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

Rapid screening tests for the assignment of X. fastidiosa genotypes to a subspecies cluster

Until now, different molecular tests can be used to assign novel X. fastidiosa isolates to subspecies clusters, among which MLST/MLSA represents the most common method. X. fastidiosa outbreaks in EU motivated the search for accurate and faster approaches to differentiate the X. fastidiosa isolates. Because MLST/MLSA requires PCR reactions and sequencing analyses, 2 independent approaches were recently developed and implemented for rapid taxonomic assignment of uncharacterized isolates: (1) single-nucleotide primer extension (SNuPE) method that allows to differentiate all subspecies and three genotypes within X. fastidiosa subsp. pauca including the typeisolate infecting olive in Italy and (2) high-resolution melting (HRM) analysis of the amplicon recovered from the gene encoding the conserved HL protein. Both assays were validated on a larger panel of isolates and proved to clearly differentiate X. fastidiosa isolates currently known to occur in the Italian, France and Spain outbreaks. These rapid approaches could represent a useful tool for prescreening of infected samples to be further analyzed by MLST or whole genome sequencing. In addition alternative genomic regions of X. fastidiosa are going to be analyzed to implement approaches aimed to assign genotypes to a subspecies cluster, with the purpose to support a rapid identification of genotypes/subspecies at interception places or when new findings occur in a pest free are

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

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

Genomic and physiological basis of resistance to Xylella fastidiosa in olive

<p>It is almost 10 years since the discovery of Xylella fastidiosa in the southern part of the region of Apulia (southern Italy) [1]. The rapid spread of the infections and the large territory interested by the epidemics, urged for the adoption of containment strategies in place of the eradication measures. In the frame of these strategies a breakthrough is represented by the discovery of traits of resistance in the cvs Leccino and FS17. Replanting olive groves with resistant cultivars is of fundamental importance to restore the landscape and the agriculture of the devastated area. Investigations on the mechanisms governing the resistance phenomena in olives, showed that, for example in Leccino, resistance appears to develop from a complex of mechanisms, involving both genomic and physiological basis that keep the bacterium population lower than in the highly susceptible cultivars such as Cellina di Nardò or Ogliarola salentina or other susceptible cultivars. Extensive or targeted gene expression studies indicated that Leccino senses the bacterium by cell wall receptors and manages to contain the induced drought stress by modulating genes involved in the sugar metabolism and water flux across membranes [2,3]. Moreover, the bacterium spread trough the xylem network is likely enhanced in the susceptible Cellina di Nardò because of its facilitated exploitations of pit membranes interconnecting xylem vessels. These studies show that Leccino is more resilient to the infection, whose physiological response to the water stress is not as extreme as in susceptible cultivars. In addition, recent genomic investigations on spontaneous seedlings or cross-bred progenies derived from Leccino, support the evidence that the genetic traits of resistance can be transferred from resistant parentals to the progenies and pave the way to widening the olive germplasm resistant to Xylella. <br>                </p><p>[1]Saponari M. et al., 2019. Phytopathology, 109, 175-186.</p><p>[2]Giampetruzzi A. et al., 2016. BMC Genomics, 17:475</p><p>[3]Sabella E. et al., 2019. Scientific Reports, 9:9602          </p&gt

Fast and Reliable Electronic Assay of a Xylella fastidiosa Single Bacterium in Infected Plants Sap

Pathogens ultra-sensitive detection is vital for early diagnosis and provision of restraining actions and/or treatments. Among plant pathogens, Xylella fastidiosa is among the most threatening as it can infect hundreds of plant species worldwide with consequences on agriculture and the environment. An electrolyte-gated transistor is here demonstrated to detect X. fastidiosa at a limit-of-quantification (LOQ) of 2 +/- 1 bacteria in 0.1 mL (20 colony-forming-unit per mL). The assay is carried out with a millimeter-wide gate functionalized with Xylella-capturing antibodies directly in saps recovered from naturally infected plants. The proposed platform is benchmarked against the quantitave polymerase chain reaction (qPCR) gold standard, whose LOQ turns out to be at least one order of magnitude higher. Furthermore, the assay selectivity is proven against the Paraburkholderia phytofirmans bacterium (negative-control experiment). The proposed label-free, fast (30 min), and precise (false-negatives, false-positives below 1%) electronic assay, lays the ground for an ultra-high performing immunometric point-of-care platform potentially enabling large-scale screening of asymptomatic plants