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Babaco is a hybrid cultivar native to the Andean region of Ecuador and Colombia, commercially attractive for its fruit. Babaco production in Ecuador faces losses from plant pathogens like babaco mosaic virus (BabMV), an RNA virus that causes chlorosis, leaf mottling, and deformation. Phylogenetic studies link BabMV to papaya mosaic virus (PapMV), alternanthera mosaic virus, and senna mosaic virus. To address this threat, we developed novel species-specific primers to detect BabMV targeting a 165 bp region of the coat protein (CP). Genus-specific primers were designed to validate the species-specific primers and attest their ability to discriminate between BabMV and its closest relatives. These primers targeted a 175 bp fragment of the CP region. The most effective sets of primers were chosen for reverse transcription polymerase chain reaction (RT-PCR) and SYBR® Green-based quantitative reverse transcription polymerase chain reaction (RT-qPCR) in symptomatic and asymptomatic babaco plants. Among 28 plants tested, 25 were positive and 3 were negative for BabMV using species-specific and genus-specific primers in RT-PCR and RT-qPCR, while the PapMV positive control was detected with the genus-specific primers and was negative for the species-specific primers. These primers represent a valuable molecular tool for detecting BabMV, potentially enhancing crop management.

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Turtlegrass virus X, which infects the seagrass Thalassia testudinum, is the only potexvirus known to infect marine flowering plants. We investigated potexvirus distribution in seagrasses using a degenerate reverse transcription polymerase chain reaction (RT-PCR) assay originally designed to capture potexvirus diversity in terrestrial plants. The assay, which implements Potex-5 and Potex-2RC primers, successfully amplified a 584 nt RNA-dependent RNA polymerase (RdRp) fragment from TVX-infected seagrasses. Following validation, we screened 74 opportunistically collected, apparently healthy seagrass samples for potexviruses using this RT-PCR assay. The survey examined the host species T. testudinum, Halodule wrightii, Halophila stipulacea, Syringodium filiforme, Ruppia maritima, and Zostera marina. Potexvirus PCR products were successfully generated only from T. testudinum samples and phylogenetic analysis of sequenced PCR products revealed five distinct TVX sequence variants. Although the RT-PCR assay revealed limited potexvirus diversity in seagrasses, the expanded geographic distribution of TVX shown here emphasizes the importance of future studies to investigate T. testudinum populations across its native range and understand how the observed fine-scale genetic diversity affects host-virus interactions.

Assuntos

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Hibiscus is not native to Colombia but well suited to its arid soil and dry climates. A single hibiscus plant from Risaralda, showing black spots on upper and lower sides of its leaves, was collected for virome analysis using meta-transcriptomic high-throughput sequencing technology. Bioinformatic analysis identified 12.5% of the total reads in the Ribo-Zero cDNA library which mapped to viral genomes. BLAST searches revealed the presence of carlavirus, potexvirus, and of known members of the genera Betacarmovirus, Cilevirus, Nepovirus, and Tobamovirus in the sample; confirmed by RT-PCR with virus-specific primers followed by amplicon sequencing. Furthermore, in silico analysis suggested the possibility of a novel soymovirus, and a new hibiscus strain of citrus leprosis virus C2 in the mixed infection. Both RNA dependent RNA polymerase and coat protein gene sequences of the potex and carla viruses shared less than 72% nucleotide and 80% amino acid identities with any alphaflexi- and betaflexi-virus sequences available in GenBank, identifying three novel carlavirus and one potexvirus species in the Hibiscus rosa-sinensis plant. The detection of physalis vein necrosis nepovirus and passion fruit green spot cilevirus in hibiscus are also new reports from Colombia. Overall, the meta-transcriptome analysis identified the complex virome associated with the black spot symptoms on hibiscus leaves and demonstrated the diversity of virus genera tolerated in the mixed infection of a single H. rosa-sinensis plant.

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The occurrence of Schlumbergera virus X (SchVX) in commercial dragon fruit fields in three provinces of Ecuador has been identified in this study. The virus was found in symptomatic and asymptomatic cladodes of the two major species (Hylocereus undatus and H. megalanthus) cultivated in the country. Symptoms in H. undatus included irregular and ring-shaped chlorotic spots that coalesce into large chlorotic patches along the cladodes, whereas small chlorotic spot symptoms on the cladodes were observed in H. megalanthus. Phylogenetic inferences based on 27 partial nucleotide sequences of the RNA-dependent RNA polymerase (RdRp) and three whole genome comparisons showed that Ecuadorean isolates from H. undatus and H. megalanthus share a most recent ancestor with isolates from Spain and Portugal. In addition, an SchVX isolate with a distinct genomic lineage was found in symptomatic H. polyrhizus plants from a single location, suggesting two independent virus introductions into the country.

