RNAi-mediated silencing of Mediterranean fruit fly (Ceratitis capitata) endogenous genes using orally-supplied double-stranded RNAs produced in Escherichia coli.

BACKGROUND: The Mediterranean fruit fly (medfly), Ceratitis capitata Wiedemann, is a major pest affecting fruit and vegetable production worldwide, whose control is mainly based on insecticides. Double-stranded RNA (dsRNA) able to down-regulate endogenous genes, thus affecting essential vital functions via RNA interference (RNAi) in pests and pathogens, is envisioned as a more specific and environmentally-friendly alternative to traditional insecticides. However, this strategy has not been explored in medfly yet. RESULTS: Here, we screened seven candidate target genes by injecting in adult medflies gene-specific dsRNA hairpins transcribed in vitro. Several genes were significantly down-regulated, resulting in increased insect mortality compared to flies treated with a control dsRNA targeting the green fluorescent protein (GFP) complementary DNA (cDNA). Three of the dsRNAs, homologous to the beta subunit of adenosine triphosphate (ATP) synthase (ATPsynbeta), a vacuolar ATPase (V-ATPase), and the ribosomal protein S13 (RPS13), were able to halve the probability of survival in only 48 h after injection. We then produced new versions of these three dsRNAs and that of the GFP control as circular molecules in Escherichia coli using a two-self-splicing-intron-based expression system and tested them as orally-delivered insecticidal compounds against medfly adults. We observed a significant down-regulation of V-ATPase and RPS13 messenger RNAs (mRNAs) (approximately 30% and 90%, respectively) compared with the control medflies after 3 days of treatment. No significant mortality was recorded in medflies, but egg laying and hatching reduction was achieved by silencing V-ATPase and RPS13. CONCLUSION: In sum, we report the potential of dsRNA molecules as oral insecticide in medfly. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Molecular characterization of a putative new cavemovirus isolated from wild chicory (Cichorium intybus).

A putative new virus with sequence similarity to members of the genus Cavemovirus in the family Caulimoviridae was identified in wild chicory (Cichorium intybus) by next-generation sequencing (NGS). The putative new virus was tentatively named "chicory mosaic cavemovirus" (ChiMV), and its genome was determined to be 7,775 nucleotides (nt) long with the typical genome organization of cavemoviruses. ORF1 encodes a putative coat protein/movement polyprotein (1,278 aa), ORF2 encodes a putative replicase (650 aa), and ORF3 encodes a putative transactivator factor (384 aa). The first two putative proteins have 46.2% and 68.7% amino acid sequence identity to the CP/MP protein (YP_004347414) and replicase (YP_004347415), respectively, of sweet potato collusive virus (SPCV). ORF3 encodes a protein with 38.5% amino acid sequence identity to the putative transactivator factor (NP_056849) of cassava vein mosaic virus (CsVMV). The new putative viral genome and those of three cavemoviruses (epiphyllum virus 4 [EpV-4], SPCV, and CsVMV) differ by 24-27% in the nt sequence of the replicase gene, which exceeds the species demarcation cutoff (>20%) for the family.

Characterization of Cowpea aphid-borne mosaic virus in Passiflora edulis 'Catarina' in the southern coast of Santa Catarina State, Brazil / Caracterização do Cowpea aphid-borne mosaic virus em Passiflora edulis cv. Catarina no litoral sul de Santa Catarina, Brasil

CABMV is a limiting virus for passion fruit crop in Brazil, its main producing country. This virus has been reported in all producing states of the country, with the state of Santa Catarina (SC) in 2017 standing as the third largest passion fruit producer. In 2017, it reached 8.4% of the national production. The southern coast is the main responsible for the increase in production, which has been supplying the domestic market. However, in that same year, this region recorded the first symptom expressions in plants and fruits. The evaluation of the samples collected in the municipalities of Sombrio, Praia Grande and São João do Sul, southern coast of SC, was performed by using a mechanical transmission to indicator plants, PTA-ELISA and RT-PCR, and by sequencing. The evaluation results were positive for CABMV and negative for CMV in PTA-ELISA. In RT-PCR, there was the formation of a 700bp ca band, expected size for Potyvirus, whose sequence comparison with those deposited in GenBank reveled 98% identity with the isolates from São Paulo State. The occurrence of the virus in the southern coast of SC did not reach a serious decrease in passion fruit production due to the union of producers, who adopted preventive management measures to control the virus, whose effect led to a consolidation of the passion fruit production chain in the region.(AU)

