Outbreak of Xylella fastidiosa subsp. pauca ST53 affecting wild and cultivated olive trees on the island of Mallorca, Spain

Phytopathologia Mediterranea 63(3): 475-480, 2024 Firenze University Press www.fupress.com/pm Phytopathologia Mediterranea The international journal of the Mediterranean Phytopathological Union ISSN 0031-9465 (print) | ISSN 1593-2095 (online) | DOI: 10.36253/phyto-15891 Short Notes Citation: Moralejo, E., Quetglas, B., Montesinos, M., Adrover, F., Olmo, D., Nieto, A., Pedrosa, A., López, M., Juan, A., Marco-Noales, E., NavarroHerrero, I., Barbé, S., Velasco-Amo, M.P., Olivares-García, C., & Landa, B.B. (2024). Outbreak of Xylella fastidiosa subsp. pauca ST53 affecting wild and cultivated olive trees on the island of Mallorca, Spain. Phytopathologia Mediterranea 63(3): 475-480. doi: 10.36253/ phyto-15891 Accepted: December 27, 2024 Published: December 30, 2024 © 2024 Author(s). This is an open access, peer-reviewed article published by Firenze University Press (https://www. fupress.com) and distributed, except where otherwise noted, under the terms of the CC BY 4.0 License for content and CC0 1.0 Universal for metadata. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Editor: Anna Maria D’Onghia, CIHEAM/Mediterranean Agronomic Institute of Bari, Italy. ORCID: EM: 0000-0003-4927-9367 DO: 0000-0002-8542-9089 AN: 0000-0001-5484-7088 EM-N: 0000-0001-7973-0345 SB: 0000-0002-2929-6410 MPV-A: 0000-0001-7176-0435 CO-G: 0009-0004-6511-2633 BBL: 0000-0002-9511-3731 Outbreak of Xylella fastidiosa subsp. pauca ST53 affecting wild and cultivated olive trees on the island of Mallorca, Spain Eduardo MORALEJO1,*, Bàrbara QUETGLAS1, Marina MONTESINOS1, Francisco ADROVER2, Diego OLMO2, Alicia NIETO2, Ana PEDROSA2, Marta LÓPEZ3, Andreu JUAN3, Ester MARCO-NOALES4, Inmaculada NAVARRO-HERRERO4, Silvia BARBÉ4, María Pilar VELASCO-AMO5, Concepción OLIVARES-GARCÍA5, Blanca B. LANDA5 1 Tragsa, Empresa de Transformación Agraria, Delegación de Baleares, 07005 Palma de Mallorca, Spain 2 Institut de Recerca i Formació Agroalimentària i Pesquera de les Illes Balears, 07009 Palma de Mallorca, Spain 3 Servicio de Agricultura, Conselleria de Medi Ambient, Agricultura i Pesca, 07006 Palma de Mallorca, Spain 4 Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), CV-315 km 10.7, 46113 Moncada, Spain 5 Institute for Sustainable Agriculture, Consejo Superior de Investigaciones Científicas (IAS-CSIC), 14004 Córdoba, Spain *Corresponding author: E-mail: [email protected] Summary. The Balearic Islands have emerged as a hotspot for the invasive plant pathogen Xylella fastidiosa (Xf). Since 2016, the Xf subsp. fastidiosa and multiplex have been detected causing almond leaf scorch and Pierce’s disease on the island of Mallorca, Spain, and a new sequence type (ST), ST80, of subsp. pauca is infecting wild and cultivated olive trees on the island of Ibiza. In addition, Xf subsp. multiplex ST81 is widespread in scrublands, and causes mild, sub-lethal dieback of wild olive trees in Menorca and Mallorca. A new outbreak is here reported of the Xf subsp. pauca in the municipality of Sencelles in the centre of Mallorca island. In early 2024, dying patches were observed in wild olive trees (Olea europaea var. europaea subsp. sylvestris). Samples from these trees were Xf-positive in different qPCR tests, and the pathogen was subsequently identified as belonging to ST53 of subsp. pauca, the same genetic variant responsible for olive quick decline syndrome in Apulia, Italy. More than 184 plants of eight hosts have tested positive for subsp. pauca within a demarcation zone of approx. 1 km radius. The identified host species include 124 wild olive trees, 40 cultivated olive trees, nine Rhamnus alaternus, six Nerium oleander, two Lavandula angustifolia, one Laurus nobilis, one Lavandula dentata and one Polygala myrtifolia. Of particular concern is detection of co-infections by Xf subsp. subsp. pauca and multiplex on plants from natural settings (wild olives, L. dentata and R. alaternus), posing potential risk of genetic recombinations. Intensive surveys are being carried out to contain the spread of ST53, and infected plants have been destroyed in the demarcated zone. Keywords. Genetic diversity, olive quick decline syndrome, invasive pathogens, MultiLocus Sequence Typing (MLST), disease outbreak. 