ABSTRACT
Plants of Senna multiglandulosa (family Fabaceae), an ornamental shrub, growing adjacent to tomato and chrysanthemum greenhouses located in San Diego, Texcoco, Estado de Mexico, had leaves with putative virus symptoms, consisting of annular or irregular chlorotic spots of different sizes (Supplementary Fig. 1a). To investigate the presence of a virus, high-throughput sequencing (HTS) was performed. Total RNA was extracted from symptomatic leaves of S. multiglandulosa plants using the SV Total RNA Isolation System Kit (Promega, USA). A portion of the RNA was sent to BGI Genomics (China) for cDNA library construction and sequencing on the DNBSEQ platform (BGI Genomics). HTS yielded 14,673,469 clean paired reads (150x2), which were assembled de novo into 91,879 contigs using SPAdes v3.15 software (Prjibelski et al. 2020). The contigs ranged from 78 to 14,534 nucleotides (nts), which were subjected to BLASTx and BLASTn analyses. A single viral contig of 9,501 nts was detected (average coverage: 56,716x per nt) representing the nearly complete genome of tobacco etch virus (TEV). The highest identity was 79.26% at the nt level (92% query coverage) with TEV isolate TEV7DA (GenBank: DQ986288; length: 9,539 nts) from the USA, and 86.67% at the amino acid (aa) level considering the polyprotein, which are higher than the species demarcation threshold (<76% nt and <82% aa) for the genus Potyvirus (Inoue-Nagata et al. 2022). Additionally, the sequence obtained from S. multiglandulosa revealed 79.21-79.37% nt identities with different TEV isolates from Solanaceae plants (Capsicum annuum, MW748496; Solanum lycopersicum, OM471966.1; Nicotiana tabacum, OL311684.1). The new TEV genome was deposited in GenBank under accession number ON110203. The results obtained by HTS were confirmed by RT-PCR with the original isolated RNA using a pair of specific primers designed from the TEV sequence (TEV-NIb-F, 5'- GCGCTTAAATGCAGACTCGG-3' and TEV-NIb-R, 5'-GTGAAAGTTCAGCAGCAAGCGCA-3') that amplify a 550-bp fragment of the RNA-dependent RNA polymerase. The obtained amplicon was sequenced by the Sanger method, and was 100% identical to the sequence generated by HTS. Subsequently, N. tabacum and N. glutinosa plants were mechanically inoculated using TEV-positive S. multiglandulosa leaves as the inoculum source. Twenty days after inoculation, light chlorotic spots and necrotic lesions were observed on N. tabacum and mosaic on N. glutinosa (Supplementary Fig. 1b-c). RT-PCR analysis confirmed the presence of TEV infection in these indicator plants. To determine the incidence of S. multiglandulosa plants showing TEV-infection symptoms, a survey (n=16) was carried out on two farms in Texcoco; the survey showed a 100% incidence of symptoms. Five survey samples were randomly selected, and the presence of TEV was confirmed by RT-PCR. The discovery of Tobacco etch virus (family Potyviridae: genus Potyvirus) in tobacco was reported in Kentucky, USA in 1928 (Valleau and Johnson, 1928), one of the most common and damaging viruses for the chili crop in Mexico (Delgado, 1974). TEV causes heavy yield loss in several Solanaceae plants and infects more than 120 species in 19 families of dicotyledons (Holmes, 1946). S. obtusifolia (originally Cassia obtusifolia) was the first legume reported as a natural host of TEV in Florida, USA (Anderson, 1954). To our knowledge, this is the first report of the natural infection of S. multiglandulosa by TEV in Mexico and the first TEV genome isolated and sequenced from a legume. S. multiglandulosa is widely distributed in 16 states in Mexico, both cultivated and naturalized, however, it is not considered native to the country (Rzedowski and Calderón, 1997). The occurrence of TEV in S. multiglandulosa represents an alternative reservoir of the virus, with an important role in the epidemiology of the disease.
ABSTRACT
Viroids are single-stranded, circular RNA molecules (234-406 nt) that infect a wide range of crop species and cause economic losses in agriculture worldwide. They are characterized by the existence of a population of sequence variants, attributed to the low fidelity of RNA polymerases involved in their transcription, resulting in high mutation rates. Therefore, these biological entities exist as quasispecies. This feature allows them to replicate within a wide range of host plants, both monocots and dicots. Viroid hosts include economically important crops such as tomato, citrus, and fruit trees such as peach and avocado. Given the high risk of introducing viroids to viroid disease-free countries, these pathogens have been quarantined globally. As discussed herein, Mexico represents a geographical landscape of viroids linked to their origin and comprises considerable biodiversity. The biological features of viroid species endemic to Mexico are highlighted in this communication. In addition, we report the phylogenetic relationships among viroid and viroid strains, their economic impact, geographical distribution, and epidemiological features, including a broad host range and possible long-distance, seed, or insect-mediated transmission. In summary, this review could be helpful for a better understanding of the biology of viroid diseases and future programs on control of movement and spread to avoid economic losses in agricultural industries.
