Grapevine Virome of the Don Ampelographic Collection in Russia Has Concealed Five Novel Viruses

Abstract

In this study, an analysis of the virome of 51 grapevines from the Don ampelographic collection named after Ya. I. Potapenko (Russia) was performed using high-throughput sequencing of total RNA. A total of 20 previously described grapevine viruses and 4 viroids were identified. The most detected were grapevine rupestris stem pitting-associated virus (98%), hop stunt viroid (98%), grapevine Pinot gris virus (96%), grapevine yellow speckle viroid 1 (94%), and grapevine fleck virus (GFkV, 80%). Among the economically significant viruses, the most present were grapevine leafroll-associated virus 3 (37%), grapevine virus A (24%), and grapevine leafroll-associated virus 1 (16%). For the first time in Russia, a grapevine-associated tymo-like virus (78%) was detected. After a bioinformatics analysis, 123 complete or nearly complete viral genomes and 64 complete viroid genomes were assembled. An analysis of the phylogenetic relationships with reported global isolates was performed. We discovered and characterized the genomes of five novel grapevine viruses: bipartite dsRNA grapevine alphapartitivirus (genus Alphapartitivirus, family Partitiviridae), bipartite (+) ssRNA grapevine secovirus (genus Fabavirus, family Secoviridae) and three (+) ssRNA grapevine umbra-like viruses 2, -3, -4 (which phylogenetically occupy an intermediate position between representatives of the genus Umbravirus and umbravirus-like associated RNAs).

Keywords: RNA-seq; Vitis vinifera; alphapartitivirus; grapevine germplasm; grapevine virome; grapevine viruses; high-throughput sequencing; secovirus; umbra-like virus.

Figure 1

(a) Conserved sequences of the 5′ terminal regions of GAPV RNA1 and RNA2; (b) genome organization of GAPV. Each genomic segment contains one ORF.

Figure 2

(a) Phylogenetic tree based on nucleotide sequences of RNA1 of GAPV (red dot) and representative members of the family Partitiviridae; (b) Fragment of phylogenetic tree based on nucleotide sequences of RNA1 of GAPV (red dot), alphapartitiviruses (blue dots), and unclassified partitiviruses. The full tree is contained in Supplementary Figure S21. The trees were constructed in MEGA11 using the maximum-likelihood method and GTR model with 1000 bootstrap replicates. Bootstrap values below 50 are not shown.

Figure 2

(a) Phylogenetic tree based on nucleotide sequences of RNA1 of GAPV (red dot) and representative members of the family Partitiviridae; (b) Fragment of phylogenetic tree based on nucleotide sequences of RNA1 of GAPV (red dot), alphapartitiviruses (blue dots), and unclassified partitiviruses. The full tree is contained in Supplementary Figure S21. The trees were constructed in MEGA11 using the maximum-likelihood method and GTR model with 1000 bootstrap replicates. Bootstrap values below 50 are not shown.

Figure 3

Pairwise identity matrix including amino acid sequences of (a) RdRp and (b) CP of GAPV and closest partitiviruses.

Figure 4

Phylogenetic trees based on nucleotide sequences of (a) RNA1 and (b) RNA2 of GSV (red dot) and members of the genera Nepovirus, Fabavirus, Comovirus, and unclassified Secoviridae. The trees were constructed in MEGA11 using the maximum-likelihood method and GTR model with 1000 bootstrap replicates.

Figure 5

(a) Putative genome organization of GSV. Cleavage sites were predicted by multiple alignments of the aa sequences with fabaviruses. Light colors indicate conserved domains predicted by InterPro; (b) conserved sequences of the 3′ terminal regions of GSV RNA1 and RNA2.

Figure 6

Pairwise identity matrix including amino acid sequences of the (a) Pro-Pol region and (b) CP of GSV and fabaviruses.

Figure 7

Putative genome organization of GULV-2, -3, -4. ORF1 and ORF2 encode RdRp, ORF3 and ORF4 encode proteins with unknown function.

Figure 8

Phylogenetic trees based on nucleotide sequences of (a) genomes and (b) RdRp of GULVs (red dots), umbraviruses, and ulaRNAs. The tree was constructed in MEGA11 using the maximum-likelihood method and GTR model with 1000 bootstrap replicates.

Figure 9

Pairwise identity matrix including amino acid sequences of ORF3 and ORF4 of GULVs, four umbraviruses, and ten ulaRNAs. OULV: opuntia umbra-like virus; FULV: fig umbra-like virus; EMaV: Ethiopia maize-associated virus; SULV: sugarcane umbra-like virus; MaULV: maize-associated umbra-like virus; MULV: maize umbra-like virus 1; GULV: grapevine umbra-like virus; JgULV: johnsongrass umbra-like virus 1; PEMV2: pea enation mosaic virus 2; OPMV: opium poppy mosaic virus; PatMMoV: patrinia mild mottle virus; CmotV: carrot mottle virus; SbaVA: strawberry associated virus A.

Figure 10

Frequency of spread of grapevine viruses and viroids in the Don ampelographic collection (percentage of the total number of collected samples). Economically significant viruses are shown as .