High Incidence of Strawberry Polerovirus 1 in the Czech Republic and Its Vectors, Genetic Variability and Recombination

In total, 332 strawberry plants from 33 different locations in the Czech Republic with or without disease symptoms were screened by RT-PCR for the presence of strawberry polerovirus 1 (SPV1) and five other viruses: strawberry mottle virus, strawberry crinkle virus, strawberry mild yellow edge virus, strawberry vein banding virus, and strawberry virus 1. SPV1 was detected in 115 tested strawberry plants (35%), including 89 mixed infections. No correlation between symptoms and the detected viruses was found. To identify potential invertebrate SPV1 vectors, strawberry-associated invertebrate species were screened by RT-PCR, and the virus was found in the aphids Aphis forbesi, A. gossypii, A. ruborum, A.sanquisorbae, Aulacorthum solani, Chaetosiphon fragaefolii, Myzus ascalonicus, and several other non-aphid invertebrate species. SPV1 was also detected in aphid honeydew. Subsequent tests of C. fragaefolii and A.gossypii virus transmission ability showed that at least 4 h of acquisition time were needed to acquire the virus. However, 1 day was sufficient for inoculation using C. fragaefolii. In conclusion, being aphid-transmitted like other tested viruses SPV1 was nevertheless the most frequently detected agent. Czech SPV1 isolates belonged to at least two phylogenetic clusters. The sequence analysis also indicated that recombination events influence evolution of SPV1 genomes.

Functional Analysis of a Viral Promoter From the Strawberry Vein Banding Virus (SVBV) Chinese Isolate

BackgroundPromoter is an important factor during gene expression in cells. In this study, we cloned a full-length promoter from the strawberry vein banding virus (SVBV) Chinese isolate and produced several its deletion mutants.MethodsThe full-length promoter of SVBV (SP1) and its three deletion mutants (SP2, SP3, and SP4) were amplified using polymerase chain reaction (PCR). The expression activities controlled by the SVBV SP1, SP2, SP3, and SP4 were evaluated using β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes.ResultsOur transient expression assays showed that the SVBV SP1 promoter as well as its three deletion mutants all expressed the reporter genes, but to very different levels. Interestingly, the expression activity driven by the SP1 promoter was much higher than that shown by the CaMV 35S promoter. After stable transformation of a GUS gene into Nicotiana tabacum plants, the transgene expression level driven by the SVBV SP1 promoter was about 2.6-fold greater than that driven by the CaMV 35S promoter. In addition, the GUS gene expression levels could be enhanced by co-infiltrating the plants with the SP1 promoter-driven vector carrying the GUS gene and the vector expressing the SVBV ORF V or ORF VI.ConclusionsThe SVBV Chinese isolate promoter SP1 is a stronger promoter than the CaMV 35S and FLt-US promoter, may be more useful for production of stable transgenic plants.

Detecção molecular e análise filogenética da sequência parcial do gene da proteína do capsídeo do vírus da faixa das nervuras do morangueiro

O morango cultivado (Fragaria x ananassa Duch. (Rosaceae) é um híbrido originado pelo cruzamento das espécies americanas Fragaria chiloensis e Fragaria virginiana e pertence à família Rosaceae. O morangueiro possui reprodução vegetativa e, por isso, é comum o acúmulo de viroses e outras doenças de difícil controle. Uma das quatro viroses mais importantes na cultura do morango é causada pelo vírus da faixa das nervuras (Strawberry vein banding virus – SVBV), que é transmitido no campo por afídeos, de maneira semipersistente. Para melhorar o conhecimento genômico, são recomendadas técnicas moleculares e a classificação de isolados de SVBV. Um dos determinantes primários da transmissibilidade e especificidade por afídeos é a proteína do capsídeo, que possui importância crítica para o estabelecimento da infecção. Neste trabalho, foi sequenciada parcialmente a proteína do capsídeo do gene do SVBV de um isolado alemão, inoculado em morangueiros e mantido em casa de vegetação no Brasil, por mais de dez anos. Foi realizada a análise filogenética comparando as sequências contidas no GenBank com o objetivo de elucidar as relações evolutivas nesta espécie. As análises filogenéticas mostraram que a sequência do isolado está mais próxima dos isolados dos EUA e Egito. Estes resultados contribuem para a melhor elucidação dos mecanismos de evolução do vírus e do patossistema em questão.

Sequencing a Strawberry Germplasm Collection Reveals New Viral Genetic Diversity and the Basis for New RT-qPCR Assays

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.

