New Host Plant Species of Grapevine Virus A Identified with Vector-Mediated Infections.

Grapevine virus A (GVA) is an economically important virus and a member of the genus Vitivirus (family Betaflexiviridae) that causes a range of symptoms with qualitative and quantitative effects on grape production. Wild and domesticated species of Vitis, including hybrids used as rootstocks, are considered important natural hosts of GVA. Mechanical transmission to some herbaceous plant species, graft transmission, and vector transmission from grape to grape by various mealybugs and soft scale insects have been reported. Under laboratory and greenhouse conditions, this study demonstrates the transmission of GVA from grapes to alternative hosts by the vine mealybug (Planococcus ficus). Results of ELISA, end-point one-step RT-PCR, and real-time RT-PCR, and in some cases electron microscopy and genome sequencing, confirmed successful transmission to three new plant species commonly found in Croatian vineyards: velvetleaf (Abutilon theophrasti), redroot pigweed (Amaranthus retroflexus), and field poppy (Papaver rhoeas), along with Chenopodium murale and the previously known host Nicotiana benthamiana, with variable infection rates. Depending on the host species, symptoms in the form of leaf reddening, yellow spots, reduced growth of lateral shoots, systemic vein clearing, foliar deformation and rugosity, and dwarfism were observed in GVA-infected plants, whereas no symptoms were observed in infected plants of A. theophrasti. Reverse transmission from these new hosts to grapevines by Pl. ficus was not successful. These results confirm four new GVA host species and open new research venues.

Biology and Ultrastructural Characterization of Grapevine Badnavirus 1 and Grapevine Virus G.

The biological characteristics of grapevine viruses, such as their transmission and host range, are important for the adoption of successful prophylaxis strategies. The aim of this study was to investigate the traits of two newly described grapevine viruses widely distributed in Croatia, grapevine badnavirus 1 (GBV-1) and grapevine virus G (GVG). The vine mealybug (Planoccocus ficus) proved to be a vector of GBV-1 and GVG capable of vine-to-vine transmission with overall experimental transmission rates of 61% and 14.6%, respectively. Transmission was also demonstrated by grafting, with an overall transmission rate of 53.8% for GBV-1 and 100% for GVG, as well as by green grafting using the T-budding technique. Symptoms of GBV-1 and GVG were not observed on the woody cylinders of the indicators LN 33, Kober 5BB, 110 Richter and cvs. Chardonnay and Cabernet Sauvignon. Seed transmission and mechanical transmission were not confirmed. Electron microscopy revealed accumulation of GBV-1 particles and viroplasms in the cytoplasm, but no alternations of the cell structure. Infection with GVG revealed the proliferation of tonoplast-associated vesicles inside phloem cells and cell wall thickening.

Incidence and Genetic Diversity of Grapevine Virus G in Croatian Vineyards.

Grapevine virus G (GVG) is a recently discovered vitivirus infecting grapevines. Historically, viruses in the genus Vitivirus have been associated with the grapevine rugose wood disease. Based on new and previously reported GVG isolates, primers and probes were developed for real-time RT-PCR. The developed assay successfully detected the virus in infected plants during dormancy and the growing season. A field study of 4327 grapevines from Croatian continental and coastal wine-growing regions confirmed the presence of GVG in 456 (~10.5%) grapevines from three collection plantations and 77 commercial vineyards, with infection rates ranging from 2% to 100%. Interestingly, the virus was confirmed only in vines considered to be Croatian autochthonous cultivars, but not in introduced cultivars. A 564-nucleotide long portion of the coat protein gene from previously known and newly characterized GVG isolates had nucleotide and amino acid identities ranging from 89% to 100% and from 96.8% to 100%, respectively. Phylogenetic analysis revealed five distinct groups, with isolates originating from the same site being close to each other, indicating possible local infection. The information presented in this manuscript sets the stage for future studies to better understand the ecology and epidemiology of GVG and the possible need for inclusion in certification schemes.

Grapevine Badnavirus 1: Detection, Genetic Diversity, and Distribution in Croatia.