O CABMV é um vírus limitante para a cultura do maracujá no Brasil, principal país produtor mundial, cuja ocorrência já foi relatada em todos os estados produtores. Em 2017, o estado de Santa Catarina (SC) foi o terceiro maior produtor de maracujá no Brasil, responsável por 8,4% da produção nacional, sendo o litoral sul o principal responsável pelo aumento da produção, garantindo o abastecimento do mercado interno. Entretanto, nesse mesmo ano, essa região registrou as primeiras expressões de sintomas em plantas e frutos. Uma avaliação das amostras coletadas nos municípios de Sombrio, Praia Grande e São João do Sul, litoral sul de Santa Catarina, foi realizada por transmissão mecânica para plantas indicadoras, PTA-ELISA, RT-PCR e sequenciamento. Os resultados foram positivos para o CABMV e negativos para o CMV, tanto em PTA-ELISA quanto RT-PCR. Na RT-PCR, houve a amplificação de bandas com ca de 700pb, tamanho esperado para o Potyvirus cuja comparação de sequências com as depositadas no GenBank revelaram 98% de similaridade com os isolados do estado de São Paulo. A ocorrência do vírus na região do litoral sul de Santa Catarina não causou quebra na produção de maracujá devido à adoção conjunta de medidas preventivas de manejo pelos produtores, fato que consolidou a cadeia produtiva do maracujá na região.(AU)

Surveys in the Chrysanthemum Production Areas of Brazil and Colombia Reveal That Weeds Are Potential Reservoirs of Chrysanthemum Stunt Viroid.

The stunting disease, incited by chrysanthemum stunt viroid (CSVd), has become a serious problem in chrysanthemum production areas worldwide. Here we identified 46 weed species from chrysanthemum fields in two producing regions of the State of São Paulo, Brazil. The mechanical inoculation of these weeds with a Brazilian CSVd isolate revealed that this viroid was able to infect 17 of these species, in addition to chrysanthemum, tomato and potato. Plants of Oxalis latifolia and chrysanthemum naturally infected with CSVd were found in chrysanthemum fields in Colombia, which is the first CSVd report in that country. Therefore, weeds have the potential to act as reservoirs of CSVd in the field. These results are the first reports of experimental CSVd infection in the following species: Amaranthus viridis, Cardamine bonariensis, Chamaesyce hirta, Conyza bonariensis, Digitaria sanguinalis, Gomphrena globosa, Helianthus annuus, Lupinus polyphyllus, Mirabilis jalapa, Oxalis latifolia, Portulaca oleracea and Catharanthus roseus. The phylogenetic analyses of the CSVd variants identified herein showed three groups with Brazilian CSVd variants distributed in them all, which suggests that Brazilian CSVd isolates may have different origins through successive introductions of infected germplasm of chrysanthemum in Brazil.

Survey of aphid population in a yellow passion fruit crop and its relationship on the spread Cowpea aphid-borne mosaic virus in a subtropical region of Brazil.

BACKGROUND: Passion fruit woodiness may be caused by Cowpea aphid-borne mosaic virus (CABMV) and is currently the major passion fruit disease in Brazil. To assess the virus-vector-host interactions, a newly introduced golden passion fruit plantation located in eastern region of São Paulo State, Brazil, was monitored. METHODS: Dissemination of CABMV was determined analyzing golden passion fruit plants monthly for 18 months by PTA-ELISA. Seasonality and aphid fauna diversity was determined by identification of the captured species using yellow sticky, yellow water-pan and green tile traps. Population composition of the aphid species was determined using the descriptive index of occurrence, dominance and general classification and overlap of species in the R program. RESULTS: Analyses of species grouping afforded to recognize 14 aphid species. The genus Aphis represented 55.42 % of the species captured. Aphid species formed two distinct clusters, one of which was characterized by the diversity of polyphagous species that presented high potential to spread CABMV. CONCLUSION: The low abundance and diversity of aphid species did not interfere negatively in the CABMV epidemiology. The genus Aphis, particularly Aphis fabae/solanella and A. gossypii, was crucial in the spread of CABMV in passion fruit orchards in the eastern State of São Paulo.

