Isolation and molecular characterization of a Xylella fastidiosa subsp. multiplex strain from almond (Prunus dulcis) in Apulia, Southern Italy

Abstract

Xylella fastidiosa is a xylem-limited phytopathogenic bacterium under regulation in the European Union as a priority pest. Given the potential risk posed by this pathogen to cultivated and ornamental plants, mandatory annual surveys and laboratory testing are required in Member States to early detect outbreaks. In the course of surveys carried out during early spring 2024 in the Apulia region (Southern Italy), X. fastidiosa subsp. multiplex was identified using quantitative real-time Polymerase Chain Reaction (qPCR), in a non-symptomatic sample from an almond tree (Prunus dulcis) in an orchard located in Santeramo in Colle, in Bari province. Multilocus sequence typing (MLST) was used to identify the subspecies and sequence type (ST) of the bacterium using the genomic DNAs extracted from the infected sample. Comparative sequence analysis of the seven MLST allele genes indicated that the obtained nucleotide sequences completely matched allele sequences of X. fastidiosa in PubMLST database corresponding to the allelic profile (Sequence Type) ST26 related to subsp. multiplex. Bacterial colonies consistent in morphology with X. fastidiosa were isolated from asymptomatic host samples and identity was confirmed by real-time PCR analysis. This is the first report of detection of X fastidiosa subsp. multiplex ST26 in the EU.

Headnote

Summary. Xylella fastidiosa is a xylem-limited phytopathogenic bacterium under regulation in the European Union as a priority pest. Given the potential risk posed by this pathogen to cultivated and ornamental plants, mandatory annual surveys and laboratory testing are required in Member States to early detect outbreaks. In the course of surveys carried out during early spring 2024 in the Apulia region (Southern Italy), X. fastidiosa subsp. multiplex was identified using quantitative real-time Polymerase Chain Reaction (qPCR), in a non-symptomatic sample from an almond tree (Prunus dulcis) in an orchard located in Santeramo in Colle, in Bari province. Multilocus sequence typing (MLST) was used to identify the subspecies and sequence type (ST) of the bacterium using the genomic DNAs extracted from the infected sample. Comparative sequence analysis of the seven MLST allele genes indicated that the obtained nucleotide sequences completely matched allele sequences of X. fastidiosa in PubMLST database corresponding to the allelic profile (Sequence Type) ST26 related to subsp. multiplex. Bacterial colonies consistent in morphology with X. fastidiosa were isolated from asymptomatic host samples and identity was confirmed by real-time PCR analysis. This is the first report of detection of X fastidiosa subsp. multiplex ST26 in the EU.

Keywords. Xanthomonadaceae, MLST, priority pest, sequence type, xylem-limited bacterium.

INTRODUCTION

Xylella fastidiosa (Xanthomonadaceae) (Wells et al., 1987) is a gram-negative plant pathogenic bacterium comprising several subspecies, which are pathogenic to a broad spectrum of host plants including agricultural crops of economic importance, ornamentals and natural vegetation (EFSA, 2023). The pathogen is limited to host plant xylem tissues (Purcell and Hopkins, 1996), leading to symptoms generally related to xylem vessel occlusion, which include scorching of leaves and dieback that vary in severity depending on the host susceptibility. The pathogen is naturally transmitted by xylem sap-feeding leafhoppers (Cicadellidae) and spittlebugs (Cercopi-dae) (Hopkins, 1989), and is spread over long distances through movement of infected plant material or infectious insect vectors (Purcell and Hopkins, 1996; Lourei-ro et al, 2024).

