Complete nucleotide sequence and genome organization of Grapevine fleck virus

The complete nucleotide sequence of Grapevine fleck virus (GFkV) genomic RNA was determined. The genome is 7564 nt in size, excluding the 3′-terminal poly(A) tail, is characterized by an extremely high cytosine content (ca. 50%), and contains four putative open reading frames and untranslated regions of 291 and 35 nt at the 5′ and 3′ ends, respectively. ORF 1 potentially encodes a 215·4 kDa polypeptide (p215), which has the conserved motifs of replication-associated proteins of positive-strand RNA viruses. ORF 2 encodes a 24·3 kDa polypeptide (p24) identified as the coat protein. ORFs 3 and 4 are located at the extreme 3′ end of the viral genome and encode proline-rich proteins of 31·4 kDa (p31) and 15·9 kDa (p16), respectively, of unknown function. Phylogenetic analysis of the viral replicase and coat protein genes showed that GFkV is related to members of the Tymovirus and Marafivirus genera. Two subgenomic RNAs were present in the GFkV preparations as ascertained by molecular hybridization. The genome organization of GFkV resembles to some extent that of tymoviruses and marafiviruses. However, differences in the biological and epidemiological behaviour, cytopathology and molecular properties (i.e. size of genomic RNA and coat protein, and number of ORFs) support the notion that GFkV is a separate virus belonging in a new genus

First report on the occurence of grapevine rupestris stem pitting-associated virus in Moroccan grapevines

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Identification of a single-stranded DNA virus associated with citrus chlorotic dwarf disease, a new member in the family Geminiviridae

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The endophytic microbiome of X. fastidiosa susceptible and resistant olives

A multi-factorial strategy is required to co-exist with X. fastidiosa infections, which are devastating olive trees in the southern area of Apulia (Italy). Observations in the outbreak area can provide information on potential approaches for containment. Olive cvs Leccino and FS17 show lessened symptoms and host lower bacterial populations (1,2) than cvs Ogliarola salentina, Cellina di Nardò and Kalamata. We are evaluating whether microbial communities inhabiting the xylem vessels of olive cvs showing different susceptibilities to X. fastidiosa -infection play a role in resistance. To explore these endophytic microbiomes, a whole-metagenome shotgun analysis is currently ongoing. X. fastidiosa -infected and healthy olive plants of the cultivars FS17, Leccino and Kalamata, were selected from the same plot to limit the influence of diverse soil composition and crop management. Shotgun sequencing of DNA extracted from the xylem tissues will be used to investigate the microbiome community by bio-informatic analysis. Moreover, efforts to isolate culturable microorganisms to be used in antagonistic assays against X. fastidiosa, will be performed. Concurrently, the X. fastidiosa-biocontrol potency of Paraburkholderia phytofirmans PsJN strain, whose beneficial effects in the reduction of symptoms in Pierce’s Disease (3) have been recently described, are under evaluation. We are testing the ability of P. phytofirmans to colonise xylem vessels and interact with X. fastidiosa in tobacco and olive

Complete Genome Sequence of the Olive-Infecting Strain Xylella fastidiosa subsp. pauca De Donno

We report here the complete and annotated genome sequence of the plant-pathogenic bacterium Xylella fastidiosa subsp. pauca strain De Donno. This strain was recovered from an olive tree severely affected by olive quick decline syndrome (OQDS), a devastating olive disease associated with X. fastidiosa infections in susceptible olive cultivars

First report of cherry virus a and plum bark necrosis stem pitting-associated virus in cherry in Chile