Grapevine badnavirus 1 (GBV-1) was recently discovered in grapevine using high throughput sequencing. In order to carry out large-scale testing that will allow for better insights into virus distribution, conventional and real-time PCR assays were developed using sequences both from previously known, and four newly characterized isolates. Throughout the growing season and dormancy, GBV-1 can be detected by real-time PCR using available tissue, with the possibility of false-negative results early in vegetation growth. GBV-1 real-time PCR analysis of 4302 grapevine samples from the Croatian continental and coastal wine-growing regions revealed 576 (~13.4%) positive vines. In the continental wine-growing region, virus incidence was confirmed in only two collection plantations, whereas in the coastal region, infection was confirmed in 30 commercial vineyards and one collection plantation. Infection rates ranged from 1.9 to 96% at the different sites, with predominantly autochthonous grapevine cultivars infected. Conventional PCR products obtained from 50 newly discovered GBV-1 isolates, containing the 375 nucleotides long portion of the reverse transcriptase gene, showed nucleotide and amino acid identities ranging from 94.1 to 100% and from 92.8 to 100%, respectively. The reconstructed phylogenetic tree positioned the GBV-1 isolates taken from the same vineyard close to each other indicating a possible local infection event, although the tree nodes were generally not well supported.

Virus and Virus-like Pathogens in the Grapevine Virus Collection of Croatian Autochthonous Grapevine Cultivars.

Grapevine collections play an important role, especially in the study of viruses and virus-like pathogens. In 2009, after an initial ELISA screening for eight viruses (arabis mosaic virus, grapevine fanleaf virus, grapevine fleck virus, grapevine leafroll-associated viruses 1, 2, and 3, and grapevine viruses A and B), a collection of 368 grapevine accessions representing 14 different Croatian autochthonous cultivars and containing single or mixed infection of viruses was established to further characterize the viral pathogens. Subsequently, Western blot, RT-PCR, cloning, and sequencing revealed that grapevine rupestris stem pitting-associated virus was frequently found in accessions of the collection, with isolates showing substantial genetic diversity in the helicase and coat protein regions. High-throughput sequencing of 22 grapevine accessions provides additional insight into the viruses and viroids present in the collection and confirms the fact that Croatian autochthonous grapevine cultivars have high infection rates and high virome diversity. The recent spread of "flavescence dorée" phytoplasma in Europe has not spared the collection. After the first symptoms observed in 2020 and 2021, the presence of phytoplasma was confirmed by LAMP in six grapevine accessions and some of them were lost. Single or multiple viruses and viroids, as well as own rooted grapevines in the collection, make the plants susceptible to various abiotic factors, which, together with the recent occurrence of "flavescence dorée", makes the maintenance of the collection a challenge. Future efforts will be directed towards renewing the collection, as 56% of the original collection has been lost in the last 13 years.

Prevalence of Virus Infections and GLRaV-3 Genetic Diversity in Selected Clones of Croatian Indigenous Grapevine Cultivar Plavac Mali.

The cultivar Plavac Mali (Vitis vinifera L.), the most important indigenous red grapevine cultivar in Croatia, was tested for the presence of 16 grapevine viruses. Thirty-five samples from the collection vineyard were tested for the presence of grapevine leafroll-associated viruses-1, -2, and -3 (GLRaV-1, GLRaV-2 and GLRaV-3, respectively), grapevine fanleaf virus (GFLV), arabis mosaic virus (ArMV), grapevine virus-A (GVA), -B (GVB), -G (GVG), -H (GVH), -I (GVI), -J (GVJ), grapevine fleck virus (GFkV), grapevine rupestris stem pitting associated virus (GRSPaV), and grapevine pinot gris virus (GPGV) by reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, standard PCR was conducted for grapevine badnavirus 1 (GBV-1) and grapevine red blotch virus (GRBV). Mixed infections were most common and GLRaV-3, the most abundant virus found in 85.71% of the vines tested, was further molecularly characterised. Different genomic variants of the heat shock protein homologue (HSP70h) were separated by cloning, detected by single-strand conformation polymorphism (SSCP) analysis, sequenced, and phylogenetically analysed. The presence of phylogenetic groups I and II was only confirmed. This study demonstrates the high virus infection rate of Plavac Mali vines and the heterogeneity of GLRaV-3 present nowadays in a collection vineyard.

Detection, Transmission, and Characterization of Grapevine Virus H in Croatia.

