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During the summer of 2022, a cluster of Madagascar periwinkle plants with white and mauve flowers were observed with foliar mild yellow mosaic symptoms on a private property in Harlingen, Cameron County, Texas. The symptoms were reproduced on mechanically inoculated periwinkle and Nicotiana benthamiana plants. Virions of 776 to 849 nm in length and 11.7 to 14.8 nm in width were observed in transmission electron microscopy of leaf dip preparations made from symptomatic periwinkle leaves. High-throughput sequencing (HTS) analysis of total RNA extracts from symptomatic leaves revealed the occurrence of two highly divergent variants of a novel Potyvirus species as the only virus-like sequences present in the sample. The complete genomes of both variants were independently amplified via reverse transcriptase PCR, cloned, and Sanger sequenced. The 5' and 3' of the genomes were acquired using random amplification of cDNA ends methodology. The assembled virus genomes were 9,936 and 9,944 nucleotides (nt) long, and they shared 99.9 to 100% identities with the respective HTS-derived genomes. Each genome encoded hypothetical polyprotein of 3,171 amino acids (aa) (362.6 kilodaltons [kDa]) and 3,173 aa (362.7 kDa), respectively, and they shared 77.3/84.4% nt/aa polyprotein identities, indicating that they represent highly divergent variants of the same Potyvirus species. Both genomes also shared below-species-threshold polyprotein identity levels with the most closely phylogenetically related known potyviruses, thus indicating that they belong to a novel species. The name periwinkle mild yellow mosaic virus (PwMYMV) is given to the potyvirus with complete genomes of 9,936 nt for variant 1 (PwMYMV-1) and 9,944 nt for variant 2 (PwMYMV-2). We propose that PwMYMV be assigned into the genus Potyvirus (family Potyviridae).
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Catharanthus , Genoma Viral , Filogenia , Doenças das Plantas , Potyvirus , Potyvirus/genética , Potyvirus/classificação , Potyvirus/isolamento & purificação , Doenças das Plantas/virologia , Genoma Viral/genética , Catharanthus/virologia , Folhas de Planta/virologia , Sequenciamento de Nucleotídeos em Larga Escala , RNA Viral/genética , Variação Genética , Nicotiana/virologia , TexasRESUMO
In the original publication [...].
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Grapevine virus A (GVA) is an economically important virus and a member of the genus Vitivirus (family Betaflexiviridae) that causes a range of symptoms with qualitative and quantitative effects on grape production. Wild and domesticated species of Vitis, including hybrids used as rootstocks, are considered important natural hosts of GVA. Mechanical transmission to some herbaceous plant species, graft transmission, and vector transmission from grape to grape by various mealybugs and soft scale insects have been reported. Under laboratory and greenhouse conditions, this study demonstrates the transmission of GVA from grapes to alternative hosts by the vine mealybug (Planococcus ficus). Results of ELISA, end-point one-step RT-PCR, and real-time RT-PCR, and in some cases electron microscopy and genome sequencing, confirmed successful transmission to three new plant species commonly found in Croatian vineyards: velvetleaf (Abutilon theophrasti), redroot pigweed (Amaranthus retroflexus), and field poppy (Papaver rhoeas), along with Chenopodium murale and the previously known host Nicotiana benthamiana, with variable infection rates. Depending on the host species, symptoms in the form of leaf reddening, yellow spots, reduced growth of lateral shoots, systemic vein clearing, foliar deformation and rugosity, and dwarfism were observed in GVA-infected plants, whereas no symptoms were observed in infected plants of A. theophrasti. Reverse transmission from these new hosts to grapevines by Pl. ficus was not successful. These results confirm four new GVA host species and open new research venues.