476 Eduardo Moralejo et alii INTRODUCTION The vector-borne plant pathogenic bacterium Xylella fastidiosa (Xf) has recently emerged as a threat to agriculture in southern Europe. Following its first detection in Apulia (Italy) in 2013, associated with the rapid decline syndrome of olive trees (Saponari et al., 2013), the European Union (EU) took decisive action with mandatory surveys to contain possible spread of the pathogen within the EU (Reg. EU 2016/2031). The three main subspecies of Xf have since then been detected, and their establishment confirmed, in several southern European countries, including the island of Corsica and the Balearic Islands (Saponari et al., 2013; Denance et al., 2017; Landa et al., 2020; Olmo et al., 2017; Carvalho-Luis et al., 2022). The Balearic Islands have become a hotspot for establishment of genetic diversity of this pathogen, which originated from North, Central and South America. Since 2016, the three main subspecies of Xf, fastidiosa, multiplex, and pauca, have been detected, along with unique genotypes such as the sequence type (ST) 81 (from subsp. multiplex) on the islands of Mallorca and Menorca, and the ST80 (from subsp. pauca) on the island of Ibiza (Olmo et al., 2021). Subspecies fastidiosa, and in particular ST1, is only known on Mallorca island, where it caused severe outbreaks of Pierce’s disease of grapevines and leaf scorch of almond (Moralejo et al., 2019; Moralejo et al., 2020). Some evidences suggest that the genotypes responsible for Pierce’s disease (ST1) and almond leaf scorch (ST1 and ST81) in Mallorca were likely a single introduction via infected plant material from California, in approx. 1993 (Moralejo et al., 2020; Velasco-Amo et al., 2022). METHODS Early in 2024, plant health inspectors in the Balearic Islands noticed an unusually severe decline in a stand of wild olive trees (Olea europaea subsp. europaea var. sylvestris) (Figure 1). Samples from different tree branches were collected, were pooled, and then sent to the Balearic Islands Official Plant Health Laboratory (LOSVIB) for analyses. Sample extracts were prepared by homogenizing 0.5 g of leaf petioles in an extraction bag (BIOREBA®) and grinding them in 5 mL (1:10 weight:volume) of phoshate buffered saline (PBS) using a semi-automated homogenizer (Homex 7; BIOREBA®). Total DNA extraction was carried out using 200 µL of each sample extract, and the EZNA HP Plant Mini kit (Omega-Biotek), which employs a CTAB-based method, as described in the EPPO protocol (EPPO, 2023). The DNA extracts were subsequently tested for the presence ofXfby quantitative real-time PCR (qPCR), according to Harper et al. (2010). Aliquots of DNA from the Xf positive samples from the wild olive tree were sent to the Institute for Sustainable Agriculture (IAS-CSIC), Córdoba, Spain, for ST determination, which was carried out by conventional MultiLocus Sequence Typing (MLST) analysis (Yuan et al., 2010). A nested-MLST (Cesbron et al., 2020) analysis was used when not enough amplification product for direct sequencing was obtained when using the conventional MLST approach. RESULTS AND DISCUSSION MLST analyses identified the presence of alleles leuA-7, petC-6, malF-16, cysG-24, holC-10, nuoL-16 and gltT-14 associated with Xf subsp. pauca ST53 in the affected wild olive tree. Additionally, the qPCR protocol of Dupas et al. (2019) validated the diagnosis as Xf subsp. pauca. After confirming the diagnosis and recognizing the potential devastating impact if this subspecies were to spread, the phytosanitary authority of the Balearic Government implemented a targeted action plan. This