Incidência de vírus em videiras no Nordeste brasileiro e caracterização molecular parcial de isolados virais locais / Incidence of viruses in grapevines in the Brazilian Northeast and partial molecular characterization of local virus isolates

Os objetivos deste trabalho foram identificar as espécies virais presentes em vinhedos comerciais de duas regiões do Nordeste do Brasil e realizar a caracterização molecular parcial de isolados de três espécies virais. A diagnose foi realizada por meio de RT-PCR em tempo real para a detecção de Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine leafroll-associated virus 2, 3 e 4 (GLRaV-2, -3 e -4), Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV) e Grapevine fanleaf virus (GFLV). Exceto para GFLV, os vírus avaliados estão amplamente disseminados nas áreas amostradas, frequentemente em altas incidências e em infecções múltiplas, de até 98% e 76,4%, na Zona da Mata e no Vale do São Francisco, respectivamente. Isolados locais de GVA, GVB e GLRaV-3 foram parcialmente caracterizados com base na sequência completa de nucleotídeos do gene da proteína capsidial e apresentaram alta porcentagem de identidade de nucleotídeos com outros isolados brasileiros: 91,2% (GVA), 99,8% (GVB) e 99,7% (GLRaV-3).

The objectives of this study were to identify viral species infecting commercial vineyards in two regions of Northeastern Brazil and perform partial molecular characterization of isolates of three virus species. The diagnosis was performed by real time RT-PCR for detection of GRSPaV, GVA, GVB, GLRaV-2, GLRaV-3, GLRaV-4, GFkV, GRVFV and GFLV. Except for GFLV, the evaluated viruses are widespread in the sampled areas, often in high incidences and in multiple infections, up to 98% and 76.4%, in the Zona da Mata and in the Vale do São Francisco regions, respectively. Local isolates of GVA, GVB and GLRaV-3, partially characterized by complete coat protein gene nucleotide sequencing, showed high percentage of nucleotide identities with other Brazilian isolates of these viruses: 91.2% (GVA), 99.8% (GVB) and 99.7% (GLRaV-3).

Detection and partial molecular characterization of Grapevine fleck virus, Grapevine virus D, Grapevine leafroll-associated virus -5 and -6 infecting grapevines in Brazil / Detecção e caracterização molecular parcial de Grapevine fleck virus, Grapevine virus D, Grapevine leafroll-associated virus -5 e -6 que infectam videiras no Brasil

Grapevine fleck, rugose wood and leafroll are three grapevine viral diseases whose causal agents (or associated viruses) respectively are Grapevine fleck virus (GFkV), Grapevine virus D (GVD) and Grapevine leafroll-associated virus 5 and 6 (GLRaV-5 and -6). The objective of this work was to perform a partial molecular characterization of local isolates of these four viral species that infect grapevines. The nucleotide and deduced amino acid sequences of complete genes of the coat protein (CP) (of GFkV), the CP and the RNA binding protein (of GVD), the CP and the partial hHSP70 gene (of GLRaV-5) and the partial hHSP70 gene (of GLRaV-6) were aligned and compared in silico with other isolates. These data extend the available information about Brazilian isolates of GFkV, GLRaV-5 and -6, and reports for the first time the GVD occurrence in Brazil.