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Flexiviridae , Hemípteros , Vírus de Plantas , Animais , Flexiviridae/genética , Vírus de Plantas/genética , NicotianaRESUMO
This is the first viral metagenomic analysis of grapevine conducted in Mexico. During the summer of 2021, 48 plants displaying virus-like symptoms were sampled in Queretaro, an important grapevine-producing area of Mexico, and analyzed for the presence of viruses via high-throughput sequencing (HTS). The results of HTS were verified by real-time RT-PCR following a standardized testing scheme (Protocol 2010). Fourteen different viruses were identified, including grapevine asteroid mosaic-associated virus (GAMaV), grapevine Cabernet Sauvignon reovirus (GCSV), grapevine fanleaf virus (GFLV), grapevine fleck virus (GFkV), grapevine Pinot gris virus (GPGV), grapevine red globe virus (GRGV), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus 1 (GSyV-1), grapevine virus B (GVB), and grapevine leafroll-associated viruses 1, 2, 3, 4 (GLRaV1, 2, 3, 4). Additionally, divergent variants of GLRaV4 and GFkV, and a novel Enamovirus-like virus were discovered. This is the first report of GAMaV, GCSV, GLRaV4, GPGV, GRGV, GRVFV, and GSyV-1 infecting grapevines in Mexico; the impact of these pathogens on production is unknown.
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Luteoviridae , Vitis , México , Incidência , Doenças das Plantas , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
High-throughput sequencing (HTS), more specifically RNA sequencing of plant tissues, has become an indispensable tool for plant virologists to detect and identify plant viruses. During the data analysis step, plant virologists typically compare the obtained sequences to reference virus databases. In this way, they are neglecting sequences without homologies to viruses, which usually represent the majority of sequencing reads. We hypothesized that traces of other pathogens might be detected in this unused sequence data. In the present study, our goal was to investigate whether total RNA-seq data, as generated for plant virus detection, is also suitable for the detection of other plant pathogens and pests. As proof of concept, we first analyzed RNA-seq datasets of plant materials with confirmed infections by cellular pathogens in order to check whether these non-viral pathogens could be easily detected in the data. Next, we set up a community effort to re-analyze existing Illumina RNA-seq datasets used for virus detection to check for the potential presence of non-viral pathogens or pests. In total, 101 datasets from 15 participants derived from 51 different plant species were re-analyzed, of which 37 were selected for subsequent in-depth analyses. In 29 of the 37 selected samples (78%), we found convincing traces of non-viral plant pathogens or pests. The organisms most frequently detected in this way were fungi (15/37 datasets), followed by insects (13/37) and mites (9/37). The presence of some of the detected pathogens was confirmed by independent (q)PCRs analyses. After communicating the results, 6 out of the 15 participants indicated that they were unaware of the possible presence of these pathogens in their sample(s). All participants indicated that they would broaden the scope of their bioinformatic analyses in future studies and thus check for the presence of non-viral pathogens. In conclusion, we show that it is possible to detect non-viral pathogens or pests from total RNA-seq datasets, in this case primarily fungi, insects, and mites. With this study, we hope to raise awareness among plant virologists that their data might be useful for fellow plant pathologists in other disciplines (mycology, entomology, bacteriology) as well.
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Malabar spinach plants (Basella alba, Basellaceae) with leaves exhibiting symptoms of mosaic, rugosity, and malformation were found in a community garden on Oahu, HI in 2018. Preliminary studies using enzyme-linked immunosorbent assay and reverse-transcription (RT)-PCR identified Basella rugose mosaic virus (BaRMV) in symptomatic plants. However, nucleotide sequence analysis of RT-PCR amplicons indicated that additional potyviruses were also present in the symptomatic Malabar spinach. High-throughput sequencing (HTS) analysis was conducted on ribosomal RNA-depleted composite RNA samples of potyvirus-positive plants from three locations. Assembled contigs shared sequences similar to BaRMV, chilli veinal mottle virus (ChiVMV), Alternanthera mosaic virus (AltMV), Basella alba endornavirus (BaEV), broad bean wilt virus 2 (BBWV2), and Iresine viroid 1. Virus- and viroid-specific primers were designed based on HTS sequencing results and used in RT-PCR and Sanger sequencing to confirm the presence of these viruses and the viroid. We tested 63 additional samples from six community gardens for a survey of viruses in Malabar spinach and found that 21 of them were positive for BaRMV, 57 for ChiVMV, 21 for AltMV, 19 for BaEV, and 14 for BBWV2. This is the first characterization of the virome from B. alba.
