Update of the Xylella fastidiosa outbreak in France: two new variants detected and a new region affected

Xylella fastidiosa is a xylem-limited bacterium native to America and classified as a priority pest for EU regulation. Since 2013, X. fastidiosa has been identified in European countries with a Mediterranean climate, such as Italy, France, Spain and Portugal, with different subspecies and sequence types (ST) detected. Since 2015 X. fastidiosa subsp. multiplex ST6 and/or ST7 has been detected in Corsica and the Provence-Alpes-Côte d’Azur in almost 70 plant species, whereas X. fastidiosa subsp. pauca ST53 has been found in only two host plants. In this study, we report two new variants, recently detected in two separated areas of the PACA region, genetically related to the subspecies multiplex and assigned to (i) ST88 detected on Polygala myrtifolia, Hebe sp., Osteospermum ecklonis, Lavandula x intermedia, Coronilla glauca and Euryops chrysanthemoides and (ii) ST89: detected on Myoporum sp. and Viburnum tinus. Both variant strains were isolated in vitro. Moreover, we report here the identification of X. fastidiosa subsp. multiplex ST6 in a new region of the South of France, Occitanie (Aude), in plants from natural and urban settings and from a nursery.

Even though the X. fastidiosa diseases have been described in America for over 100 years, X. fastidiosa has only recently been detected in Europe. Several subspecies were reported from the natural settings, agricultural and urban areas in Italy (pauca and multiplex) (EPPO, 2019b;Saponari et al., 2013), in France (multiplex and pauca) (Denancé et al., 2017), in Spain (fastidiosa, pauca and multiplex) (Landa, 2017) and in Portugal (multiplex) (EPPO, 2019b). In Italy, the subspecies pauca is responsible for Olive Quick Decline Syndrom (OQDS), and is causing dramatic economic losses in olive cultivation in Apulia (Olivicola Italia, 2019;Saponari et al., 2013). A recent study evaluated the impact of the strain causing OQDS on the major olive-cultivation European countries (Italy, Greece and Spain), using a spatially explicit bio-economic model (Schneider et al., 2020). They highlighted the importance of applying surveillance and adapting measures in infected areas to slow the spread of the disease.
Up to now, 87 sequence types (ST) of X. fastidiosa have been reported all around the world, and the corresponding partial sequences of seven housekeeping genes related to X. fastidiosa were registered in the PubMLST database (Jolley et al., 2018) dedicated to X. fastidiosa (https://pubmlst.org/organisms/xylellafastidiosa). Among the 39 STs belonging to subspecies multiplex, two new variants were reported in Europe during the last four years: (i) ST81 in Balearic Islands, Spain, (Landa, 2017), and later in California, USA (O'Leary et al., 2020); (ii) ST87 in Tuscany, Italy, .
In 2015, the subspecies multiplex ST6 and ST7 genotypes were detected in France in Corsica and in PA-CA regions (Alpes-Maritimes and Var), and annual official surveys with plant and vector samplings confirmed the presence and dissemination of these STs in Corsica and PACA regions (Cunty et al., 2020;Denancé et al., 2017). The presence of the subspecies pauca ST53 genotype was reported in only one location, in Menton in PACA region, in 2015 (Denancé et al., 2017) and four years later at the same location on one olive tree (Cunty et al., 2020). In 2020, a new region, Occitanie (Aude), was found to be impacted by the presence of X. fastidiosa subspecies multiplex (EPPO, 2020). The methods applied to detect the bacterium and identify the ST in plants are in line with the EPPO protocol PM7/24 (EPPO, 2019a). Briefly, the detection is performed using the real-time PCR Harper and the identification of the ST by applying an MLST scheme of seven housekeeping genes and using the PubMLST database to identify allele numbers and ST (EPPO, 2019a;Harper et al., 2010;Yuan et al., 2010).
The aim of this study was to provide an overview of the current X. fastidiosa situation in France. We report the identification of X. fastidiosa subsp. multiplex (i) two new variants in the PACA region, ST88 in Var and ST89 in Alpes-Maritimes, and (ii) ST6 in a new French area, Aude in Occitanie region, in different plant species, including the new host plant Perovskia abrotanoides.
