Draft Genome Sequence Resources of Three Strains (TOS4, TOS5, and TOS14) of Xylella fastidiosa Infecting Different Host Plants in the Newly Discovered Outbreak in Tuscany, Italy.

An outbreak of Xylella fastidiosa was discovered in late 2018 in northern Italy affecting several plant species. Multilocus sequence typing analyses detected the presence of strains clustering in X. fastidiosa subsp. multiplex and harboring a hitherto uncharacterized sequence type, ST87. Three cultured strains (TOS4, TOS5, and TOS14) were subjected to high-throughput sequencing and the draft genomes assembled. Phylogenetic analysis conclusively indicated that they belong to the subspecies multiplex. The genetic information generated for these newly discovered strains further supports the evidence that sequence types are associated with the emergence of X. fastidiosa in Europe, posing major challenges for predicting the main threatened European and Mediterranean crops and plant species.

Genome-Wide Analysis Provides Evidence on the Genetic Relatedness of the Emergent Xylella fastidiosa Genotype in Italy to Isolates from Central America.

Xylella fastidiosa is a plant-pathogenic bacterium recently introduced in Europe that is causing decline in olive trees in the South of Italy. Genetic studies have consistently shown that the bacterial genotype recovered from infected olive trees belongs to the sequence type ST53 within subspecies pauca. This genotype, ST53, has also been reported to occur in Costa Rica. The ancestry of ST53 was recently clarified, showing it contains alleles that are monophyletic with those of subsp. pauca in South America. To more robustly determine the phylogenetic placement of ST53 within X. fastidiosa, we performed a comparative analysis based on single nucleotide polymorphisms (SNPs) and the study of the pan-genome of the 27 currently public available whole genome sequences of X. fastidiosa. The resulting maximum-parsimony and maximum likelihood trees constructed using the SNPs and the pan-genome analysis are consistent with previously described X. fastidiosa taxonomy, distinguishing the subsp. fastidiosa, multiplex, pauca, sandyi, and morus. Within the subsp. pauca, the Italian and three Costa Rican isolates, all belonging to ST53, formed a compact phylotype in a clade divergent from the South American pauca isolates, also distinct from the recently described coffee isolate CFBP8072 imported into Europe from Ecuador. These findings were also supported by the gene characterization of a conjugative plasmid shared by all the four ST53 isolates. Furthermore, isolates of the ST53 clade possess an exclusive locus encoding a putative ATP-binding protein belonging to the family of histidine kinase-like ATPase gene, which is not present in isolates from the subspecies multiplex, sandyi, and pauca, but was detected in ST21 isolates of the subspecies fastidiosa from Costa Rica. The clustering and distinctiveness of the ST53 isolates supports the hypothesis of their common origin, and the limited genetic diversity among these isolates suggests this is an emerging clade within subsp. pauca.

Hitching a ride on vesicles: cauliflower mosaic virus movement protein trafficking in the endomembrane system.

The transport of a viral genome from cell to cell is enabled by movement proteins (MPs) targeting the cell periphery to mediate the gating of plasmodesmata. Given their essential role in the development of viral infection, understanding the regulation of MPs is of great importance. Here, we show that cauliflower mosaic virus (CaMV) MP contains three tyrosine-based sorting signals that interact with an Arabidopsis (Arabidopsis thaliana) µA-adaptin subunit. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide. The presence of at least one of the three endocytosis motifs is essential for internalization of the protein from the plasma membrane to early endosomes, for tubule formation, and for CaMV infection. In addition, we show that MP colocalizes in vesicles with the Rab GTPase AtRAB-F2b, which is resident in prevacuolar late endosomal compartments that deliver proteins to the vacuole for degradation. Altogether, these results demonstrate that CaMV MP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes.

Caulimoviridae tubule-guided transport is dictated by movement protein properties.

Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway.

Olea Europaea Geminivirus: A Novel Bipartite Geminivirid Infecting Olive Trees.

In 2014, high-throughput sequencing of libraries of total DNA from olive trees allowed the identification of two geminivirus-like contigs. After conventional resequencing of the two genomic DNAs, their analysis revealed they belonged to the same viral entity, for which the provisional name of Olea europaea geminivirus (OEGV) was proposed. Although DNA-A showed a genome organization similar to that of New World begomoviruses, DNA-B had a peculiar ORF arrangement, consisting of a movement protein (MP) in the virion sense and a protein with unknown function on the complementary sense. Phylogenetic analysis performed either on full-length genome or on coat protein, replication associated protein (Rep), and MP sequences did not endorse the inclusion of this virus in any of the established genera in the family Geminiviridae. A survey of 55 plants revealed that the virus is widespread in Apulia (Italy) with 91% of the samples testing positive, although no correlation of OEGV with a disease or specific symptoms was encountered. Southern blot assay suggested that the virus is not integrated in the olive genome. The study of OEGV-derived siRNA obtained from small RNA libraries of leaves and fruits of three different cultivars, showed that the accumulation of the two genomic components is influenced by the plant genotype while virus-derived-siRNA profile is in line with other geminivirids reported in literature. Single-nucleotide polymorphism (SNP) analysis unveiled a low intra-specific variability.