Subject(s)
Citrus , Solanum lycopersicum , Viroids , Viroids/genetics , Phylogeny , Mexico/epidemiologyABSTRACT
En plantaciones de alstroemeria (Alstroemeria L.) de Villa Guerrero, Estado de México, se han detectado plantas con síntomas similares a los inducidos por geminivirus en otros cultivos hortícolas. En dichas plantaciones también se ha observado la presencia de la mosquita blanca, considerada como el vector más eficiente de estos virus. El objetivo del presente trabajo fue detectar la presencia de geminivirus en plantas de alstroemeria. Mediante PCR, usando los iniciadores MotCP2118/MotCP2123, se obtuvo un segmento de ~600pb, similar al del control positivo correspondiente a chile infectado con el begomovirus pepper huasteco yellow vein virus (PHYVV, antes pepper huasteco virus) en plantas sintomáticas, mientras que en las asintomáticas la detección fue negativa. Plantas de Nicotiana glutinosa, N. benthamiana, N. rustica, N. tabacum var. xanthi y Datura stramonium inoculadas por biobalística con ADN total obtenido de alstroemerias con síntomas y positivas a PHYVV mediante PCR, mostraron mosaicos leves y deformación de hojas, mientras que en plantas de Capsicum annuum se observaron mosaicos, necrosis en nervaduras y abultamientos en hojas. Con el ADN de estas plantas también se obtuvieron bandas correspondientes al PHYVV, pero en las monocotiledóneas bombardeadas, incluyendo alstroemeria, no fue detectado el fragmento. La secuencia de oligonucleótidos de los productos de PCR mostró 98% de homología con el begomovirus PHYVV. Aunque no fue posible reproducir en alstroemeria los síntomas observados en campo, sí se evidenció mediante PCR la presencia de un geminivirus similar al PHYVV en tejido de plantas sintomáticas.
In alstroemeria (Alstroemeria L.) plantations located in Villa Guerrero, Mexico State, plants with symptoms similar to those induced by geminivirus in other horticultural crops have been detected. In addition, the presence of whiteflies, which are considered the most efficient vectors of these viruses, has been observed in these plantations. The goal of this work was to detect the presence of this geminivirus species in alstroemeria plants. By means of PCR analysis using primers MotCP2118/MotCP2123, a fragment of ~600pb similar to the amplicon obtained from PHYVV-infected positive control was amplified only from symptomatic plants. Nicotiana glutinosa, N. benthamiana, N. rustica, N. tabacum var. xanthi and Datura stramonium plants were inoculated by bombardment with total DNA obtained from symptomatic alstroemerias and positive to PHYVV by means of PCR. Inoculated plants showed mild mosaics and deformation of leaves, whereas in the leaves of Capsicum annum plants, mosaics, vein necrosis and blisters were observed. Using DNA from these plants as template in PCR, amplicons corresponded to PHYVV were also obtained; however, in bombarded monocotyledons, including alstroemeria, this fragment was not detected. The sequence of oligonucleotides from the PCR products showed 98% homology to PHYVV geminivirus. Even though symptoms presented by alstroemeria plants in the field were not reproduced, the presence of a geminivirus similar to PHYVV in tissue of symptomatic plants was evidenced through PCR.
Em plantações de alstroemeria (Alstroemeria L.) de Villa Guerrero, Estado do México, se tem detectado plantas com sintomas similares aos induzidos por geminivírus em outros cultivos hortícolas. Em ditas plantações também tem sido observada a presença da mosquinha branca, considerada como o vetor mais eficiente destes vírus. O objetivo do presente trabalho foi detectar a presença de geminivírus em plantas de alstroemeria. Mediante PCR, usando os iniciadores MotCP2118/MotCP2123, obteve-se um segmento de ~600pb, similar ao do controle positivo correspondente a chile infectado com o begomovírus pepper huasteco yellow vein virus (PHYVV, antes pepper huasteco virus) em plantas sintomáticas, enquanto que nas assintomáticas a detecção foi negativa. Plantas de Nicotiana glutinosa, N. benthamiana, N. rustica, N. tabacum var. xanthi e Datura stramonium inoculadas por biobalística com DNA total obtido de alstroemerias com sintomas e positivas a PHYVV mediante PCR, mostraram mosaicos leves e deformação de folhas, enquanto que em plantas de Capsicum annuum se observaram mosaicos, necrose em nervaduras e protuberâncias em folhas. Com o DNA destas plantas também se obtiveram bandas correspondentes ao PHYVV, mas nas monocotiledóneas bombardeadas, incluindo alstroemeria, não foi detectado o fragmento. A sequência de oligonucleotídeos dos produtos de PCR mostrou 98% de homologia com o begomovírus PHYVV. Embora não foi possível reproduzir em alstroemeria os sintomas observados em campo, sím foi evidenciada, mediante PCR, a presença de um geminivírus similar ao PHYVV em tecido de plantas sintomáticas.