included increasing surveys and intensive sampling in the area of the new outbreak. Additional measures were also established to those already in place from the current Regulation (EU) 2016/2031. These included analyses to determine the subspecies present in all the Xf-positive samples, in collaboration with the Valencian Institute of Agricultural Research (IVIA), using the qPCR protocols developed by Dupas et al. (2019) and Hodgetts et al. (2021). Since the first detection in January 2024 and up to December 2024, a total of 1,328 samples from within the disease outbreak area have been analyzed. This has shown that 184 plants were infected by Xf subsp. pauca, Figure 1. A group of wild olive trees with severe symptoms of quick decline syndrome. This photograph was taken at focus 0 near the municipality of Sencelles, Mallorca Island, Spain, where Xylella fastidiosa subsp. pauca ST53 was found for the first time in January 2024. 477 Xylella fastidiosa subsp. pauca ST53 on olive trees on the island of Mallorca, Spain and eight different hosts tested positive for Xf subsp. pauca, from within a demarcated zone (approx. 1 km radius) around the first disease detection. The infected host species include 124 wild olive trees, 40 cultivated olive trees, nine Rhamnus alaternus plants, six of Nerium oleander, two of Lavandula angustifolia, and one each of Laurus nobilis, Lavandula dentata and Polygala myrtifolia (Table 1). Despite the numerous cases of Xf subsp. pauca infections detected (Table 1), all cases analyzed by MLST, up to present, and within the initial focus of the outbreak belong to the ST53 (Figure 2). However, given that Xf subsp. fastidiosa and Xf subsp. multiplex were already established in the area, it was not surprising to find some mixed infections by these two subspecies in some individual plants. Eleven percent of Xf-positive samples were of mixed infections, with 26 cases of subspp. pauca/multiplex, one of subspp. pauca/fastidiosa and two of subspp. multiplex/fastidiosa (Table 1). The multiple infections probably may increase risk of genetic recombinations in the pathogen and the formation of new sequence types with different virulence and/or host ranges (Potnis et al., 2019). Although it is premature to draw definitive conclusions, the ST53 strain identified in Mallorca exhibited a level of virulence comparable to that associated with the olive quick decline syndrome observed in Apulia, based on the observed higher severity of symptoms in both wild and cultivated olive trees, compared to those induced by Xf subsp. multiplex ST81. Efforts are currently underway (December 2024) to isolate the bacterium, to enable complete genome sequencing and carry out comparative analysis of the pathogen’s compete genome with those of ST53 strains from Apulia. A new procedure has been established based on targeted sequence capture enrichment of Xf in combination with high-throughput sequencing using the Illumina platform. This is being implemented to provide additional genomic information (Velasco-Amo et al., 2021). Initial results based on approx. 126 genes have shown close genetic relatedness of the ST53 strain infecting the wild olive in Mallorca with all other ST53 strains isolated from Costa Rica and Italy (M.P. Velasco-Amo and B.B. Landa; unpublished results). A complicating factor in containment of ST53 strains is the large population of wild olive trees throughout Mallorca, which may facilitate transmission of the pathogen to cultivated olive trees. Tight wild olive tree population networks can be dense reservoirs of Xf, and provide effective dispersal pathways to commercial olive plantations. It is also unclear whether previous infections of wild olive trees by ST81 of Xf. subsp. multiplex will have influence subsequent infections by ST53. It is estimated that more than 50% of wild olive trees are infected with ST81 (Olmo et al., 2021), so any interaction between ST53 and ST81 in co-infections, regardless