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Potyvirus , Viroides , Havaí , Potyvirus/genética , Primers do DNA , Ensaio de Imunoadsorção EnzimáticaRESUMO
Pawpaw (Asimina triloba) trees exhibiting stunting and foliar mosaic, chlorosis, or distortions were observed in New York. In 2021, leaf samples from two symptomatic trees and a sapling, as well as two asymptomatic trees, were tested for the presence of viruses and viroids by high-throughput sequencing (HTS) using total RNA after ribosomal RNA depletion. HTS sequence information revealed tobacco ringspot virus (TRSV) and tomato ringspot virus (ToRSV) in symptomatic but not in asymptomatic leaves. HTS reads and de novo-assembled contigs covering the genomes of both viruses were obtained, with a higher average read depth for RNA2 than RNA1. The occurrence of TRSV and ToRSV was confirmed in the original leaf samples used for HTS and 12 additional trees and saplings from New York and Maryland in 2022 by RT-PCR combined with Sanger sequencing, and DAS-ELISA. Single infections by TRSV in 11 of 14 trees and dual infections by TRSV and ToRSV in 3 of 14 trees were identified. The nucleotide sequence identity of partial gene fragments of TRSV and ToRSV was high among pawpaw isolates (94.9-100% and 91.8-100%, respectively) and between pawpaw isolates and isolates from other horticultural crops (93.6-100% and 71.3-99.3%, respectively). This study is the first to determine the virome of pawpaw.
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The biological characteristics of grapevine viruses, such as their transmission and host range, are important for the adoption of successful prophylaxis strategies. The aim of this study was to investigate the traits of two newly described grapevine viruses widely distributed in Croatia, grapevine badnavirus 1 (GBV-1) and grapevine virus G (GVG). The vine mealybug (Planoccocus ficus) proved to be a vector of GBV-1 and GVG capable of vine-to-vine transmission with overall experimental transmission rates of 61% and 14.6%, respectively. Transmission was also demonstrated by grafting, with an overall transmission rate of 53.8% for GBV-1 and 100% for GVG, as well as by green grafting using the T-budding technique. Symptoms of GBV-1 and GVG were not observed on the woody cylinders of the indicators LN 33, Kober 5BB, 110 Richter and cvs. Chardonnay and Cabernet Sauvignon. Seed transmission and mechanical transmission were not confirmed. Electron microscopy revealed accumulation of GBV-1 particles and viroplasms in the cytoplasm, but no alternations of the cell structure. Infection with GVG revealed the proliferation of tonoplast-associated vesicles inside phloem cells and cell wall thickening.
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Badnavirus , Flexiviridae , Vitis , Doenças das Plantas , BiologiaRESUMO
Watermelon mosaic virus (WMV, genus Potyvirus, family Potyviridae) is a species of considerable economic importance to cucurbit crops worldwide (Keinath et al. 2017). This virus has a wide host range that includes more than 170 plant species from 27 families (Dong et al. 2017; Lecoq et al. 2011). In 2018, leaves of coriander (Coriandrum sativum) plants in a student garden (C-SG) at UC Davis, and in a home garden in Davis, CA (C-Pet) (~1.1 miles apart) showed symptoms of light green mottling and crumpling. Symptomatic leaves from each location were weakly positive with the general potyvirus immunostrip test (Agdia, Elkhart, IN). In RT-PCR tests with total RNA extracts (RNeasy Plant Mini Kit Qiagen, Germantown, MD) of these leaves and the potyvirus degenerate primer pair CIFor/CIRev (Ha et al. 2008), the expected-size ~0.7 kb fragment was amplified. These fragments were gel-purified and sequenced, and a BLASTn search revealed highest identities of 91.6% (C-SG) and 97.9% (C-Pet) with the sequence of an isolate of WMV from watermelon in the U.S. (TX29, KU246036). Thus, these isolates are designated WMV-C-SG-18 and WMV-C-Pet-18. Mechanical inoculation experiments were next performed with sap prepared with symptomatic coriander leaf tissue in ice-cold 0.01 M phosphate buffer (pH 7.0) in a 1:4 wt/vol ratio. First, to obtain pure isolates, sap was inoculated onto celite-dusted leaves of Chenopodium quinoa plants (3-4 leaf stage). As expected for WMV, leaves inoculated with sap of each isolate developed chlorotic local lesions ~9 d post-inoculation (dpi) (Moreno et al. 2004). One lesion for each isolate was excised, ground in phosphate buffer, and the sap was mechanically inoculated onto leaves of Nicotiana benthamiana plants. By ~14 dpi, newly emerged leaves showed mild mottling and crumpling, and were weakly positive with the potyvirus immunostrip test. To confirm that these plants were only infected with WMV, total RNA was extracted from symptomatic leaves and used for high throughput sequencing (HTS) (Soltani