Assuntos

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An emerging virus isolated from papaya (Carica papaya) crops in northwestern (NW) Argentina was sequenced and characterized using next-generation sequencing. The resulting genome is 6667-nt long and encodes five open reading frames in an arrangement typical of other potexviruses. This virus appears to be a novel member within the genus Potexvirus. Blast analysis of RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes showed the highest amino acid sequence identity (67% and 71%, respectively) with pitaya virus X. Based on nucleotide sequence similarity and phylogenetic analysis, the name papaya virus X is proposed for this newly characterized potexvirus that was mechanically transmitted to papaya plants causing chlorotic patches and severe mosaic symptoms. Papaya virus X (PapVX) was found only in the NW region of Argentina. This prevalence could be associated with a recent emergence or adaptation of this virus to papaya in NW Argentina.

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Our group works on the detection and characterization of cassava viruses, supporting projects that involve large scale pathogen surveillance activities and resistance screening assays in multiple and remote locations. In order to comply with these applications, nucleic acid isolation protocols need to be cost effective, adjusted for samples that will stand long distance transport and harsh storage conditions, while maximizing the yield and quality of the nucleic acid extracts obtained. The method we describe here has been widely used and validated using different downstream tests (including, but not limited to, Rolling Circle Amplification and Illumina and Nanopore sequencing), but is currently unpublished. The protocol begins with milligram amounts of dry leaf samples stored in silica gel, does not require liquid Nitrogen nor phenol extraction and produces an average of 2.11 µg of nucleic acids per mg of dry tissue.•DNA purity estimations reveal OD260/280 ratios above 2.0 and OD260/230 ratios above 1.7, even for samples stored in silica gel for several months.•The high quality of the extracts is suitable for detection of DNA and RNA viruses, with high efficiency.•We suggest this method could be used as part of a gold standard kit for virus detection in cassava.

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Several potexviruses (Family Alphaflexiviridae) have been reported infecting cassava (Manihot esculenta Crantz) in the Americas. They were isolated from severely diseased plants during the last 30-40 years and include: Cassava common mosaic virus (CsCMV), Cassava Caribbean mosaic virus (CsCaMV), Cassava Colombian symptomless virus (CsCSV) and Cassava virus X (CsVX). However, their definitive classification as distinct species remains unresolved for several reasons, including the lack of sequence data and unavailability of samples from original isolates. This complicates disease diagnostics, cassava germplasm exchange certification, evaluation of virus cleaning protocols and epidemiological studies. Furthermore, a recently detected novel alphaflexivirus, indicates that cassava-infecting potexviruses may be more diverse. To solve the identity of these viruses, we started indexing samples from different parts of Colombia using different sets of PCR primers, antisera available and inoculation to indicator plants. Results show that there are three major phylogenetic groups of potexviruses infecting cassava, and they correspond to CsCMV, CsVX and the newly identified Cassava new alphaflexivirus (CsNAV). Bioassays and sequence analysis established that isolates of CsNAV and CsVX cause latent infections in different cassava landraces, they are not efficiently transmitted to the indicator plant Nicotiana benthamiana and they lack the gene 3 of the conserved potexviral 'triple gene block' (TGB). In contrast, all isolates of CsCMV (which have a characteristic potexvirus genome arrangement) caused Cassava Common Mosaic Disease (CCMD) in single infections and were efficiently transmitted to N. benthamiana. Although phylogenetic analysis of the replicase sequence placed CsNAV and CsVX as members of the Potexvirus genus, their distinct genome arrangement and biological characteristics suggest they can be considered as members of a separate taxonomic group.

