The dynamics of Xylella fastidiosa infections in the context of the endophytic microbiome was studied in field-grown plants of the susceptible and resistant olive cultivars Kalamata and FS17. Whole metagenome shotgun sequencing (WMSS) coupled with 16S/ITS rRNA gene sequencing was carried out on the same trees at two different stages of the infections: In Spring 2017 when plants were almost symptomless and in Autumn 2018 when the trees of the susceptible cultivar clearly showed desiccations. The progression of the infections detected in both cultivars clearly unraveled that Xylella tends to occupy the whole ecological niche and suppresses the diversity of the endophytic microbiome. However, this trend was mitigated in the resistant cultivar FS17, harboring lower population sizes and therefore lower Xylella average abundance ratio over total bacteria, and a higher α-diversity. Host cultivar had a negligible effect on the community composition and no clear associations of a single taxon or microbial consortia with the resistance cultivar were found with both sequencing approaches, suggesting that the mechanisms of resistance likely reside on factors that are independent of the microbiome structure. Overall, Proteobacteria, Actinobacteria, Firmicutes, and Bacteriodetes dominated the bacterial microbiome while Ascomycota and Basidiomycota those of Fungi.

Olive quick decline syndrome (OQDS) is a devastating disease of olive trees in the Salento region, Italy. This disease is caused by the bacterium Xylella fastidiosa, which is widespread in the outbreak area; however, the “Leccino” variety of olives has proven to be resistant with fewer symptoms and lower bacterial populations than the “Ogliarola salentina” variety. We completed an empirical study to determine the mineral and trace element contents (viz; ionome) of leaves from infected trees comparing the two varieties, to develop hypotheses related to the resistance of Leccino trees to X. fastidiosa infection. All samples from both cultivars tested were infected by X. fastidiosa, even if leaves were asymptomatic at the time of collection, due to the high disease pressure in the outbreak area and the long incubation period of this disease. Leaves were binned for the analysis by variety, field location, and infected symptomatic and infected asymptomatic status by visual inspection. The ionome of leaf samples was determined using inductively coupled plasma optical emission spectroscopy (ICP-OES) and compared with each other. These analyses showed that Leccino variety consistently contained higher manganese (Mn) levels compared with Ogliarola salentina, and these levels were higher in both infected asymptomatic and infected symptomatic leaves. Infected asymptomatic and infected symptomatic leaves within a host genotype also showed differences in the ionome, particularly a higher concentration of calcium (Ca) and Mn levels in the Leccino cultivar, and sodium (Na) in both varieties. We hypothesize that the ionome differences in the two varieties contribute to protection against disease caused by X. fastidiosa infection.

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.

Xylella fastidiosa subsp. pauca strain De Donno has been recently identified as the causal agent of a severe disease affecting olive trees in a wide area of the Apulia Region (Italy). While insights on the genetics and epidemiology of this virulent strain have been gained, its phenotypic and biological traits remained to be explored. We investigated in vitro behavior of the strain and compare its relevant biological features (growth rate, biofilm formation, cell–cell aggregation, and twitching motility) with those of the type strain Temecula1. The experiments clearly showed that the strain De Donno did not show fringe on the agar plates, produced larger amounts of biofilm and had a more aggregative behavior than the strain Temecula1. Repeated attempts to transform, by natural competence, the strain De Donno failed to produce a GFP-expressing and a knockout mutant for the rpfF gene. Computational prediction allowed us to identify potentially deleterious sequence variations most likely affecting the natural competence and the lack of fringe formation. GFP and rpfF- mutants were successfully obtained by co-electroporation in the presence of an inhibitor of the type I restriction–modification system. The availability of De Donno mutant strains will open for new explorations of its interactions with hosts and insect vectors.

This study investigated in-vitro the non-lethal effects of N-acetylcysteine (NAC) on Xylella fastidiosa subspecies pauca strain De Donno (Xf-DD) biofilm. This strain was isolated from the olive trees affected by the olive quick decline syndrome in southern Italy. Xf-DD was first exposed to non-lethal concentrations of NAC from 0.05 to 1000 µM. Cell surface adhesion was dramatically reduced at 500 µM NAC (−47%), hence, this concentration was selected for investigating the effects of pre-, post- and co-treatments on biofilm physiology and structural development, oxidative homeostasis, and biofilm detachment. Even though 500 µM NAC reduced bacterial attachment to surfaces, compared to the control samples, it promoted Xf-DD biofilm formation by increasing: (i) biofilm biomass by up to 78% in the co-treatment, (ii) matrix polysaccharides production by up to 72% in the pre-treatment, and (iii) reactive oxygen species levels by 3.5-fold in the co-treatment. Xf-DD biofilm detachment without and with NAC was also investigated. The NAC treatment did not increase biofilm detachment, compared to the control samples. All these findings suggested that, at 500 µM, NAC diversified the phenotypes in Xf-DD biofilm, promoting biofilm formation (hyper-biofilm-forming phenotype) and discouraging biofilm detachment (hyper-attachment phenotype), while increasing oxidative stress level in the biofilm.

Wisteria vein mosaic virus (WVMV) is a member of the genus Potyvirus, found to be associated with the Wisteria mosaic disease (WMD). In 2022, severe symptoms resembling WMD were observed on the foliage of a Chinese wisteria (Wisteria sinensis) tree growing in Apulia (Italy). The presence of WVMV was ascertained by RT-PCR using the universal degenerate primers NIb2F/NIb3R in the NIb gene. Next, we derived the complete sequence of the CP gene. MAFFT pairwise alignment of the two target regions showed a high amino acid sequence identity of isolate Bari with other WVMV isolates, confirming they belonged to the same species. NIb phylogenetic analysis placed WVMV Bari within the lineage identified in the genus Potyvirus as the bean common mosaic virus supergroup. Closer analysis based on the CP gene showed that WVMV Bari was part of a sub-clade gathering all WVMV isolates within a larger clade clustering closely related species. An exploratory analysis of the genetic diversity and possible mechanisms underlying the evolution of geographic populations of WVMV was attempted. The analysis was based on the N-terminal coding region of CP, available for 20 isolates from Europe, Asia, and Oceania. A high diversity, mainly found in the European population, led to the identification of 16 different haplotypes. Based on the dN/dS ratio, the target region appears to be under negative selective pressure. Neutrality tests indicated a potential population expansion in Europe and a recent decrease in Asia. The two populations exhibited a high level of gene flow. We found that WVMV Bari may have originated from a recombination event in the NIb gene. To date, the presence of WVMV had never been reported on W. sinensis in Italy, and no molecular information was available on any Italian isolate. Our report draws attention to the further spread of WVMV in the European territory and its rising threat to the ornamental nursery sector.