Mancha das nervuras, lenho rugoso e enrolamento das folhas são três doenças virais da videira, cujos agentes causais (ou vírus associados) são o Grapevine fleck virus (GFkV), o Grapevine virus D (GVD) e os Grapevine leafroll-associated virus 5 e 6 (GLRaV-5 e -6), respectivamente. O objetivo deste trabalho foi realizar a caracterização molecular parcial de isolados locais dessas quatro espécies virais que infectam videira. As sequências de nucleotídeos e de aminoácidos deduzidos dos genes completos da proteína capsidial (CP) (do GFkV), da CP e da RNA binding protein (do GVD), da CP do GLRaV-5 e parte do gene codificador da hHSP70 do GLRaV-5 e -6 foram alinhadas e comparadas in silico com sequências de outros isolados. Os dados obtidos expandem a informação existente sobre isolados brasileiros de GFkV, GLRaV-5 e - 6 e relatam pela primeira vez a ocorrência do GVD no Brasil.

Ribosomal protein L5 and transcription factor IIIA from Arabidopsis thaliana bind in vitro specifically Potato spindle tuber viroid RNA.

Potato spindle tuber viroid (PSTVd) contains an element of tertiary structure -loop E- also present in eukaryotic 5S rRNA. The ribosomal protein L5 and transcription factor IIIA (TFIIIA) from Arabidopsis thaliana bind 5S rRNA in vitro and in vivo, mediating different functions that include nucleocytoplasmic transport and transcription activation, respectively. We show that A. thaliana L5 and TFIIIA also bind PSTVd (+) RNA in vitro with the same affinity as they bind 5S rRNA, whereas the affinity for a chloroplastic viroid is significantly lower. These two proteins might participate in the synthesis and delivery of PSTVd RNA in vivo.

Detecção e caracterização molecular dos genes da proteína capsidial de ilarvírus e ampelovírus que infectam fruteiras temperadas / Detection and molecular characterization of ilarvirus and ampelovirus coat protein genes infecting temperate fruit trees

Dentre os principais patógenos que incidem em fruteiras temperadas, destacam-se o Prune dwarf virus (PDV), o Apple mosaic virus (ApMV) e o Grapevine leafroll-associated virus 1 (GLRaV-1). Neste trabalho foram realizadas a detecção e a caracterização molecular dos genes da proteína capsidial de isolados destas três espécies virais. RNAs totais foram extraídos de amostras de folhas de pessegueiros, macieiras e videiras e, nas reações de RT-PCR, foram utilizados oligonucleotídeos específicos para cada espécie viral. Os cDNAs amplificados foram clonados e sequenciados. Foram verificadas altas identidades entre as sequências de nucleotídeos dos genes da proteína capsidial dos isolados brasileiros de PDV, ApMV e GLRaV-1 e isolados de outros países, independente da origem geográfica e da hospedeira. O peso molecular da proteína capsidial destes vírus foi estimado por meio de Western blot em cerca de 24kDa (PDV), 26kDa (ApMV) e 39kDa (GLRaV-1).

Among the main pathogens infecting temperate fruit trees are Prune dwarf virus, Apple mosaic virus and Grapevine leafroll-associated virus 1. In this work the detection and molecular characterization of the coat protein genes of isolates from these viral species were carried out. Total RNA was extracted from peach, apple and grapevine leaves and RT-PCR reactions were performed using specific primers to each virus. The amplified cDNA fragments were cloned and sequenced. High identities were observed between coat protein nucleotide sequences of Brazilian isolates of PDV, ApMV and GLRaV-1 and isolates from other countries, independently from geographic origin and host. Coat protein molecular weights of these viruses were estimated by Western blot to be ca. 24kDa (PDV), 26kDa (ApMV) and 39kDa (GLRaV-1).

Detecção e identificação molecular de vírus associados a videiras sintomáticas e assintomáticas / Molecular detection and identification of virus associated with symptomatic and symptomless grapevines