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El Potato virus X (PVX) es uno de los virus más limitantes del cultivo de la papa en el mundo. Es transmitido solamente por contacto y por tubérculo-semilla. Su control se fundamenta en la siembra de tubérculos certificados por su sanidad viral y en la disponibilidad de metodologías de diagnóstico altamente sensibles. En este trabajo se evaluó la prevalencia del PVX en cuatro diferentes tejidos de tubérculos de Solanum tuberosum subsp. andigena var. Diacol-Capiro y S. phureja var. Criolla Colombia utilizando pruebas de DAS-ELISA para 128 submuestras y de RT-qPCR para 32 grupos de submuestras (4 submuestras/grupo). Los resultados de las pruebas serológicas indicaron la presencia de PVX en el 6,25 y 50% de las submuestras analizadas para la variedad Diacol-Capiro y Criolla Colombia, respectivamente; mientras que los niveles de prevalencia del PVX utilizando la detección por RT-qPCR fueron del 93,75%, independientemente de la variedad de papa y del tejido evaluado. Los valores promedio del ciclo umbral (Ct) en las RT-qPCR fueron de 25,6 (Ct=18,02 a 34,49) y el análisis de las curvas de desnaturalización permitió identificar dos variantes del virus con valores de Tm de 79,5±1°C y 83,7±1°C. La secuenciación de los amplicones obtenidos por RT-qPCR para los controles positivos y para dos de las muestras, confirmó su naturaleza viral. Estos resultados señalan unos muy altos niveles de prevalencia de PVX en el material de siembra de papa en Antioquia y la necesidad de fortalecer los programas de certificación de semilla con pruebas de detección como RT-qPCR.

Potato virus X (PVX) is one of the most important virus affecting potato crops worldwide. The virus is only transmitted mechanically and through tuber-seeds. Control of PVX is based on the usage of certified tubers, which in turn depends on the availability of sensitive diagnostic tests that allow its direct detection on seeds. In this work, the prevalence of PVX in four different tuber tissues of Solanum tuberosum subsp. andigena var. Diacol-Capiro and S. phureja var. Criolla was evaluated using DAS-ELISA (128 subsamples) and RT-qPCR (4 subsamples per group). DAS-ELISA revealed the presence of PVX in 6.25 and 50% of Diacol-Capiro and Criolla Colombia subsamples; in contrast, RT-qPCR detected PVX in 93.75% of the samples independent of the potato variety or type of tissue. Ct values were in the 18.02 to 34.49 range with a mean value of 25.6. Melting curve analysis allowed the identification of two virus variants with Tm values of 79.5±1°C and 83.7±1°C. Sanger sequencing of the positive controls and two of the samples confirmed RT-qPCR amplicons to be PVX. These results reveal a high level of prevalence of PVX in potato tuber seeds used in Antioquia and the need to strengthen seed certification programs in Colombia through RT-qPCR detection assays.

RESUMO

En este estudio se determinaron las relaciones filogenéticas y los niveles de variación de aislamientos de PVX obtenidos en tejidos foliares de plantas de Solanum tuberosum subsp. andigena var. Diacol-Capiro y S. phureja var. Criolla Colombia en Antioquia, utilizando métodos de secuenciación de nueva generación (NGS) y de Sanger. Inicialmente, se detectó el PVX mediante DAS-ELISA (Agdia-PSA10000), confirmándose su presencia en ocho de las muestras por Inmunocaptura-RT-PCR en tiempo real (IC-RT-qPCR). Los resultados de las pruebas serológicas indicaron la infección de PVX en 14,7 % y 13,3 % de las muestras de Diacol-Capiro y Criolla Colombia, respectivamente. Su identidad fue confirmada por IC-RT-qPCR, con valores de ciclo umbral (Ct) de 15,04 a 27,59 y dos temperaturas de fusión (Tm) (Tm1 = 80,3 °C ± 0,5 y Tm2 = 83,3 °C ± 0,5), encontrándose así dos variantes de PVX en Antioquia. Utilizando NGS se detectó el PVX en bajos niveles de infección en las muestras de Criolla Colombia, siendo posible obtener contigs parciales para todos los ORFs del genoma viral. Con NGS no se detectó el virus en las muestras de Diacol-Capiro evaluadas. Los análisis filogenéticos realizados con base en secuencias de cápside y replicasa viral separaron los aislamientos de PVX de Antioquia en dos grupos, relacionados con el clado Eurasiático (I) de este virus. Los altos niveles de infección de PVX detectados en los cultivos de papa de Antioquia y la ocurrencia de al menos dos variantes, enfatizan en la necesidad de fortalecer los programas de certificación de tubérculos-semilla de papa, como principal herramienta para el control de este virus.

