Identification of grapevine virus G and grapevine virus H in Sardinia, Italy

Summary. Grapevine virus G (GVG) and grapevine virus H (GVH) (genus Vitivirus ) are recently discovered viruses. Analysis of 38 samples from Sardinian grapevine cultivars for the presence of GVG and GVH was carried out using RT-PCR. All samples were also tested for grapevine Pinot gris virus (GPGV) using RT-PCR, and for grape-vine leafroll virus -1, -2 and -3, grapevine virus A (GVA) and B (GVB), arabis mosaic virus (ArMV), grapevine fanleaf virus (GFLV) and grapevine fleck virus (GFkV) using multiplex RT-PCR. GVG was confirmed in four vines, and GVH was detected in only one sample. In phylogenetic analyses of the coat protein (CP) region, the Sardinian GVG isolates clustered separately from isolates from Croatia and New Zealand. The Sardinian GVH isolate clustered with most sequences from other countries, but with greater affinity to isolates from California (USA) for the CP region, whereas it clustered with isolates from Croatia in the RNA-dependent RNA polymerase (RdRp) region. In addition to GVG and GVH, many samples were coinfected with GVA, viruses from the leafroll complex, and GPGV. This is the first record of GVG and GVH occurring in Italy.

To verify occurrence of new grapevine viruses in Sardinia (Italy), particularly GVG and GVH, a survey was conducted of several grapevine varieties randomly collected from different vineyards in this region.
All samples were tested for the presence of GVG and GVH using the OneStep RT-PCR kit (Qiagen), with 0.5 µM of each specific primer (Supplementary Table 1) and the supplied mastermix, following the manufacturer's recommendations.The RT-PCR conditions applied in the Mastercycler (Eppendorf) included the reverse transcription step at 52°C for 30 min, an initial activa-tion step at 95°C for 15 min, 35 cycles at 94°C for 30 s, 55°C for 45 s, and 72ºC for 1 min, followed by a final extension at 72°C for 7 min.In addition, all samples were tested for GPGV by RT-PCR using specific primers (Supplementary Table 1), and by multiplex RT-PCR, to simultaneously test the samples for the presence of GLRaV-1, GLRaV-2, GLRaV-3, GVA, GVB, ArMV, GFLV and GFkV.Multiplex PCR was carried out according to published protocols (Faggioli et al., 2012).
The PCR products obtained, including partial coat protein (CP) and partial RNA-dependent RNA polymerase (RdRp) genes, were Sanger sequenced in both directions at Macrogen Europe (Amsterdam, The Netherlands), and analyzed using MEGA X (Kumar et al., 2018) and BioEdit software, version 7.2.5 (Hall, 1999).
Phylogenetic trees for GVG and GVH were generated using the sequences from the Sardinian isolates and sequences available in GenBank, including the GVN isolate (GenBank.MZ68235) for rooting the GVG tree and the GVM isolate (GenBank.MK492703) for rooting the GVH trees (Supplementary Table 2).Phylogenetic analyses were carried out using the aligned sequences, and trees were generated using the maximum likelihood method with the MEGA X program.A bootstrap analysis with 1,000 replicates was performed to estimate statistical support for the different tree branches, using a minimum of 50% as a threshold.
Four of the 38 grapevines/samples tested were positive for GVG, and one sample was positive for GVH (Table 1).The four GVG-positive samples were detected only with primers specific to the CP region, whereas the same samples were negative with primer pairs targeting the RdRp region.The failure to detect the virus was probably due to the genetic variability of the Sardinian isolates in the respective regions of the genomes.
In several studies investigating the effects of GVA, GVB, and GVD on plants (Sciancalepore et al., 2006;Blouin et al., 2018b;Rosa et al. 2011), combined infections were found to have greater negative effects on symptoms than single virus infections.Sardinian grapevines positive for GVG were coinfected with GVA in two samples, GLRaV-1 in one sample, GLRaV-2 in one sample, and GPGV in three samples, whereas coinfections with GLRaV-3 were present in all the samples (Table 1).Presence of other viruses included in the study was not confirmed in the vine infected with GVH.
Phylogenetic analyses indicated that all Sardinian GVG CP sequences formed a separate clade.The four Sardinian GVG isolates had nucleotide similarities from 98.3% to 100%.In addition, CP sequences clustered in separate clades based on the location of the vineyards, in Narbolia and Olmedo (Figure 1).Sardinian GVG isolates showed the greatest nt identity (93.57%) with the Croatian VD-102 isolate (Vončina and Almeida 2018).
The GVH phylogenetic trees (Figures 2 and 3) generated using the partial CP and RdRp sequences showed the greatest CP nucleotide similarity of 99.72% with isolate GC5462 (GenBank acc.no.MK838926) from the United States of America, but a grapevine accession originating from Romania (Diaz-Lara et al., 2019).In the RdRp region, the Sardinian GVH isolate showed greatest nucleotide identity (98.31%) with the Babica plosnata isolate from Croatia (Jagunić et al., 2021).
This study is the first to record the presence of GVG and GVH in Italy.GVG was found in two different areas of Sardinia, in one area in the red grape 'Carignano', and one in the white grape 'Vermentino'.GVH Table 1.RT-PCR detection of different viruses in different grapevine in Sardinia, in cvs.'Cannonau'(CN) located in Alghero (AHO) and Narbolia (NAR), 'Vermentino' located in Olmedo (OLM) and Narbolia (NAR), and 'Vernaccia' (VRN) and 'Carignano' (CRG) located in Narbolia (NAR).Vines positives for GVG and GVH are indicated in bold font.