Since 2015 more than 24,000 plants were sampled and analysed in the PACA region within the framework of the surveillance. A sample was considered positive when the Cq value was lower or equal to 38, as described by Harper et al. (2010). Among the Harperpositive X. fastidiosa samples, two new MLST profiles, from two distinct locations, were identified. MLST performed on samples of Polygala myrtifolia and Hebe sp., both collected in 2020 in the same area in Saint-Raphaël (Var), revealed a new allele malF_19, similar to malF_13 (T at position 301 and A at position 486) and the sequence was registered on Genbank (accession number OL353884). The MLST profile, such as: leuA_3, petC_3, malF_19, cysG_7, holC_3, nuoL_3 and gltT_3, was assigned to ST88 ( Fig. 1). This profile varied in only one gene from the ST7 previously found in this region. Strain isolation was performed on these positive plant samples, using petioles, midribs or young shoots dilacerated and macerated in sterile water, plated on modified PWG (PWGm) medium and incubated at 28°C according to EPPO standard PM 7/24 (EPPO, 2019a). After 10-15 days, typical colonies were recovered (strain named LSV52.37) from P. myrtifolia (presenting leaf scorch) with the same MLST profile as the one obtained directly from plant macerate. In some plant samples received in 2021 from Saint-Raphaël, ST88 was found, including Osteospermum ecklonis, Lavandula x intermedia, Coronilla glauca and Euryops chrysanthemoides. This indicated the potential maintenance of this variant over time at this location. Moreover, a sample of P. myrtifolia was found to be coinfected by ST7 and ST88. Additionally, another variant was detected in Villeneuve-Loubet (Alpes-Maritimes) in 2020. A new allele nuoL_23, similar to nuoL_3 (T at position 185), was identified and the sequence was registered on Genbank (accession number OL353885). The MLST profile, such as: leuA_6, petC_3, malF_5, cysG_18, holC_4, nuoL_23 and gltT_7, was assigned to ST89 (Fig. 1). This profile was very different from ST6 or ST7 usually found in PACA, such as only petC_3 was in common. The ST89 was detected directly on DNA extracted from samples of Myoporum sp. and Viburnum tinus, both collected in 2020 in the same area. One strain (strain named LSV52.52) was isolated from Myoporum sp. (presenting leaf scorch) with same MLST profile. Interestingly, this MLST profile was previously found at the same location in 2018 only on one sample of Veronica elliptica, but the isolation of the strain was unsuccessful. However, this confirmed the maintenance of this variant over time at this location. In order to evaluate the phylogenetic relationships of the 87 STs available on PubMLST database and the two new variants ST88 and ST89 identified here, the sequences of the seven housekeeping genes were concatenated, aligned, and the phylogenetic networks were retrieved using Splits Tree4 (Huson & Bryant, 2005) (Fig. 2). Both new STs grouped with strains belonging to the subspecies multiplex. The ST88 was closely related to STs already found in Europe, such as ST6 (France, mainland Spain), ST7 (France, Balearic Islands, Portugal), ST81 (Balearic Islands) and ST87 (Italy) (Landa, 2017;Saponari et al., 2019), while ST89 was closer related to STs found in USA such as ST41 and ST43 (Nunney et al., 2013).
