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

Establishment of an experimental field to explore the differential olive cultivar response to Xylella fastidiosa infection

While different sources of natural resistance to Xylella fastidiosa (Xf) have been described in grapevines and citrus, lack of consolidated information exists on the wide panel of cultivars characterizing the vast olive germplasm. Preliminary observations on few cultivars, support the evidence that differential cultivar responses to Xf infections may exist. To explore the response of a larger panel of cultivars, in April 2015, an experimental olive plot, located within the Xf-heavily affected olive groves, was established in the Apulia Region (Italy). Twenty-four trees for each of the ten different cultivars were planted in randomized blocks. Each tree was caged with 15-20 specimens of Philaenus spumarius collected from the neighboring infected olive groves. Upon removing the cages, the trees are then continuously exposed to the natural vector populations occurring in the area. Nine and 12-months after planting, the trees were sampled, tested for Xf and inspected for symptoms. The first data confirmed the infectivity of the vector populations occurring in the Apulian contaminated area and the Xf susceptibility of the olive cultivars tested. Almost 50% of the trees tested positive, with an infection incidence ranging from 25% (Leccino) to 78% (Koroneiki). Symptoms of shoot dieback started to appear 1-year after planting, limitedly on few replicates of Cellina di Nardò. In April 2016, the number of cultivars has been increased up to 30. Periodical surveys for symptoms and quantitative analyses to monitor the differential bacterial titer and expression of target genes involved in the host response, are underway

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

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

Low Temperature Plasma Strategies for <i>Xylella fastidiosa</i> Inactivation

The quarantine bacterium Xylella fastidiosa was first detected in Salento (Apulia, Italy) in 2013 and caused severe symptoms in olives, leading to plant death. The disease, named Olive Quick Decline Syndrome (OQDS), is caused by the strain “De Donno” ST53 of the subspecies pauca of this bacterium (XfDD), which is spread by the insect Philaenus spumarius. The epidemic poses a serious threat to the agricultural economy and the landscape, as X. fastidiosa infects several plant species and there is yet no recognized solution. Research on OQDS is focused on finding strategies to control its spread or mitigate its symptoms. As a perspective solution, we investigated the efficacy of the low-temperature plasma and plasma-activated water to kill bacterial cells. Experiments were conducted in vitro to test the biocidal effect of the direct application of a Surface Dielectric Barrier Discharge (SDBD) plasma on bacteria cells and Plasma Activated Water (PAW). PAW activity was tested as a possible biocidal agent that can move freely in the xylem network paving the way to test the strategy on infected plants. The results showed a high decontamination rate even for cells of XfDD embedded in biofilms grown on solid media and complete inactivation in liquid culture medium

Genome-wide analysis provides evidence on the genetic relatedness of the emergent Xylella fastidiosa genotype in Italy to isolates from Central America

First, third, and fifth authors: Università degli Studi di Bari Aldo Moro, Dipartimento di Scienze del Suolo della Pianta e degli Alimenti, via Amendola 165/A, Bari, Italy; second, fourth, seventh, and tenth authors: Consiglio Nazionale delle Ricerche, Istituto per la Protezione Sostenibile delle Piante, Bari, via Amendola 122/D, Bari, Italy; sixth author: Department of Environmental Science, Policy and Management, University of California, Berkeley, CA, 94720-3114; eighth author: Institute for Sustainable Agriculture, Consejo Superior de Investigaciones Científicas, 14004 Córdoba, Spain; and ninth author: Centro de Investigación en Enfermedades Tropicales (CIET), Facultad de Microbiología, Universidad de Costa Rica, 2060 San José, Costa Rica.The work was supported by funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 635646: POnTE (Pest Organisms Threatening Europe).Peer reviewe