Following the first confirmed report of X. fastidiosa in the European Union (EU) in 2013, in Salento, Apulia Region, Southern Italy (Saponari et al, 2013), where a strain of X. fastidiosa subsp. pauca Sequence Type (ST) 53 (Giampetruzzi et al., 2015; Giampetruzzi et al., 2017) was found to cause the Olive Quick Decline Syndrome (OQDS) (Martelli, 2016; Saponari et al, 2017), EU emergency measures against plant pests were updated with the new plant health Regulation (EU) 2016/2031 (European Commission, 2016). Under this regulation, X. fastidiosa became a priority pest (European Commission, 2019), and has been since subjected to mandatory annual surveys by Member States to prevent its entrance and spread within the EU (European Commission, 2020; European Commission, 2024). As a result of extensive survey activities, X. fastidiosa has also been detected in France, Spain, and Portugal (Denance et al., 2017; Olmo et al., 2017; Marco-Noales et al., 2021; Carvalho-Luis et al., 2022; EFSA, 2023), in which several sequence types (STs) of the bacterium belonging to different subspecies were identified on various plant species. More recently, X. fastidiosa subsp. multiplex ST87 has been found in the Tuscany region of Italy (Saponari et al., 2019), and a new outbreak of the subspecies fastidiosa ST1 emerged in a location in the province of Bari in Apulia (Cornara et al., 2024).

Official inspections performed for the detection and identification of the bacterium and its subspecies are regulated by Commission Implementing Regulation (EU) 2020/1201, amended and corrected by Commission Implementing Regulation (EU) 2024/2507), that specify which molecular tests must be used for the identification of X. fastidiosa and its subspecies.

Inspections are based on visual surveys, and collection of representative plant samples for pathogen diagnosis to species level by real-time Harper PCR (Harper et al., 2010, erratum 2013). Following the diagnostic confirmation of the positive detection of X. fastidiosa in previously free areas or in new plant host species, multilo-cus sequence typing (MLST) analysis (Yuan et al., 2010) is the most common test used for the assignment of positive samples to subspecies and Sequence Type (ST). Real-time PCR methods based on Dupas et al. (2019) and Hodgetts et al. (2021) can also be used for subspecies assignment (CI Regulation (EU) 2024/2507).

This paper reports identification of X. fastidiosa subsp. multiplex ST26 in a non-symptomatic sample from an almond tree (Prunus dulcis) in an orchard in Apulia, in the province of Bari (Southern Italy) (Europhyt outbreak notification n. 2549). This discovery occurred in the context of the regional surveillance program for X. fastidiosa associated with OQDS, enforced by the Plant Health Service of the Apulia Region, and carried out by the Regional Agency for Irrigation and Forestry Activities (ARIF).

MATERIALS, METHODS, AND RESULTS

Samples were randomly collected in March 2024, from a site that included orchards in the municipal territory of Santeramo in Colle, a few tens of kilometres from the west of the X. fastidiosa subsp. pauca ST53 demarcated area (DA). The plant material delivered to the laboratory of the Department of Soil, Plant and Food Sciences, University of Bari (Italy) consisted of mature lignified branches from non-symptomatic almond trees, which were refrigerated until testing.

Samples were prepared by debarking the hardwood cuttings and scraping the exposed surfaces of the wooden tissues with a sterile razor blade. From each sample, 0.5 g of wood shavings were placed in extraction bags (BIOREBA ) and then ground in 5 mL (1:10 weight:volume) of CTAB extraction buffer using a semi-automated homogenizer (Homex 7, BIOREBA*). Total nucleic acids were extracted using a cetyltrimethylam-monium bromide (CTAB) based method (Loconsole et al, 2014; EPPO, 2023). Samples from an OQDS-infect-ed and a non-infected plant were included in the DNA extraction as positive (PIC) and negative (NIC) isolation controls (EPPO, 2023).

DNA extracts were analysed by quantitative polymerase chain reaction (qPCR) assays carried out according to Appendix 5 of the EPPO Diagnostic Standard for X. fastidiosa PM 7/24 (5) based on the protocol of Harper et al. (2010, erratum 2013). Total nucleic acids of PIC and NIC were run alongside the samples, and a negative amplification control (NAC) was included. A positive amplification control (PAC) consisting of a suspension at a known concentration of X. fastidiosa subsp. pauca ST53 cells was also included in the same plate of the qPCR assay. According to the guidelines issued by the Plant Health Service of the Apulia Region (DDS no. 31 of 13 May 2022) for the monitoring and eradication of the pathogenic bacterium at regional level, samples that produced by qPCR (Harper et al., 2010) a quantification cycle (Cq) <32 were considered positive, while samples that did not exhibit exponential amplification were considered negative. If the Cq was greater than 32 the result was considered undetermined, and the sample was re-tested.