Antagonistic activity of olive endophytic bacteria and of Bacillus spp. strains against Xylella fastidiosa.

Strains of Xylella fastidiosa subsp. pauca characterized by a specific genotype, the so called sequence type "ST53", have been associated with a severe disease named Olive Quick Decline Syndrome (OQDS). Despite the relevant research efforts devoted to control the disease caused by X. fastidiosa, so far there are no therapeutic means able to cure the infected host plants. As such, the aim of this study was the identification of antagonistic bacteria potentially deployable as bio-control agents against X. fastidiosa. To this end, two approaches were used, i.e. the evaluation of the antagonistic activity of: i) endophytic bacteria isolated from olive trees located in an infected area but showing mild or no symptoms, and ii) Bacillus strains, as they are already known as bio-control agents. Characterization of endophytic bacterial isolates revealed that the majority belonged to different species of the genera Sphingomonas, Methylobacterium, Micrococcus and Curtobacterium. However, when they were tested in vitro against X. fastidiosa ST53 none of them showed antagonistic activity. On the contrary, when strains belonging to different species of the genus Bacillus were included in these tests, remarkable antagonistic activities were recorded. Some B. velezensis strains also produced culture filtrates with inhibitory activity against X. fastidiosa ST53. Taking also into account that two of these B. velezensis strains (namely strains D747 and QST713) are already registered and commercially available as bio-control agents, our results pave the way for further studies aimed at the development of a sustainable bio-control strategy of the OQDS.

Detection of Citrus tristeza virus and Coinfecting Viroids.

Citrus can host a number of important vector- and graft-transmissible pathogens which cause severe diseases. Citrus disease management and clean stock programs require pathogen detection systems which must be economical and sensitive to maintain a healthy citrus industry. Rapid diagnostic tests for simultaneous detection of major graft-transmissible disease agents enable reduction of cost and time. The genetic and biological features of viruses and viroids can vary according to the strains/variants, with severe and mild strains described within the same species. The use of diagnostic tests that can allow to selectively discriminate severe strain(s) is a powerful tool to intercept the most harmful strains and to reduce the need for biological indexing. Moreover a combination of these detection methods will facilitate the studies on the interactions between CTV and viroids, a research topic only partially explored so far.

A Negative-Stranded RNA Virus Infecting Citrus Trees: The Second Member of a New Genus Within the Order Bunyavirales.

A new RNA virus has been identified from a sweet orange tree in southern Italy. This virus, tentatively named citrus virus A (CiVA), has a bipartite genome composed of (i) a negative-stranded (ns) RNA1, encoding the viral RNA-dependent RNA polymerase (RdRp), and (ii) an ambisense RNA2, coding for the putative movement protein (MP) and nucleocapsid protein (NP), with the two open reading frames separated by a long AU-rich intergenic region (IR) adopting a hairpin conformation. CiVA genomic RNAs and the encoded proteins resemble those of the recently discovered citrus concave gum-associated virus (CCGaV). This CCGaV, a nsRNA virus associated with the ancient citrus concave gum disease, has been proposed as the representative member of a new genus tentatively named Coguvirus. Molecular and phylogenetic analyses presented here support the classification of CiVA, and likely of other two recently described nsRNA viruses infecting plants, in this new genus. By showing that the evolutionary origin of the MP of all the putative coguviruses likely differs from that of their respective RdRp and NP, this study also provides evidence of a likely modular genome evolution for these viruses. Moreover, phylogenetic data support the proposal that, during the evolutionary history of nsRNA viruses, the plant-infecting viruses most likely emerged from an invertebrate-infecting ancestor several times as independent events. CiVA was identified in a field sweet orange tree not showing any obvious symptom and was graft-transmitted to sweet orange, grapefruit, rough lemon and Dweet tangor indicator plants that did not developed symptoms. The capacity of infecting citrus hosts of several species was also confirmed by a preliminary survey that identified orange, mandarin, clementine and lemon trees as natural hosts of CiVA in several fields of southern Italy, again without any obvious association with specific symptoms.