et al. 2021) at the Foundation Plant Services at UC Davis. The HTS analyses revealed infection with only WMV, i.e., no other viral contigs were identified, and allowed for determination of the complete sequences (~10,000 nt) of WMV [US-CA-C-SG-18] and WMV [US-CA-C-Pet-18] with GenBank accession numbers: OM746964 and OM746965, respectively. Whole genome sequence comparisons revealed that the sequences are 99.0% identical, and 97.3% identical to the sequence of WMV TX29. Sap from symptomatic N. benthamiana leaves infected with each isolate was mechanical inoculated onto leaves of coriander plants (30-35 d old). Newly emerged leaves developed epinasty, crumpling and light green mottling by 14 dpi, and WMV infection was confirmed by RT-PCR with the WMV-specific primer pair WMV-UNI-1F and WMV-UNI-1R (Kim et al. 2019). Thus, Koch's postulates were fulfilled for this leaf mottling disease of coriander. Furthermore, the isolates from coriander induced stunting and distortion and mosaic in leaves of melon, pumpkin and squash plants by 7 dpi, whereas watermelon plants developed stunting and small leaves with mild mottling by 20 dpi. Similar results were obtained with sap prepared from infected coriander leaves. Thus, infected coriander plants are a potential inoculum source for cucurbits via several aphid vectors (Keinath et al. 2017). This is the first report of a mottle disease of coriander caused by WMV, and adds to the wide host range of the virus.
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Grapevine collections play an important role, especially in the study of viruses and virus-like pathogens. In 2009, after an initial ELISA screening for eight viruses (arabis mosaic virus, grapevine fanleaf virus, grapevine fleck virus, grapevine leafroll-associated viruses 1, 2, and 3, and grapevine viruses A and B), a collection of 368 grapevine accessions representing 14 different Croatian autochthonous cultivars and containing single or mixed infection of viruses was established to further characterize the viral pathogens. Subsequently, Western blot, RT-PCR, cloning, and sequencing revealed that grapevine rupestris stem pitting-associated virus was frequently found in accessions of the collection, with isolates showing substantial genetic diversity in the helicase and coat protein regions. High-throughput sequencing of 22 grapevine accessions provides additional insight into the viruses and viroids present in the collection and confirms the fact that Croatian autochthonous grapevine cultivars have high infection rates and high virome diversity. The recent spread of "flavescence dorée" phytoplasma in Europe has not spared the collection. After the first symptoms observed in 2020 and 2021, the presence of phytoplasma was confirmed by LAMP in six grapevine accessions and some of them were lost. Single or multiple viruses and viroids, as well as own rooted grapevines in the collection, make the plants susceptible to various abiotic factors, which, together with the recent occurrence of "flavescence dorée", makes the maintenance of the collection a challenge. Future efforts will be directed towards renewing the collection, as 56% of the original collection has been lost in the last 13 years.
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Grapevine virus infectious clones are important tools for fundamental studies, but also because of their potential for translational applications for grapevine improvement. Although several grapevine virus infectious clones have been developed, there has been difficulty in directly infecting mature grapevine plants, and many of the viruses used still cause disease symptoms in grapevine plants, making them less likely candidates for biotechnological applications in grapes. Here, we developed an improved Agrobacterium tumefaciens infiltration method that can be used to deliver DNA plasmids and viral infectious clones directly into approximately 20- to 40-cm-high (above soil) greenhouse-grown grapevine plants. We also developed infectious clones for two isolates of grapevine geminivirus A (GGVA): Longyan (China; GenBank accession KX570611; GGVA-76) and Super Hamburg (Japan; GenBank accession KX570610; GGVA-93). Neither virus caused any obvious symptoms when inoculated to plants of grapevine varieties Colombard, Salt Creek, Cabernet Sauvignon, and Vaccarèse. However, the two GGVA isolates induced different symptom severity and viral titer in Nicotiana benthamiana plants. The two GGVA isolates used here were found to accumulate to different titers in different parts/branches of the infected grapevine plants. The GGVA infectious clones and the improved grapevine infiltration technique developed here provide new, valuable tools that can be applied to grapevine plants, possibly even for translational applications such as disease management and desired trait improvements.