The qPCR assays for individual trees revealed presence of X. fastidiosa in one almond tree identified with the code ID 19107. The Cq value produced for this sample by qPCR was 28,54. Both the negative (NIC and NAC) and positive (PIC and PAC) controls produced the respective expected results.

Consequently, an aliquot of DNA of this sample was used for multilocus sequence typing (MLST) analysis (Yuan et al., 2010), according to the Appendix 16 of the EPPO Diagnostic Standard PM 7/24 (5) (EPPO, 2023), to further characterize the X. fastidiosa genotype detected outside the X. fastidiosa DA. Amplicons with the expected size were sequenced by Macrogen Inc., Seoul, South Korea. Allele sequences were then assembled by BioEdit Sequence Alignment Editor version 7.2.5 software and analyzed using the PubMLST database (http:// pubmlst.org/xfastidiosa/) to identify allele types. Allele

sequences amplified from the infected almond tree had 100% nucleotide identity with those of alleles leuA_5, petC_3, malF_3, cysG_3, holC_6, nuoL_3, and gltT_5 corresponding to the ST26 genotype belonging to X. fastidiosa subsp. multiplex (EPPO, 2023). The sequences obtained for the MLST alleles were deposited in Gen-Bank under accession numbers: leuA allele 5, PQ535574; petC allele 3, PQ535575; malF allele 3, PQ535576; cysG allele 3, PQ535577; holC allele 6, PQ515132; nuol allele 3, PQ535578; and gltT allele 5, PQ535579. A phyloge-netic network, inferred through the concatenation of the MLST sequences of all X. fastidiosa (STs) retrieved from the PubMLST database (Jolley et al, 2018), and conducted using the Neighbor-Net method implemented in Splits Tree4 (version 4.12.2) (Huson and Bryant, 2006), indicated that the genotype ST26 shared close similarity to a complex of strains of subsp. multiplex (Figure 1).

An aliquot of the DNA sample extracted from the infected almond tree was also analyzed by real-time tetraplex PCR assay (Dupas et al., 2019) to further confirm the isolated bacterium subspecies. This test was carried out using the reaction volumes and amplification conditions validated in the test performance study for the X. fastidiosa subspecies identification previously con-

Figure 2. Results from multiplex real-time PCR based on primer sets reported in Dupas et al. (2019). The blue exponential curves were generated by the primer-probe set, labelled with fluorophore FAM, which detected bacteria at species levels in the positive controls, and in the sample ID 19107. The green exponential curve was generated by the primer-probe set, labelled with HEX, which specifically detected subsp. fastidiosa in the positive control (Xff-Pos_ Ctrl). The orange exponential curves were generated by the primer-probe set, labelled ROX, which detected the subsp. multiplex in the positive control Xfm-Pos, and in the sample ID 19107. The violet exponential curve was generated by the primer-probe set, labelled Cy5, which detected subsp. pauca in the positive control Xfp-Pos. Negative amplification controls (NIC-Neg_Ctrl and NTC-Neg_Ctrl) are also indicated. Quantification cycles are indicated on the x axis, and relative fluorescence units (RFUs) are indicated on the y axis.

ducted by the Official Laboratories of the Italian National Plant Protection Organization (NPPO), with coordination of the National Reference Laboratory, constituted by the Council for Agricultural Research and Economics, Research Centre for Plant Protection and Certification (CREA-DC) (Pucci et al, 2023). For the assay PACs were provided by CREA-DC and consisted of DNA extracted from plant samples infected by isolates of X. fastidiosa subsp. fastidiosa, subsp. multiplex or subsp. pauca. The tetraplex real-time PCR assay detected and identified subsp. multiplex in the sample ID 19107, producing a Cq value of 27.47 (Figure 2). All DNAs extracted from the PAC tested positive for the corresponding target subspecies of X. fastidiosa when using the appropriate subspecies-specific primers and probes, while no amplification reaction occurred for the other subspecies. No signal amplification was observed for the NAC.