In this study, the phylogenetic relationships and molecular variability of PVX isolates from leaf samples of Solanum tuberosum subsp. andigena var. Diacol-Capiro and S. phureja var. Criolla Colombia in Antioquia were analyzed. Sequences were obtained using Next Generation Sequencing (NGS) of bulk samples and Sanger sequencing. DAS-ELISA (Agdia-PSA10000) revealed infection levels of 14.7 % and 13.3 % leaf samples of Diacol-Capiro and Criolla Colombia, respectively. The presence of PVX was further confirmed by IC-RT-qPCR in eight samples, which resulted in Ct values in the 15.04-27.59 range and two melting temperatures (Tm1 = 80.3 °C ± 0.5 and Tm2 = 83.3 °C ± 0.5). These results suggest the presence of at least two PVX variants in Antioquia. Using NGS, PVX was detected at low levels in leaf samples of Criolla Colombia, which resulted in contigs for most ORFs of the viral genome; NGS did not detect PVX in Diacol-Capiro samples. Phylogenetic analysis using capsid and replicase sequences separated PVX isolates into two groups within the Eurasian class (I). The high levels of PVX infection detected in potato crops in Antioquia and the presence of at least two variants highlight the need to strenghten current tuber seed certification programs aimed at controlling the spread of this virus.

RESUMO

ABSTRACT Sixty-seven tulip samples intercepted from the Netherlands by the Brazilian Agriculture Ministry, between 2004 and 2006, and two samples from São Paulo local market, Brazil, were assayed by serological and biological techniques, as well as by electron microscopy observations, for virus screening. In bulbs from the Netherlands potexviruses were detected in five samples and tobamoviruses in other three. Symptoms induced in some differential hosts were similar to those caused by Tobacco mosaic virus (TMV), while serological results indicated an infection byTulip virus X. In two tulip samples from local flower shops, a Potyviridae was identified based on the presence of flexuous particles and cytoplasmic cylindrical inclusions. Mechanical transmission tests to potyvirus hosts in the Amaranthaceae, Chenopodiaceae and Solanaceae species were negative, making possible to exclude a possible infection by Turnip mosaic viru, a common virus species in tulips. Although TVX could be detected in intercepted tulip bulbs from the Netherlands, the virus is only reported in Scotland, Japan and USA.

RESUMO Sessenta e sete amostras de tulipas interceptadas da Holanda pelo Ministério da Agricultura, Pecuária e Abastecimento, entre os anos de 2004 e 2006, e duas amostras, adquiridas no comércio local de São Paulo, foram avaliadas para a presença de vírus por meio de ensaios biológicos, sorológicos e observações ao microscópio eletrônico. Nos bulbos provenientes da Holanda, foram detectados potexvírus em cinco amostras e tobamovírus em duas outras. Os sintomas induzidos em hospedeiras diferenciadoras indicaram a presença de Tobacco mosaic virus (TMV), enquanto que os resultados de testes sorológicos permitiram a identificaçãoo Tulip virus X (TVX). Nas duas amostras adquiridas no comércio local foram detectados Potyviridae, uma vez que partículas alongadoflexuosas e inclusões cilíndricas estavam presentes. Ensaios de transmissão mecânica desses vírus para espécies de Amaranthaceae, Chenopodiaceae e Solanaceae foram negativos, permitindo deste modo ser excluída uma possível infecção pelo Turnip mosaic virus, uma espécie frequente em tulipas. Apesar do TVX ter sido detectado em tulipas interceptadas da Holanda, esse vírus foi relatado apenas na Escócia, Japão e Estados Unidos.

RESUMO

We report the results of a survey for the presence of Papaya ringspot virus (PRSV) along the coasts of the Gulf of Mexico and the Pacific Ocean, in 15 federal states of Mexico that account for over 98% of the national papaya production. More than 80 locations were visited in 58 counties. Out of a total of 267 papaya leaf samples, 157 tested positive for PRSV. We tested for the presence of three other viruses because of the occurrence of severe, atypical symptoms in plantations. Only Papaya mosaic virus (PapMV) was detected. PRSV was present in every county. PapMV was less frequent, but its overall distribution was almost identical. PRSV and PapMV occurred in single or mixed infections of papaya and other host species that could function as virus reservoirs. We investigated the diversity of the coat protein (CP) sequences of 36 PRSV isolates. The amino acid sequence divergence among all isolates ranged from 0.4 to 9.9%, and was comparable to that found in other regions of the world. In contrast to most of these world regions, there is a clear correlation between CP sequence variation and the geographical origins of the virus isolates.