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Agrobacterium tumefaciens , Geminiviridae , Nicotiana , Agrobacterium tumefaciens/genética , Células Clonais , Geminiviridae/genética , Doenças das PlantasRESUMO
The destructive citrus disease, Huanglongbing (HLB) or citrus greening, continues to devastate Florida's citrus industry. A hemipteran insect, the Asian citrus psyllid (ACP), disperses Candidatus Liberibacter asiaticus, one of the putative bacterial pathogens of HLB. This study builds upon ongoing research utilizing high-throughput sequencing to analyze the virome of ACP populations collected from citrus groves throughout Florida. Following the widespread detection of sequences aligning to the genome of citrus tristeza virus (CTV) across consecutive years in the Florida ACP virome, we continued to detect a pervasive amount of CTV in Florida ACPs during subsequent years. Simultaneously, we also detected mixed infections of CTV strains in pooled ACPs from different Florida regions. Predating the HLB epidemic, CTV has been present in Florida for many years and our results confirm its widespread and diverse persistence in Florida citrus groves through a unique lens, the ACP. CTV presence in the ACP likely results from feeding on CTV-infected citrus trees in Florida citrus groves, which may help to understand an overlapping presence of CTV and HLB, both endemic citrus pathosystems in the state, and their role in future integrated pest management strategies.
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The production of common bean (Phaseolus vulgaris L.) is adversely affected by virus-like diseases globally, but little is known about the occurrence, distribution, and diversity of common bean-infecting viruses in Zambia. Consequently, field surveys were conducted during the 2018 season in 128 fields across six provinces of Zambia and 640 common bean leaf tissue samples were collected with (n = 585) or without (n = 55) symptoms. The prevalence of symptomatic fields was 100%, but incidence of symptomatic plants ranged from 32 to 67.5%. Metagenomic analyses of nine composite samples and a single plant sample of interest revealed the occurrence of isolates of Bean common mosaic necrosis virus, Bean common mosaic virus, Cowpea aphid-borne mosaic virus, Peanut mottle virus, Southern bean mosaic virus (SBMV), Cucumber mosaic virus, Phaseolus vulgaris alphaendornavirus 1 (PvEV-1), PvEV-2, Ethiopian tobacco bushy top virus (ETBTV), and a novel strain of Cowpea polerovirus 1 (CPPV1-Pv) of 5,902 nt in length. While CPPV1-Pv was consistently detected in mixed infection with ETBTV and its satellite RNA molecule, based on results of mechanical transmission assays it does not appear to be involved in disease etiology, suggesting that its role may be limited to being a helper virus for the umbravirus. Screening of the survey samples by real-time PCR for the viruses detected by high-throughput sequencing revealed the prevalence of single (65.2% or 417/640) over mixed (1.9% or 12/640) infections in the samples. SBMV was the most frequently detected virus, occurring in â¼29.4% (188/640) of the samples and at a prevalence rate of 58.6% (75/128) across fields. The results showed that diverse virus species are present in Zambian common bean fields and the information will be useful for the management of common bean viral diseases.
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Luteoviridae , Phaseolus , Vigna , Luteoviridae/genética , Doenças das Plantas , Vírus de Plantas , ZâmbiaRESUMO
Huanglongbing is caused by Candidatus Liberibacter asiaticus (CLas) and transmitted by Diaphorina citri. D. citri harbors various insect-specific viruses, including the Diaphorina citri flavi-like virus (DcFLV). The distribution and biological role of DcFLV in its host and the relationship with CLas are unknown. DcFLV was found in various organs of D. citri, including the midgut and salivary glands, where it co-localized with CLas. CLas-infected nymphs had the highest DcFLV titers compared to the infected adults and CLas-free adults and nymphs. DcFLV was vertically transmitted to offspring from female D. citri and was temporarily detected in Citrus macrophylla and grapefruit leaves from greenhouse and field. The incidences of DcFLV and CLas were positively correlated in field-collected D. citri samples, suggesting that DcFLV might be associated with CLas in the vector. These results provide new insights on the interactions between DcFLV, the D. citri, and CLas.
