showed that these viruses are widely distributed in Kenya. Phylogenetic analyses of nucleotide sequences showed that MCMV, MYDV-RMV and MSV are similar to isolates from East Africa and other parts of the world. Single nucleotide polymorphism, nucleotide and polyprotein sequence alignments identified three genetically distinct groups of SCMV in Kenya. Variation mapped to sequences at the border of NIb and the coat protein. Partial genome sequences were obtained for other four potyviruses and one polerovirus. CONCLUSION: Our results uncover the complexity of the maize lethal necrosis epidemic in Kenya. MCMV, SCMV, MSV and MYDV-RMV are widely distributed and infect both maize and sorghum. SCMV population in Kenya is diverse and consists of numerous strains that are genetically different to isolates from other parts of the world. Several potyviruses, and possibly poleroviruses, are also involved.

Complete genomic sequence of a novel macluravirus, alpinia oxyphylla mosaic virus (AloMV), identified in Alpinia oxyphylla.

A macluravirus, tentatively named alpinia oxyphylla mosaic virus (AloMV), was identified in Alpinia oxyphylla, and its complete genomic sequence determined. The positively single-stranded RNA genome is comprised of 8213 nucleotides excluding the poly (A) tail, and contains one large open reading frame encoding a polyprotein of 2,626 amino acids. Blastp search showed that the polyprotein of AloMV shared 48%~68% aa sequence identities with other reported macluraviruses. Phylogenetic analysis based on the nucleotide sequence of the polyprotein showed that AloMV, together with all other macluraviruses, clustered into the same group most closely related to cardamom mosaic virus, sharing 66.3% nt and 68% aa sequence identities, respectively. These data above suggest that AloMV represents an isolate of a putative new member within the genus Macluravirus.

Complete genome sequence of yam chlorotic necrosis virus, a novel macluravirus infecting yam.

The complete genome sequence of a novel member of the genus Macluravirus was determined from yam plants with chlorotic and necrotic symptoms in China. The genomic RNA consists of 8,261 nucleotides (nt) excluding the 3'-terminal poly(A) tail, containing one long open reading frame (ORF) encoding a large putative polyprotein of 2,627 amino acids. Its genomic structure is typical of macluraviruses, which lack the P1 protein, N-terminal HC-Pro, and D-A-G motif for aphid transmission that are found in potyviruses. The virus shares 56.3-63.8% sequence identity at the genome sequence level and 49.7-63.9% at the polyprotein sequence level with other members of the genus Macluravirus. Phylogenetic analysis based on the complete polyprotein sequence of representative members of the family Potyviridae clearly places the virus within the genus Macluravirus. These results suggest that the virus, tentatively named "yam chlorotic necrosis virus" (YCNV), should be considered a member of a novel species in the genus Macluravirus.

Temperature and Alternative Hosts Influence Aceria tosichella Infestation and Wheat Streak Mosaic Virus Infection.

Wheat streak mosaic, caused by Wheat streak mosaic virus (WSMV; family Potyviridae), is the most important and common viral disease of wheat (Triticum aestivum L.) in the Great Plains of North America. WSMV is transmitted by the wheat curl mite (WCM; Aceria tosichella). We evaluated how mean daily temperatures, cumulative growing degree-days, day of the year, and surrounding alternative host identity affected WCM infestation and WSMV infection of wheat from late summer through early autumn in Montana, United States. Cumulative growing degree-days, warm mean daily temperatures (i.e., >10°C), and surrounding alternative hosts interacted to alter risk of WCM infestation and WSMV infection. Wheat surrounded by Bromus tectorum L. and preharvest volunteer wheat had WCM infestation and WSMV infection rates of 88% in years when the mean daily temperature was 15°C in October, compared with 23% when surrounded by bare ground, and <1% when the temperature was 0°C regardless of surrounding alternative host. Mean daily temperatures in the cereal-growing regions of Montana during autumn are marginally conducive to WCM population growth and movement. As the region continues to warm, the period of WCM movement will become longer, potentially increasing the frequency of WSMV outbreaks.