A propagação vegetativa da videira favorece infecções virais múltiplas, com expressão diferencial de sintomas em função da combinação da cultivar ou espécie da hospedeira com a espécie viral. Os objetivos deste trabalho foram detectar e identificar as espécies virais presentes em duas espécies/cultivares de videira: uma sintomática e outra assintomática. DsRNA de ambas as amostras foi submetido à RT-PCR com 17 pares de oligonucleotídeos específicos para a detecção de Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine fleck virus (GFkV), Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine chrome mosaic virus (GCMV), Rupestris stem pitting-associated virus (RSPaV), Grapevine leafroll-associated virus 1 a 4 (GLRaV-1 a -4), além de três pares de oligonucleotídeos degenerados. Pelo menos um fragmento amplificado, por par de oligonucleotídeos, foi clonado e sequenciado. Plantas sintomáticas e assintomáticas mostraram infecções múltiplas por RSPaV, GLRaV-2 e/ou GLRaV-3. As sequências de nucleotídeos obtidas para sete isolados de RSPaV, três de GLRaV-2 e dois de GLRaV-3 apresentaram identidades superiores a 90 por cento com espécies homólogas e permitiram a definição das possíveis estirpes presentes nas amostras infectadas. Esses resultados evidenciam a necessidade da diagnose viral baseada em testes específicos para determinar a condição sanitária da videira.

The vegetative propagation of grapevine facilitates multiple viral infections, with different symptoms which vary according to combinations of cultivar or host species with viral species. The aims of this research were to detect and identify the viral species infecting two grapevine species/cultivars: one symptomatic and one symptomless. DsRNA from both samples was assayed by RT-PCR using 17 pairs of specific primers for detection of the Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine fleck virus (GFkV), Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine chrome mosaic virus (GCMV), Rupestris stem pitting-associated virus (RSPaV) and Grapevine leafroll-associated virus 1-4 (GLRaV-1 to -4), besides three degenerate primer pairs. For each primer pair at least one amplicon was cloned and sequenced. Symtomatic and symptomless plants were multiple infected by RSPaV, GLRaV-2 and/or GLRaV-3. The nucleotide sequences of seven isolates of RSPaV, three of GLRaV-2 and two of GLRaV-3 showed identities higher than 90 percent with the homologous viral species and allowed to identify possible viral strains in infected samples. These results highlight the necessity of viral diagnosis based on specific assays to determine grapevine sanitary status.

Produção de antissoro policlonal utilizando a proteína capsidial recombinante do Rupestris stem pitting-associated virus / Production of polyclonal antiserum using recombinant coat protein of Rupestris stem pitting -associated virus

O Rupestris stem pitting-associated virus (RSPaV) é o agente causal das caneluras do lenho da videira. Este trabalho teve como objetivo produzir antissoro policlonal a partir da proteína capsidial (CP) recombinante do RSPaV e avaliar a sua especificidade e sensibilidade. O gene da CP do RSPaV, com 780pb, foi previamente caracterizado. Esse gene foi subclonado no sítio de restrição EcoRI, no vetor de expressão pRSET-B e o plasmídeo recombinante foi utilizado para induzir a expressão da CP em Escherichia coli. A CP, ligada a uma cauda de seis histidinas, foi purificada por meio de cromatografia de afinidade em coluna de Ni-NTA a partir do extrato de proteínas totais extraídas de E. coli. A identidade da proteína purificada foi confirmada em SDS-PAGE e Western blot, utilizando-se anticorpos comerciais contra a cauda de seis histidinas. A CP recombinante expressada in vitro apresentou massa molecular de cerca de 31kDa. A proteína purificada foi quantificada e 2,55mg foram utilizados para a imunização de um coelho. O antissoro policlonal obtido reagiu com diferentes isolados deste vírus, extraídos de videiras em ELISA indireto.

RSPaV is the causal agent of pitting in the grapevine woody cylinder. The aim of this research was to produce polyclonal antiserum against recombinant RSPaV coat protein (CP) and evaluate its specificity and sensibility. The CP gene (780bp) of RSPaV was previously characterized. This gene was subcloned into the EcoRI site of the pRSET-B expression vector and the recombinant plasmid was used to induce the expression of the CP in E. coli cells. The CP, fused to a 6-His-tag, was purified from E. coli total protein extract by affinity chromatography using Ni-NTA resin. Identity of the purified protein was confirmed by SDS-PAGE and Western blot, using antibodies against the histidine tail. The in vitro-expressed recombinant CP presented a MW of ca. 31kDa. The purified protein was quantified and 2.55mg used for the immunization of a rabbit. The obtained polyclonal antiserum reacted with different RSPaV isolates extracted from grapevines in indirect ELISA.