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Citrus/microbiologia , Flavivirus/genética , Hemípteros/virologia , Insetos Vetores/virologia , Liberibacter/genética , Ninfa/virologia , Animais , DNA Bacteriano/genética , Feminino , Hemípteros/microbiologia , Insetos Vetores/microbiologia , Intestinos/microbiologia , Intestinos/virologia , Liberibacter/patogenicidade , Ninfa/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , RNA Viral/genética , Glândulas Salivares/microbiologia , Glândulas Salivares/virologia , Simbiose/fisiologiaRESUMO
Olea europaea geminivirus (OEGV) from olive accessions in Italy was characterized recently. OEGV was also detected during routine high-throughput sequencing screening of olive (cv. Leccino) material, and its complete bipartite genome segments were sequenced and shown to be 100% identical to those of the isolate from Italy. Using two pairs of newly designed primers targeting the AV1 and BV1 genes, OEGV was detected in randomly sampled olive trees from the U.S. Department of Agriculture National Clonal Germplasm Repository (USDA-NCGR) (21.4% or 6/28), commercial and residential settings in California (47.6% or 10/21), and an orchard in Texas (60% or 30/50). The cuttings for the USDA-NCGR-positive trees originated from the former Serbia and Montenegro, Spain, Italy, and Greece. Comparative analysis of the directly sequenced gene fragments from randomly selected samples showed that OEGV isolates from the different sources were 100% identical to each other. The results indicate that OEGV spread was likely facilitated by inadvertent movement of contaminated olive germplasm.
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Geminiviridae , Olea , Geminiviridae/genética , Sequenciamento de Nucleotídeos em Larga Escala , Texas/epidemiologia , ÁrvoresRESUMO
This report describes the partial (nearly complete) genome sequence of a novel reo-like virus tentatively named Diaphorina citri Cimodo-like virus. This putative virus has 10 double-stranded RNA segments and was detected in Asian citrus psyllid (Diaphorina citri) populations collected from Florida commercial citrus groves.
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Viruses are considered of major importance in strawberry (Fragaria × ananassa Duchesne) production given their negative impact on plant vigor and growth. Strawberry accessions from the National Clonal Germplasm Repository were screened for viruses using high throughput sequencing (HTS). Analyses of sequence information from 45 plants identified multiple variants of 14 known viruses, comprising strawberry mottle virus (SMoV), beet pseudo yellows virus (BPYV), strawberry pallidosis-associated virus (SPaV), tomato ringspot virus (ToRSV), strawberry mild yellow edge virus (SMYEV), strawberry vein banding virus (SVBV), strawberry crinkle virus (SCV), strawberry polerovirus 1 (SPV-1), apple mosaic virus (ApMV), strawberry chlorotic fleck virus (SCFaV), strawberry crinivirus 4 (SCrV-4), strawberry crinivirus 3 (SCrV-3), Fragaria chiloensis latent virus (FClLV) and Fragaria chiloensis cryptic virus (FCCV). Genetic diversity of sequenced virus isolates was investigated via sequence homology analysis, and partial-genome sequences were deposited into GenBank. To confirm the HTS results and expand the detection of strawberry viruses, new reverse transcription quantitative PCR (RT-qPCR) assays were designed for the above-listed viruses. Further in silico and in vitro validation of the new diagnostic assays indicated high efficiency and reliability. Thus, the occurrence of different viruses, including divergent variants, among the strawberries was verified. This is the first viral metagenomic survey in strawberry, additionally, this study describes the design and validation of multiple RT-qPCR assays for strawberry viruses, which represent important detection tools for clean plant programs.