Stone fruits rank third among the most important crop species in Chile, after grapevine and apple. Specifically, cherry (Prunus avium L.) cultivation have increased during the last 10 years, making of Chile the most important exporter in the Southern hemisphere. Nineteen cherry samples collected in the spring of 2016 were subjected to high-throughput sequencing (HTS) analyses. Small RNA extracts were obtained following the protocol described by Giampetruzzi et al. (2012). Sequencing libraries were prepared using TruSeq smallRNA library preparation kit (Illumina Inc.) and sequenced on Illumina HiScanSQ platform. Trimming and de novo assembly of sequenced reads using CLC genomics workbench v7.0 were carried out, and the obtained contigs were analyzed with BLAST. One sample presented 131 contigs that showed homology with the Plum bark necrosis stem pitting-associated virus (PBNSPaV) reference sequence with accession no. EF546442. The alignment of nine PBNSPaV complete genome references allowed the design of two primer pairs specific for the RdRp gene (PBN-RdRp-F 5?-CTTATTATTGTGCTGAAGTTGATCT-3?/PBN-RdRp-R 5?-TGGAAAAGTATTGAGTCATCACC-3?) and a partial region of CP gene (PBN-CP-F 5?-GAGGCAATGGATGAGGAA-3?/PBN-CP-R 5?-TCTTCCACCGGACTGATTA-3?) to be used in RT-PCR. The RdRp (KY887573) and CP (KY887574) sequences amplified from isolate 10381 shared 99 and 97% identity with reference isolate WH1 (KJ792852) from China and the PBNSPaV type-strain (EF546442) from the U.S.A., respectively. In addition, the HTS analysis showed that 14 out of 19 cherry samples have several contigs showing homology with Cherry virus A (CVA) reference sequence. Two primer pairs (CVAF1 5?-CAATGTTGTTGACAATTCCCAC-3?/CVAR1 5?-CCTACATGAATTTGACCTAAACAAA-3?; CVAF2 5?-ACTGCAGAGAAAACAACTGCC-3?/CVAR2 5?-AGGCCCCTTCTTATCTCGTT-3?) were designed based on the alignment of CVA complete genomes database with the sequences obtained from Chilean isolates. CVA infection was confirmed via RT-PCR in all 14 cherry trees using both primer pairs. BLASTn analysis of the two amplification products of CVA from isolate 10596 (KY887575, KY887577) showed 99% of identity with the isolate Lambert (KU215410) from Czech Republic and the same amplicons obtained from isolate 10395 (KY887576, KY887578) showed 99% of identity with the isolate ChTA12 from China (KT310083). To our knowledge, this is the first report of CVA and PBNSPaV infecting cherry in Chile and South America. Further analyses are in progress in order to determine the prevalence of these viruses in the main cherry producing areas of Chile

Surface Plasmon Resonance Assay for Label-Free and Selective Detection of Xylella Fastidiosa

Xylella fastidiosa is among the most dangerous plant bacteria worldwide causing a variety of diseases, with huge economic impact on agriculture and environment. A surveillance tool, ensuring the highest possible sensitivity enabling the early detection of X. fastidiosa outbreaks, would be of paramount importance. So far, a variety of plant pathogen biomarkers are studied by means of surface plasmon resonance (SPR). Herein, multiparameter SPR (MP-SPR) is used for the first time to develop a reliable and label-free detection method for X. fastidiosa. The real-time monitoring of the bioaffinity reactions is provided as well. Selectivity is guaranteed by biofunctionalizing the gold transducing interface with polyclonal antibodies for X. fastidiosa and it is assessed by means of a negative control experiment involving the nonbinding Paraburkholderia phytofirmans bacterium strain PsJN. Limit of detection of 105 CFU mL 1 is achieved by transducing the direct interaction between the bacterium and its affinity antibody. Moreover, the binding affinity between polyclonal antibodies and X. fastidiosa bacteria is also evaluated, returning an affinity constant of 3.5   107m 1, comparable with those given in the literature for bacteria detection against affinity antibodies

Transcriptome profiling of two olive cultivars in response to infection by the CoDiRO strain of Xylella fastidiosa subsp. pauca

Background: The recent Xylella fastidiosa subsp. pauca (Xfp) outbreak in olive (Olea europaea) groves in southern Italy is causing a destructive disease denoted Olive Quick Decline Syndrome (OQDS). Field observations disclosed that Xfp-infected plants of cv. Leccino show much milder symptoms, than the more widely grown and highly susceptible cv. Ogliarola salentina. To determine whether these field observations underlie a tolerant condition of cv. Leccino, which could be exploited for lessening the economic impact of the disease on the local olive industry, transcriptional changes occurring in plants of the two cultivars affected by Xfp were investigated. Results: A global quantitative transcriptome profiling comparing susceptible (Ogliarola salentina) and tolerant (Leccino) olive cultivars, infected or not by Xfp, was done on messenger RNA (mRNAs) extracted from xylem tissues. The study revealed that 659 and 447 genes were differentially regulated in cvs Leccino and Ogliarola upon Xfp infection, respectively, whereas 512 genes were altered when the transcriptome of both infected cultivars was compared. Analysis of these differentially expressed genes (DEGs) shows that the presence of Xfp is perceived by the plants of both cultivars, in which it triggers a differential response strongly involving the cell wall. Up-regulation of genes encoding receptor-like kinases (RLK) and receptor-like proteins (RLP) is the predominant response of cv. Leccino, which is missing in cv. Ogliarola salentina. Moreover, both cultivars react with a strong re-modelling of cell wall proteins. These data suggest that Xfp elicits a different transcriptome response in the two cultivars, which determines a lower pathogen concentration in cv. Leccino and indicates that this cultivar may harbor genetic constituents and/or regulatory elements which counteract Xfp infection. Conclusions: Collectively these findings suggest that cv. Leccino is endowed with an intrinsic tolerance to Xfp, which makes it eligible for further studies aiming at investigating molecular basis and pathways modulating its different defense response