The occurrence of strawberry virus 1 infecting strawberry in Shandong province, China

Strawberry (Fragaria × ananassa Duch.) is one of the most important horticultural plants worldwide with high economic and nutritional value. Strawberry associated virus 1 (SaV1) is a putative Cytorhabdovirus isolated from strawberry in Fujian province, China (Ding et al., 2019). Strawberry virus 1 (StrV-1) is another putative Cytorhabdovirus characterized from F. ananassa and F. vesca in Czech Republic (Fránová et al., 2019). The complete genomes of isolates of SaV1 and StrV-1 share 79 to 98% nucleotide (nt) identities. In August 2020, foliar chlorotic spots or streaks were observed in four strawberry cultivars (cv. Honeoye, Mibao, 8128 and All Star) in Yantai, Shandong province, China. To identify the associated viruses, symptomatic leaves from two plants of each cultivar (8 samples) were pooled for high-throughput sequencing (HTS). Total RNA was extracted from the composite sample and used for constructing a cDNA library after ribosomal RNA (rRNA)-depletion. Sequencing was carried out on Illumina Hiseq 4000 (Novogene, China). Raw reads were filtered, trimmed and de novo assembled as described previously (Grabherr et al., 2013; Zhou et al. 2020). The resulting contigs were screened by BLASTn and BLASTx against GenBank database. Subsequent analyses indicated the presence of strawberry vein banding virus, strawberry pallidosis associated virus and strawberry mottle virus in the analyzed sample, which had been reported previously in strawberry (Martin and Tzanetakis, 2013; Shi et al., 2018; Bhagwat et al., 2016). Besides, five contigs ranging from 266 to 6,057 nt were obtained. They shared 87 to 91% nt sequence identity with StrV-1 isolate B (GenBank accession no. MK211271). To confirm StrV-1 infection in the strawberry plants, total RNA was isolated from all eight samples using RNAprep Pure Plant Plus Kit (Tiangen, China). Reverse transcription polymerase chain reaction (RT-PCR) was conducted with two pairs of specific primers StrVp1 (Forward: 5ʹ-CATTACTGAAGCATTCCGTG-3′/Reverse: 5ʹ-AGATATCACGCACAGTGAC-3ʹ), and StrVp2 (Forward: 5ʹ-TTGCGCGAAGCGGATGTCCG-3′/Reverse: 5ʹ-GGCTGCCAGAGCGTTGGATG-3ʹ), targeting nt positions 70-1,231 and 7,825-9,348 of StrV-1 isolate B, respectively. Fragments with the expected sizes were amplified from two samples of cv. All Star. The amplicons were cloned, sequenced, and deposited in GenBank under accession no. MW419123-124 and MW645247-248. Both protein encoding sequences shared 91 to 92% and 80 to 84% nt identities with the corresponding sequences of StrV-1 isolate B and SaV1, respectively, indicating that the isolates from this study are genetic variants of StrV-1 and distantly related to SaV1. Crude sap was prepared by homogenizing leaf tissues of StrV-1 infected strawberry in 0.02 mol/L sodium phosphate buffer with 0.45% (w/v) sodium diethyldithiocarbamate thihydrate, then gently rubbed onto five healthy Nicotiana benthamiana plants. Neither the inoculated leaves nor the systemically infected leaves showed obvious symptoms seven days post inoculation. However, StrV-1 was detected by RT-PCR in all five N. benthamiana plants as described above. In addition, a survey of strawberry greenhouses was conducted in August 2020 and approximately 10% of plants in a 667 m2 greenhouse in Yantai had StrV-1-like symptoms. To the best of our knowledge, this is the first report of the occurrence of StrV-1 infecting strawberry in Shandong province, China. Our findings expand the geographic range and genetic diversity of StrV-1 and indicate it could be a potential virus threat to strawberry production in China.

Evaluation of Various Sources of Viral Infection in Strawberry Fields of Quebec, Canada

The decline of cultivated strawberry (Fragaria × ananassa Duchesne ex Rozier; Rosaceae) observed in the province of Quebec, Canada, between 2012 and 2014 was mostly caused by persistent viruses: strawberry mild yellow edge virus (SMYEV) (Potexvirus; Alphaflexiviridae) and strawberry crinkle virus (SCV) (Cytorhabdovirus; Rhabdoviridae); and semi-persistent viruses: strawberry mottle virus (SmoV) (Secoviridae), strawberry vein banding virus (SVBV) (Caulimovirus; Caulimoviridae), and strawberry pallidosis virus (SPaV) (Crinivirus: Closteroviridae) transmitted by insect vectors. The objective of this study was to determine the sources of viral contamination in commercial strawberry fields in Quebec. Specifically, we wished to 1) determine the prevalence of persistent viruses in winged strawberry aphid Chaetosiphon fragaefolii (Cockerell) (Hemiptera: Aphididae) specimens captured; 2) determine the prevalence of all viruses in wild strawberry Fragaria virginiana Miller plants near commercial plantings; and 3) evaluate the viral contamination of strawberry transplants obtained from nurseries and tested before and after planting in commercial strawberry fields. Results indicated high percentage (38%) of the aphids (n = 205) and high percentage (67%) of F. virginiana patches (n = 12) were infected by strawberry viruses. Ultimately, our results showed a low percentage (5%) of the plants from various nurseries (n = 56) were infected before planting, whereas a third (29%) of the healthy exposed plants in the fields (n = 96) became rapidly infected by insect vectors within a year of having been planted. This study provides significant insights on the relative importance of the various sources of contamination in Quebec strawberry fields: C. fragaefolii versus F. virginiana versus nurseries versus post-nursery infections through exposure to virus-carrying insects.