A survey of recently discovered vitiviruses was performed on 113 Croatian autochthonous grapevine cultivars from the national collection "Jazbina" using one-step RT-PCR. The presence of grapevine virus H (GVH) was confirmed in nine (7.9%) cultivars and grapevine virus G in eight (7.1%), while the presence of grapevine viruses I and J were not detected. GVH was transmitted by the vine mealybug (Planococcus ficus) from a source plant to grapevine seedlings with a 10.5% transmission rate using a combination of 10 first and second instars per plant with 48 and 72 h of acquisition and inoculation access period, respectively. Transmission correlated with the presence of grapevine leafroll-associated virus 3 (GLRaV-3) in the GVH-source plant and recipient seedlings. No alternative GVH host was identified. A comparison of 356 nt fragments of the RdRP and CP coding regions showed nucleotide identity between the Croatian GVH isolates in the range of 95.5-99.2% and 97.5-99.4% and amino acid identity between 95.8 and 100% and between 98.3 and 100%, respectively. Comparison with foreign isolates revealed nucleotide sequence similarity in the RdRP and CP between 94 and 100% and between 97.7-100%, respectively. To the best of our knowledge, this is the first report of GVH in Croatia and the first identification of the vine mealybug as a vector of GVH.

Screening of some Croatian autochthonous grapevine varieties reveals a multitude of viruses, including novel ones.

Next-generation sequencing of total RNA samples from four Croatian autochthonous grapevine varieties revealed the presence of a novel virus in two grapevine accessions. The complete genome sequence of a novel virus, tentatively named "grapevine badnavirus 1" (GBV-1), was reconstructed from a de novo-assembled contig. GBV-1 has a genome of 7,145 nucleotides containing three ORFs with sequence similarity to other badnaviruses. In addition, several other viruses and viroids, including grapevine virus G, grapevine virus K/D, grapevine virus T, grapevine Roditis leaf discoloration-associated virus, grapevine yellow speckle viroids 1 and 2, and hop stunt viroid were detected and identified for the first time in Croatian grapevines in the course of this study.

Infection and Colonization of Nicotiana benthamiana by Grapevine leafroll-associated virus 3.

Grapevine leafroll disease is an increasing problem in all grape-growing regions of the world. The most widespread agent of the disease, Grapevine leafroll-associated virus 3 (GLRaV-3), has never been shown to infect species outside of the genus Vitis. Virus transmission to several plant species used as model systems was tested using the vine mealybug, Planococcus ficus. We show that GLRaV-3 is able to infect Nicotiana benthamiana. Working with GLRaV-3 infected N. benthamiana revealed distinct advantages in comparison with its natural host Vitis vinifera, yielding both higher viral protein and virion concentrations in western blot and transmission electron microscopy observations, respectively. Immunogold labelling of thin sections through N. benthamiana petioles revealed filamentous particles in the phloem cells of GLRaV-3 positive plants. Comparison of assembled whole genomes from GLRaV-3 infected V. vinifera vs. N. benthamiana revealed substitutions in the 5' UTR. These results open new avenues and opportunities for GLRaV-3 research.

Viral Diversity in Autochthonous Croatian Grapevine Cultivars.

A survey was conducted on nine autochthonous grapevine cultivars grown along the Croatian coastal region. In total, 48 vines (44 from germplasm collection, 4 from vineyards) originating from 23 sites were tested for 26 viruses using molecular methods. Results revealed high infection rates with Grapevine leafroll-associated virus 3 (GLRaV-3); Grapevine virus A (GVA, both 91.7%); Grapevine fleck virus (GFkV, 87.5%); and Grapevine rupestris stem pitting-associated virus (GRSPaV, 83.3%). Other detected viruses were: Grapevine fanleaf virus (GFLV); Grapevine leafroll-associated viruses 1, 2, and strains of 4 (GLRaV-1, GLRaV-2, GLRaV-4); Grapevine viruses B, D, F (GVB, GVD, GVF); Grapevine red globe virus (GRGV); Grapevine vein feathering virus (GVFV); Grapevine Syrah virus 1 (GSyV-1); and Grapevine Pinot gris virus (GPGV). No virus-free vine was found. Mixed infections were determined in all vines, the number of viruses in a single vine ranged from three to nine. GLRaV-3 variant typing confirmed presence of group I, II, and III. Four vines with leaf deformation and mottling were positive for GPGV. Seven viruses (GLRaV-4-like group, GVD, GVE, GVF, GRGV, GSyV-1, and GVFV) were detected for the first time in Croatia. This survey confirmed the deteriorated sanitary status of autochthonous Croatian grapevine cultivars.

Chronic fungal meningitis caused by Aureobasidium proteae.

We present a case of chronic meningitis due to the mold Aureobasidium proteae. Clinical features, the disease course, as well as the diagnostic methods and optimal treatment options are discussed. This case confirms the neuroinvasiveness of A. proteae and introduces it as a new human pathogen.