Attempts were made to isolate the bacterium. Ligni-fied portions (length 4 to 8 cm) recovered from cuttings of non-symptomatic host plant branches that had tested positive by qPCR were surface sterilized in a laminar flow hood by soaking for 2 min in 2% (v/v) sodium hypochlorite solution and 2 min in 70% ethanol, and then rinsed three times each for 2 min in sterile distilled water. The tissue portions were then each cut in half, squeezed with sterile pliers by pressing the external ends, and the freshly cut faces were blotted onto buffered cysteine-yeast extract

(BCYE) medium (Wells et al., 1981). The inoculated plates were then incubated at 28 C in the dark for at least 30 d and were periodically observed using a light microscope for appearance of colonies with morphological characteristics typical of X. fastidiosa (Wells et al, 1981; 1987) (Figure 3). Typical colonies were re-isolated and their identity as X. fastidiosa was confirmed using the qPCR assay of Harper et al. (2010, erratum 2013).

DISCUSSION

The subsp. multiplex of X. fastidiosa is native to North America (Nunney et al, 2019), and is known to have a wide plant species host range. This includes peach {P. persicd), plum (P. domestica), almond (P. dulcis), and several forest and shade trees (Schaad et al, 2004; Nunney et al, 2013; Nunney et al, 2019). Strains of X fastidiosa subsp. multiplex have also been reported in associations with olive (Krugner et al, 2014), and with grapevine (Almeida and Purcell, 2003). To date, in the European Union, strains belonging to the ST6, ST7, ST81 and ST87 of X. fastidiosa subsp. multiplex have been reported on almond and other hosts, including cultivated and ornamental species in Corsica, mainland France, the Balearic Islands, in Spain, and in Italy in Tuscany and Lazio (EFSA, 2022; Trkulja et al, 2022). The strain ST26 identified in Apulia is distinct from those found in other Italian and European regions, indicating that ST26 has been introduced from a different and unknown location.

Xylella fastidiosa subsp. multiplex ST26 has been previously detected only on P. domestica in Brazil, where it was thought to have been introduced from the North America (Coletta-Filho et al, 2017). ST26 is reported to mainly affect stone fruit trees (Prunus spp.), particularly plum (Coletta-Filho et al, 2017; Nunney et al, 2019).

After the notification of detection of X. fastidiosa multiplex ST26 to the competent Plant Health Service of the Apulia Region, a DA was established, and emergency control measures have been carried out to limit the spread of the bacterium to surrounding areas, in accordance with the legislative provisions under Regulation (EU) 2020/1201 (European Commission, 2020) amended by Regulation (EU) 2024/2507 (European Commission, 2024). A monitoring campaign is currently underway (in 2024) to determine the extent of the epidemic outbreak. Further research is required to consider the host range of ST26, seasonal development of the leaf scorch symptoms this strain causes, and the presence of infective vectors.

FUNDING

Funding for the plant diagnostic and testing service was received from Regione Puglia, in the framework of the Agreement with the Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, "Servizio di analisi di laboratorio ufficiali per rilevare la presenza di Xylella fastidiosa sul territorio della Regione Puglia". Research on characterization of the pathogen strain was supported by Agritech National Research Center, funded by European Union Next Generation EU (Piano Nazionale Di Ripresa E Resilienza (PNRR) - Missione 4 Componente 2, Investimento 1.4 - D.D 1032 17/06/2022, CN00000022).

ACKNOWLEDGEMENTS

The authors thank Dr Giuseppe Incampo, Daniele Cornacchia, Adriano Pacifico and Antonio Ceglie (University of Bari, Italy) for conducting some of the analyses described in this paper. Support of the Regional Agency for Irrigation and Forestry Activities (ARIF) is also acknowledged.

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