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Determinants of Xylella fastidiosa host specificity

Periodic Reporting for period 2 - XYL-EID (Determinants of Xylella fastidiosa host specificity)

Reporting period: 2018-10-01 to 2019-09-30

The plant pathogenic bacterium Xylella fastidiosa poses a serious threat to agriculture in Europe and especially to the Mediterranean basin. It displays both a broad host range of 563 host plant species and a wide vector range of over a hundred of vector insect species (1,2). While it has been known to be present in the Americas for decades causing severe damage in several crops of economic importance such as grapes, citrus and coffee, its first confirmed detection in Europe is relatively recent. It was first described in Southern Italy (Apulia) in 2013, where it is devastating olive trees and triggering an economic, social and political crisis. Reports of the presence of different strains in France, Germany, Spain, Portugal and Northern Italy were soon to follow (3). Although X. fastidiosa infected plant species have been eradicated in Germany, this is far to be the case in some regions of Italy, France and Spain which have been officially declared areas under containment (Corsica, France; Balearic Islands, Spain; Southern Apulia, Italy) (4). The presence of several strains and even subspecies in both France and Spain points towards several introductions in these two countries. The analysis of a limited number of X. fastidiosa genes (MLST data) suggest that the outbreak in Apulia, Italy is the result of the introduction and subsequent establishment of a single strain belonging to ST53 (X. fastidiosa subsp. pauca) – which differs from the strain(s) present in Central Italy (5). These different strains are able to infect different host plant species (i.e. have different host ranges) that remain impossible to predict.

Despite its importance as a plant pathogen, there are no hypotheses on the determinants of X. fastidiosa host specificity. Because of its recent introduction in Europe, where host plants as well as the environment represent novel opportunities for expansion, it is of paramount importance to learn how X. fastidiosa adapts. This information could then be used in an attempt to limit its impact where it is already present, as well as its potential threat to risk areas where it could be disseminated. This project aims to address this very important knowledge gap, notably by studying the recent adaptation of this bacterium to olive trees in Southern Italy.
Multilocus sequence typing (MLST) that is based on the comparison of the nucleotide sequences of usually seven housekeeping genes has been used to genetically discriminate strains as well as to detect recombination events since its development on X. fastidiosa by Scally et al. in 2005 (6). We first tried to test whether these data could be used to predict X. fastidiosa host jumps by using all the MLST data available. A comparative host–pathogen coevolutionary analysis revealed that this bacterium’s host shifts could not be inferred using these data (7).

As recombination is believed to play a major role in X. fastidiosa’s host specificity, we then sought to determine whether we could link recombination patterns to a specific host plant species. However, by using a data set of 72 genomes belonging to different subspecies, no such correlation could be made (8).

We then chose to study a population with incipient host adaptation by focusing on the current disease epidemic in Apulia to identify loci involved in the early stages of host adaptation. By doing so, we hoped to identify loci under positive selection, hypothesizing that those are important for host adaptation and, ultimately, host specificity.
We sampled 74 twigs from X. fastidiosa infected olive trees from the beginning of the outbreak (2013) until 2017 across the affected area. The majority of the samples were first isolated before being sent for Illumina sequencing. Only a few single nucleotide polymorphisms (SNPs) were detected when comparing the whole-genome sequences of these isolates to the genome of the reference Apulian strain De Donno (9), further confirming that a single introduction is at the origin of this outbreak in Italy (unpublished data). Genomic comparison of these Apulian isolates with three Costa Rican ones corroborated that the strain present in Apulia originates from Central America. These genomic comparisons have enabled us to identify a few mutations that differentiate the Apulian isolates infecting olive trees from the Costa Rican one infecting coffee (unpublished results). The genes affected by these mutations are going to be further examined by reverse genetics in order to determine whether they are involved in X. fastidiosa adaptation to olive trees in Apulia. Moreover, we are currently looking at the presence of genes under positive selection in the Apulian data set.

Dissemination activities

We have presented the results of this project in several conferences, workshops and seminars, of which some where attended not only by researchers but also by stakeholders (among which farmers, agri-cooperatives, nurseries and the European Commission). For instance, I attended and presented my results (poster presentations) at the two European conferences on X. fastidiosa (2017 and 2019) organized notably by the European Food Safety Authority (EFSA).
I was also invited to give a talk at the Phytoma-España conference on X. fastidiosa in order to impart our knowledge on what we have learned so far from X. fastidiosa’s genomic analyses.
The presence of temporal signal in this data set, which corresponds to a five-year sampling effort, has enabled us to determine the introduction date of X. fastidiosa in Apulia (unpublished results). Furthermore, we are currently modeling its early spread in this region by using a phylogeographic approach. We believe that these analyses will provide new insights into the X. fastidiosa epidemic in Southern Italy.
This is the first comprehensive study which aims at tackling the long-lasting question of the determinants of X. fastidiosa host specificity.

References:

1. European Food Safety Authority (EFSA). Update of the Xylella spp. host plant database. EFSA Journal 16, (2018).
2. Serio, F. D. et al. Collection of data and information on biology and control of vectors of Xylella fastidiosa. EFSA Journal 102 (2019).
3. EFSA Panel on Plant Health (PLH) et al. Update of the Scientific Opinion on the risks to plant health posed by Xylella fastidiosa in the EU territory. EFSA Journal 200 (2019) doi:10.2903/j.efsa.2019.5665.
4. EFSA Panel on Plant Health (EFSA PLH Panel) et al. Updated pest categorisation of Xylella fastidiosa. EFSA Journal 16, (2018).
5. Marchi, G. et al. First detection of Xylella fastidiosa subsp. multiplex DNA in Tuscany (Italy). Phytopathologia Mediterranea 57, (2018).
6. Scally, M., Schuenzel, E. L., Stouthamer, R. & Nunney, L. Multilocus sequence type system for the plant pathogen Xylella fastidiosa and relative contributions of recombination and point mutation to clonal diversity. Applied and Environmental Microbiology 71, 8491–8499 (2005).
7. Sicard, A. et al. Xylella fastidiosa : Insights into an emerging plant pathogen. Annual Review of Phytopathology 56, (2018).
8. Vanhove, M. et al. Genomic diversity and recombination among Xylella fastidiosa subspecies. Appl Environ Microbiol AEM.02972-18 aem;AEM.02972-18v1 (2019) doi:10.1128/AEM.02972-18.
9. Giampetruzzi, A. et al. Complete genome sequence of the olive-infecting strain Xylella fastidiosa subsp. pauca De Donno. Genome announcements 5, e00569–17 (2017).
Geographic distribution of X. fastidiosa isolates collected between 2014 in 2017 in Italy