After Corsica and PACA, Occitanie is the third region of France most impacted by the presence of X. fastidiosa. In Occitanie region (south-west of France), approximately 5000 plant samples, (representing more than 200 plant species) symptomatic or asymptomatic (nursery, natural settings, agricultural or urban areas), were analysed for the presence of X. fastidiosa from August 2020 to August 2021. Among these samples, 132 were found to be X. fastidiosa positive in the real-time PCR Harper et al. (2010) with Cq values ranging from 19.66 to 38. Among the 132 Harper-positive samples, 120 were typed as multiplex, and for 98 the ST identification succeeded, all being ST6. Based on EU list (https://eur-lex.europa. eu/legal-content/fr/TXT/?uri=CELEX%3A32020R1201 ), one new host plant was reported: Perovskia abrotanoides, which was asymptomatic. Moreover, the bacterium was first detected on Lavandula x intermedia (variété 'Grosso') in a nursery, and then in the natural settings on: Coronilla valentina (4) (2), Spartium junceum (38), Spartium sp. (29). It was interesting to notice that no P. myrtifolia was found to be infected with X. fastidiosa in this new infected region, in contrast with the Corsica and PACA regions. It is linked to the fact that P. myrtifolia is very scarce as an ornamental in Occitanie (Aude) due to lower winter temperatures at this area. Strain isolation was performed, as described above, and three strains presenting typical colonies were recovered from three different plant genera: Lavandula x intermedia (variété 'Grosso'), L. nobilis and Spartium sp., for which Ct value on plant extract were 28.83; 23.42 and 19.87, respectively. The strains identified were confirmed applying real-time PCR Fig. 1 Map representing the location of positive plants to Xylella fastidiosa detection (south-west: Occitanie; south-east: PACA), and the location of two new variants ST88 and ST89. The map was adapted from R Shiny dedicated for X. fastidiosa (EAS unit, Anses) Harper and typed by MLST as ST6 (allele number 3 for the seven housekeeping genes), named LSV52.20; LSV52.39 and LSV52.40, respectively and stored at −80°C. Same MLST profiles were obtained on strains and on plant macerates from which these strains were isolated. Up to now in Occitanie, only ST6 has been identified in different plants, mainly in S. junceum, Spartium sp. and P. dulcis coming from the wild environment and Lavandula sp. coming from a nursery and urban areas. Moreover, M. sativa had been found positive. In North America, cultivated alfalfa was already identified as a plant reservoir of X. fastidiosa subspecies fastidiosa, and the epidemiological role of alfalfa in the spread of the bacterium in the case of Pierce's disease was confirmed when alfalfa were located surrounding vineyards (Hewitt & Houston, 1941). Assays performed in the framework of the H2020 POnTE project (artificial inoculations of alfalfa with different strains of X. fastidiosa), confirmed the capability of a strain ST6, isolated from Corsica, to maintain and multiply in alfalfa (data not shown). It is therefore relevant to pursue surveillance of this crop in France. Fig. 2 Phylogenetic relationships of the 87 sequence types (STs) of Xylella fastidiosa available on PubMLST database and the two new variants ST88 and ST89 identified in these study, revealed using Splits Tree4 (Huson & Bryant, 2005). The subspecies were highlighted by squares, and focus was done on the top of the figure for the ST88 and ST89, which were surrounded by grey circles In summary, we revealed the presence of ST88 and ST89, two new STs of the subspecies multiplex, in the PACA region, on different host plants and at two different geographical locations, which are maintained over time. Based on the MLST results, we can hypothesize that in France ST88 and ST89 result from two distinct events. ST88 and ST7 belong to the same clonal complex as there are only two different nucleotides on only one gene out of the seven analysed by MLST. Both STs were found in the same location, and also in co-infection, suggesting that they share the same ecological niche. As the subspecies multiplex was found to exhibit a high number of recombination events (Vanhove et al., 2019) it is possible that other variants may be found in the PA-CA region. The strain ST89 is very different from the STs already present in the PACA region, and in Europe, as it belongs to a clonal complex including ST41 and ST43, reported only in the USA (Nunney et al., 2013). The presence of ST89 probably results from a new introduction, possibly from America. For the strains of ST6 identified in the Occitanie region, it will be interesting to go further and identify whether these strains are closely related to the French ST6 or the Spanish ST6, which is more closely related to French ST7 than to French ST6 (Landa et al., 2020). So, work is in progress on the genomic analyses of ST88 and ST89 strains, and also on the ST6 strains isolated in the Occitanie, to improve the knowledge of the subspecies diversity, and to give new insights into the origins and dates of introduction of X. fastidiosa subsp. multiplex strains present in France, and in Europe.