Samples
was detected only in one sample of 'Carignano'.As mentioned byDiaz-Lara et al. (2019), none of the novel grapevine vitiviruses have been associated with diseases.It may be important, however, to monitor infection by these viruses in grapevines, to check for any impacts on vine performance and production.ACKNOWLEDGMENTSThis research was supported by a project grant from the University of Sassari (Fondo di Ateneo per la ricer-ca 2020).Part of this research received funds from the Croatian Science Foundation, grant number IP-2018-01-1305 project "Ecology and characterization of two novel viruses infecting grapevine-ENVISaGE".LITERATURE CITED Abou-Ghanem N., Saldarelli P., Minafra A., Buzkan N., Castellano M.A, Martelli G.P., 1997.Properties of grapevine virus D, a novel putative trichovirus.Journal of Plant Pathology 79(1): 15-25.

Figure 1 .
Figure1.Phylogenetic tree generated from partial nucleotide sequences of the CP region of GVG isolates.Bootstrap values were obtained using 1000 replicates.Only branches with support greater than 50% are indicated.Sardinian isolates detected in this study are indicated by black dots.The phylogenetic tree was reconstructed using the maximum likelihood algorithm implemented in MEGA X software.Four isolates from Croatia (CRO) and three from New Zealand (NZ) were selected from the GenBank database.The GVN sequence (MZ682351) was used as the rooting outgroup.

Figure 2 .
Figure2.Phylogenetic tree generated from partial nucleotide sequences of the GVH CP region.Bootstrap values were obtained using 1000 replicates.Only branches having a support greater than 50% are indicated.The Sardinian isolate detected in this study is indicated by a black dot.The phylogenetic tree was reconstructed using the maximum likelihood algorithm implemented in the MEGA X software.Nineteen isolates from different countries located in the collection in the United States of America (USA) and eight isolates from Croatia (CRO) were selected from GenBank and used to construct the tree.The GVM sequence (MK492703) was used as the rooting outgroup.

Figure 3 .
Figure3.Phylogenetic tree generated from partial nucleotide sequences of the GVH RdRp region.Bootstrap values were obtained using 1000 replicates.Only branches having a support above 50% are indicated.The Sardinian GVH isolate detected in this study is indicated by a black dot.The phylogenetic tree was reconstructed using the maximum likelihood algorithm implemented in the MEGA X software.Seventeen isolates from different countries (United States of America -USA, Portugal -PT and Croatia -CRO) were selected from GenBank and used to construct the tree.A GVM sequence (MK492703) was used as the rooting outgroup.