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Fragaria/virologia , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Doenças das Plantas/virologia , Vírus de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Mapeamento Cromossômico , Genoma Viral , Metagenômica , Filogenia , Vírus de RNA/classificação , Reprodutibilidade dos TestesRESUMO
Development of High-Throughput Sequencing (HTS), also known as next generation sequencing, revolutionized diagnostic research of plant viruses. HTS outperforms bioassays and molecular diagnostic assays that are used to screen domestic and quarantine grapevine materials in data throughput, cost, scalability, and detection of novel and highly variant virus species. However, before HTS-based assays can be routinely used for plant virus diagnostics, performance specifications need to be developed and assessed. In this study, we selected 18 virus-infected grapevines as a test panel for measuring performance characteristics of an HTS-based diagnostic assay. Total nucleic acid (TNA) was extracted from petioles and dormant canes of individual samples and constructed libraries were run on Illumina NextSeq 500 instrument using a 75-bp single-end read platform. Sensitivity was 98% measured over 264 distinct virus and viroid infections with a false discovery rate (FDR) of approximately 1 in 5 positives. The results also showed that combining a spring petiole test with a fall cane test increased sensitivity to 100% for this TNA HTS assay. To evaluate extraction methodology, these results were compared to parallel dsRNA extractions. In addition, in a more detailed dilution study, the TNA HTS assay described here consistently performed well down to a dilution of 5%. In that range, sensitivity was 98% with a corresponding FDR of approximately 1 in 5. Repeatability and reproducibility were assessed at 99% and 93%, respectively. The protocol, criteria, and performance levels described here may help to standardize HTS for quality assurance and accreditation purposes in plant quarantine or certification programs.
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Sequenciamento de Nucleotídeos em Larga Escala , Doenças das Plantas/virologia , Vírus de Plantas/genética , Vitis/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , RNA Viral , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Eriophyid mites are commonly found on the leaf surface of different plant species. In the present study, a novel virus associated with an eriophyid mite species was detected using high-throughput sequencing (HTS) of total RNA from fruit tree leaves, primarily growing under greenhouse conditions. The complete genome sequence was characterized using rapid amplification of cDNA ends followed by Sanger sequencing, revealing a genome of 8885 nucleotides in length. The single positive-stranded RNA genome was predicted to encode typical conserved domains of members of the genus Iflavirus in the family Iflaviridae. Phylogenetic analysis showed this virus to be closely related to the unclassified iflavirus tomato matilda associated virus (TMaV), with a maximum amino acid sequence identity of 59% in the RNA-dependent RNA polymerase domain. This low identity value justifies the recognition of the novel virus as a potential novel iflavirus. In addition to a lack of graft-transmissibility evidence, RT-PCR and HTS detection of this virus in the putative host plants were not consistent through different years and growing seasons, raising the possibility that rather than a plant virus, this was a virus infecting an organism associated with fruit tree leaves. Identification of Tetra pinnatifidae HTS-derived contigs in all fruit tree samples carrying the novel virus suggested this mite as the most likely host of the new virus (p-value < 1e-11), which is tentatively named "eriophyid mite-associated virus" (EMaV). This study highlights the importance of a careful biological study before assigning a new virus to a particular plant host when using metagenomics data.
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Frutas/parasitologia , Ácaros/virologia , Vírus de RNA de Cadeia Positiva/classificação , Árvores/parasitologia , Sequência de Aminoácidos , Animais , Frutas/virologia , Genoma Viral/genética , Metagenômica , Filogenia , Extratos Vegetais , Folhas de Planta/parasitologia , Folhas de Planta/virologia , Vírus de RNA de Cadeia Positiva/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA , Árvores/virologiaRESUMO
Citrus yellow-vein disease (CYVD) was first reported in California in 1957. We now report that CYVD is associated with a virus-like agent, provisionally named citrus yellow-vein associated virus (CYVaV). The CYVaV RNA genome has 2,692 nucleotides and codes for two discernable open reading frames (ORFs). ORF1 encodes a protein of 190 amino acid (aa) whereas ORF2 is presumably generated by a -1 ribosomal frameshifting event just upstream of the ORF1 termination signal. The frameshift product (717 aa) encodes the RNA-dependent RNA polymerase (RdRp). Phylogenetic analyses suggest that CYVaV is closely related to unclassified virus-like RNAs in the family Tombusviridae. Bio-indexing and RNA-seq experiments indicate that CYVaV can induce yellow vein symptoms independently of known citrus viruses or viroids.