of its intensity, could have important epidemiological implications (Jeger and Bragard, 2019). Epidemiological models have shown that the most effective strategy for preventing spread of Xf is to decelTable 1. Host plant species, numbers of plants analysed, and numbers infected by different subspecies Xylella fastidiosa (Xf), in single or in mixed infections, in the outbreak of subspecies pauca around the locality of Sencelles (Mallorca island, Spain) Hosts Total analyzed Xf positivea% Xylella fastidiosa subspeciesb pauca mutliplex fastidiosa pauca/ multiplex pauca/ fastidiosa multiplex/ fastidiosa Laurus nobilis 6 2 33 1 1     Lavandula angustifolia 8 8 100 2 6     Lavandula dentata 6 5 83  4  1   Nerium oleander 22 6 27 6      Olea europaea var. europaea subsp. europaea 57 41 72 37 1  3   Olea europaea var. europaea subsp. sylvestris 929 155 17 106 29 2 18   Polygala myrtifolia 1 1 100    1   Prunus dulcis 8 1 13  1     Rhamnus alaternus 190 26 14 5 8 6 3 1 2 Salvia rosmarinus 23 19 83 19     Thirteen other plant species 78 -        Totals 1328 264 19.88 157 69 8 26 1 2 a Diagnoses performed using the qPCR test of Harper et al. (2010). b Subspecies assignation based on the qPCR tests as described by Dupas et al. (2019) and Hodgetts et al. (2021). 478 Eduardo Moralejo et alii erate transmission chains (White et al., 2020; GiménezRomero et al., 2023). Accordingly, in Mallorca, the decision has been taken to eradicate infected plants wherever feasible. Although this is a difficult and arguably unattainable objective, the attempt can have significant longterm impacts. It is anticipated that populations of the principal insect vector of Xf, Philaenus spumarius, will progressively decline in Mallorca in response to rising temperatures associated with climate change (GiménezRomero et al., 2024). Unsuitable conditions for the vector may become significant in a period of approx. 15 to 20 years. Climate change could be beneficial for containing the outbreak, provided that exponential infection rates can be reduced. Appropriate implementation of these actions will alleviate the economic consequences of Xf for the Mallorcan olive production sector in the medium term. FUNDING This research was funded by Projects E-RTA201700004-C06-02 from AEI-INIA Spain, and BeXyl (Beyond Xylella, Integrated Management Strategies for Mitigating Xylella fastidiosa impact in Europe; Grant ID No. 101060593), from the European Union Horizon Action Programme ‘Food, Bioeconomy Natural Resources, Agriculture and Environment’. ACKNOWLEDGEMENTS The authors of this paper thank the owners of the studied orchards, and especially Anita van der Werf, who has always been kind and willing to collaborate with the inspectors and technicians of the Mallorcan plant health services. Figure 2. Map of the outbreak focus of Xylella fastidiosa subsp.paucaST53 in the municipality of Sencelles, near the centre of Mallorca Island, Spain. The Xf pauca-positive plants were found within a radius of < 1 km. Both squares with amplified views show Xf-positive plants within the natural vegetation, formed mainly by wild olive trees near farms where other Xf subsp. pauca ST53 positive plants were found within olive orchards. The positive samples of Xf subsp. pauca ST53 plants were mixed with other infected with subsp. multiplex, and to a lesser extent with subsp. fastidiosa. In a few cases, co-infections by Xf subsp. pauca ST53 and subsp. multiplex were found in the same plant (Table 1). 479 Xylella fastidiosa subsp. pauca ST53 on olive trees on the island of Mallorca, Spain LITERATURE CITED Carvalho-Luis C., Rodrigues J.M., Martins L.M., 2022. Dispersion of the bacterium Xylella fastidiosa in Portugal. Journal of Agricultural Science and Technology. A 12(1): 35–41. https://doi.org/10.17265/21616256/2022.01.005 Cesbron S., Dupas E., Beaurepère Q., Briand M., MontesBorrego M., Velasco-Amo MdP., Landa B.B., Jacques M-A., 2020. 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