Coding-Complete Genome Sequence of a Black Queen Cell Virus Isolate from Honey Bees (Apis mellifera) in Italy

In this study, we documented the complete coding genome sequence of a Black queen cell virus (BQCV) isolate from honey bees in Italy. This genome sequence illustrates a high similarity with other BQCV isolates reported worldwide and could provide insights into BQCV genome phylogeny and divergence

A new variant of Xylella fastidiosa subspecies multiplex detected in different host plants in the recently emerged outbreak in the region of Tuscany, Italy

The vector-borne bacterial pathogen Xylella fastidiosa is widely distributed in the Americas; in the last decade it has emerged as a serious threat for agricultural crops, natural environment and landscape in Europe. Following the first EU outbreak in 2013 in southern Italy, associated with a severe disease in olive trees, annual mandatory surveys are now in place in the Member States, leading to the discovery of bacterial outbreaks in different countries. Among the latest findings, an outbreak has been reported in the Italian region of Tuscany, with infections identified in seven different plant species. In this work, we report the isolation and the genetic characterization of isolates associated with this newly discovered outbreak. Multilocus sequence typing approach revealed the occurrence of isolates harbouring a new sequence type, denoted ST87, genetically related to strains of subsp. multiplex, but different from the genotypes of this subspecies previously characterized in Europe. Five cultured strains were successfully recovered from four of the seven host plants, an important achievement for advancing the studies on genomics and pathogenicity of these isolates and thus assess their potential threat for European agriculture

Characterization of Citrus tristeza virus isolates recovered in Syria and Apulia (southern Italy) using different molecular tools

Summary. Citrus tristeza virus (CTV) is the causal agent of the most important virus disease of citrus. CTV isolates differing in biological and molecular characteristics have been reported worldwide. Recently, CTV was detected in Syria in citrus groves from two Governorates (Lattakia and Tartous) and several CTV outbreaks have been reported in Apulia (southern Italy) since 2003. To molecularly characterize the CTV populations spreading in Syria and Italy, a number of isolates from each region was selected and examined by different molecular approaches including: Multiple Molecular Markers analysis (MMM), real time RT-(q)PCR, single strand conformation polymorphism (SSCP) of the major coat protein (CP) gene (P25), and sequence analysis of the CP (P25), P18, P20 and RdRp genes. SSCP analysis of CP25 yielded two distinct simple patterns among the Syrian isolates and three different patterns in the Italian isolates. Based on MMM analysis, all Syrian CTV isolates were categorized as VT-like genotype, whereas the Italian isolates reacted only with the markers specific for the T30 genotype. These findings were also confirmed by RT-qPCR and by sequencing analysis of four genomic regions. The Italian isolates had nucleotide identities which varied: from 99.5 to 99.8 for the CP gene; from 97.4% to 98.3% for the P18 gene; from 98.6% to 99.8% for the P20 and from 97.8% to 99.1% for the partial RdRp sequenced. High sequence identity was found for all genomic regions analyzed between the Syrian isolates (from 98.9% to 99.6%). These results show that the CTV populations spreading in Apulia and Syria are associated with different genotypes, indicating different potential impacts on the citrus trees in the field. Since in both areas the introduction of the virus is relatively recent, infected plants resulted to contain a single and common genotype, suggesting that CTV is spreading from the first outbreaks by aphids or local movement of autochthonous infected plant material

Survey of the sanitary status of ancient native grapevine cultivars from Apulia (Italy) by real-time PCR

A study of the sanitary status of ancient native grapevine cultivars and biotypes of Apulia (Italy) was performed in the framework of the project Re.Ge.Vi.P. (“Recovery of Apulian grape germplasm”), A total of 80 native table and wine grapes (the majority of which described in historical pre-philloxera reports) were recovered throughout the Apulian territory and their dormant canes used for virus screening by ELISA for the presence of Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine leafroll-associated virus-1, -2 and -3 (GLRaV-1, -2 and -3), Grapevine fanleaf virus (GFLV), Grapevine fleck virus (GFkV) and Arabis mosaic virus (ArMV), using commercial kits (Agritest s.r.l., Italy). Certified plant propagation material of grapevine should be free of these viruses according to the Italian Law. Selected accessions were propagated and those positive for single or multiple virus infections were submitted to sanitation. Overall, at least three plantlets deriving from distinct apex excisions (totally 242) were transferred to the greenhouse after acclimatization and were tested after 60 days by REALTIME-RT-PCR for all the viruses above indicated Over 242 apexes tested, GFLV and GFKV were the most frequently detected viruses showing 9.5% and 8.2% of infection rate, respectively, followed by GVA (4.9%), GRLaV3 (2.9%) and GVB (1.6%). Furthermore, all plants reacted negatively to GLRaV-1, GLRaV-2 as well as ArMV which is known to be rare in the Mediterranean area, also because the low occurrence of its vector, Xyphinema diversicaudatum. Mixed infections by more than one virus were found in some plants assuming the failure of sanitation procedure to eliminate these viruses. The persistence of infection by GVA, GVB, GLRaV3 and GFkV in some apexes, which was not detected by the preliminary ELISA screening of source plants, underlines the higher sensitivity of Realtime-RT-PCR to detect these viruses

Xylella fastidiosa subsp. pauca and multiplex elicit differential responses in a susceptible olive cultivar

Trabajo presentado en la 3rd European Conference on Xylella fastidiosa (Building knowledge, protecting plant health), celebrada online el 29 y 30 de abril de 2021.The emergence of Xylella fastidiosa in Europe revealed the occurrence of bacterial strains belonging to different subspecies and unraveled the vulnerability of some typical European plant species. Among these, olive was found infected in different European outbreaks, with infections caused by strains harboring different sequence types (STs) and belonging to subspecies pauca and multiplex. In this work, we compared under greenhouse conditions, the response of the olive cultivar “Cellina di Nardò” to infections caused by the strains De Donno (subsp. pauca, ST53) and ESVL (subsp. multiplex, ST6), as this cultivar is one of the most susceptible in the Italian outbreak. The data showed that: (i) strain De Donno systemically colonized the plants faster than the strain ESLV; (ii) 1.5 years post-inoculation, the bacterial population detected in the plants systemically infected was ~25 times higher for strain De Donno than ESLV; (iii) a severe shoot dieback were detected on the De Donno-infected plants, whereas up to 3 years post-inoculation no dieback and desiccations have been recorded on the ESLV-infected plants. This pathogenicity test was complemented with an RNA-seq analysis. Nine libraries, three for each condition (ESVL, De Donno, mock inoculated), were sequenced by Ilumina technology and sequences mapped against the Olea europeae cultivar Picual genome for the analysis of differential gene expression (DEG). The output of this study showed that, respect to the 6,200 genes differentially expressed (FDR<1.00E-05) in De Donno-infected plants, only 550 (FDR<1.00E-05) genes had an altered expression in the ESVL-infected plants. The present data showed that strains of the two subspecies elicit differential host responses, which were biologically and molecularly attenuated in the ESVL infections with respect to the aggressive De Donno infections and provide useful insights for exploring the molecular basis of Xylella-olive interaction.Peer reviewe

Evolutionary analysis of Citrus tristeza virus outbreaks in Calabria, Italy: two rapidly spreading and independent introductions of mild and severe isolates

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