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# Development of A Nested-MultiLocus Sequence Typing Approach for A Highly Sensitive and Specific Identification of Xylella fastidiosa Subspecies Directly from Plant Samples

Sophie Cesbron, Enora Dupas, Quentin Beaurepère, Martial Briand, Miguel Montes-Borrego, Maria del Pilar Velasco-Amo, Blanca Landa, Marie-Agnès Jacques

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Sophie Cesbron, Enora Dupas, Quentin Beaurepère, Martial Briand, Miguel Montes-Borrego, et al. Development of A Nested-MultiLocus Sequence Typing Approach for A Highly Sensitive and Specific Identification of Xylella fastidiosa Subspecies Directly from Plant Samples. Agronomy, 2020, 10 (8), pp.1099. 10.3390/agronomy10081099. [hal-05056348](https://hal.inrae.fr/hal-05056348v1)

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https://hal.inrae.fr/hal-05056348v1

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Article

# Development of A Nested-MultiLocus Sequence Typing Approach for A Highly Sensitive and Specific Identification of *Xylella fastidiosa* Subspecies Directly from Plant Samples

Sophie Cesbron<sup>1,*</sup> <sup>1,2,●</sup>, Enora Dupas<sup>1,2,●</sup>, Quentin Beaurepère<sup>1</sup>, Martial Briand<sup>1</sup>, Miguel Montes-Borrego<sup>3</sup>, Maria del Pilar Velasco-Amo<sup>3</sup>, Blanca B. Landa<sup>3</sup> and Marie-Agnès Jacques<sup>1</sup>

Received: 9 June 2020; Accepted: 25 July 2020; Published: 29 July 2020

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**Abstract:** Identification of sequence types (ST) of *Xylella fastidiosa* based on direct MultiLocus Sequence Typing (MLST) of plant DNA samples is partly efficient. In order to improve the sensitivity of *X. fastidiosa* identification, we developed a direct nested-MLST assay on plant extracted DNA. This method was performed based on a largely used scheme targeting seven housekeeping gene (HKG) loci (*cysG*, *gltT*, *holC*, *leuA*, *malF*, *nuoL*, *petC*). Samples analyzed included 49 plant species and two insect species (*Philaenus spumarius*, *Neophilaenus campestris*) that were collected in 2017 (106 plant samples in France), in 2018 (162 plant samples in France, 40 plant samples and 26 insect samples in Spain), and in 2019 (30 plant samples in Spain). With the nested approach, a significant higher number of samples were amplified. The threshold was improved by 100 to 1000 times compared to conventional PCR. Using nested-MLST assay, plants that were not yet considered hosts tested positive and revealed novel alleles in France, whereas for Spanish samples it was possible to assign the subspecies or ST to samples considered as new hosts in Europe. Direct typing by nested-MLST from plant material has an increased sensitivity and may be useful for epidemiological purposes.

**Keywords:** *Xylella fastidiosa*; direct typing technique; MLST; nested PCR

## 1. Introduction

*Xylella fastidiosa* (*Xf*) is the causal agent of several devastating diseases of plants in the Americas and this pathogen was recently detected in Europe, where it causes a severe disease in olive trees in Italy and is present in several other regions. This species encompasses three well recognized subspecies, namely *fastidiosa*, *multiplex*, and *pauca* [1,2] but other subspecies are currently described [3]. The subspecies *fastidiosa* occurs in North and Central America and was recently detected in Spain (https://gd.eppo.int/taxon/XYLEFA/). It infects a wide range of trees, ornamentals, and other perennials and includes strains responsible for the well-known Pierce's disease on grapevine [3,4]. The subspecies *multiplex* is present in North and South America and in Europe (https://gd.eppo.int/taxon/XYLEFA/) and

<footnote n="1">¹ IRHS-UMR1345, INRAE, Institut Agro, Université d'Angers, SFR 4207 QuaSaV, 49071 Beaucouzeé, France; enora.dupas@inrae.fr (E.D.); quentin.beaurepere@inrae.fr (Q.B.); martial.briand@inrae.fr (M.B.); marie-agnes.jacques@inrae.fr (M.-A.J.)</footnote>

<footnote n="2">² Plant Health Laboratory, French Agency for Food, Environmental and Occupational Health & Safety, 49000 Angers, France</footnote>

<footnote n="3">³ Institute for Sustainable Agriculture, Consejo Superior de Investigaciones Científicas (IAS-CSIC), 14004 Córdoba, Spain; mmontes@ias.csic.es (M.M.-B.); mpvelasco@ias.csic.es (M.d.P.V.-A.); blanca.landa@ias.csic.es (B.B.L.)</footnote>

<footnote>* Correspondence: sophie.cesbron@inrae.fr</footnote>
is associated with scorches and dieback of a wide range of trees and ornamentals [3]. The subspecies *pauca* is mostly found in South and Central America on *Citrus* spp. and *Coffea* spp. [5]), but has been recently detected also in olive trees in Spain (https://gd.eppo.int/taxon/XYLEFA/), Brazil [6], Argentina [7], and Italy [8]. Its host range includes also ornamentals and other trees [3]. Altogether more than 560 plant species are hosts of *Xf* [3]. This member of the *Xanthomonadaceae* family inhabits the xylem of its host plants [9] and is naturally transmitted by insects from plants to plants.

A range of detection tests has been proposed for *Xf* [10]. Several immunological methods are available [10]. However, such methods have high limits of detection (LoDs) that are close to 10⁴ to 10⁵ cells.mL⁻¹ [10]. End point and also quantitative PCR (qPCR) are nowadays widely used, with a better sensivity as the LoD is around 10² cells.mL⁻¹ for several qPCR tests [11–14]. The Harper’s qPCR test is often used in Europe for its high sensitivity. Several tests based on isothermal amplification have also been reported [12,14–16]. The Harper’s test has also been successfully transferred to be used in digital PCR [17]. Some of these tests were designed to detect only one subspecies. This is the case of the nested PCR test proposed by [18] for detecting CVC (Citrus Variegated Chlorosis) strains (subspecies *pauca*) in sharpshooters and citrus plants and also of the qPCR test targeting oleander leaf scorch strains (that are included in the subspecies *fastidiosa*) [19]. Other tests were designed to detect and discriminate two or more subspecies [16,20].

Precise identification of *Xf* at an infraspecific level is essential for epidemiological and surveillance analyses, and to allow a proper description of the population structure and their dynamics. The widely used MultiLocus Sequence Typing (MLST) scheme designed for *Xf* [21,22] is based on amplification by conventional PCR and sequencing of seven HKG (housekeeping gene) fragments (loci), either from strains or from plant samples [23]. For each locus, the different sequence variants are considered as distinct alleles. The combination of allele numbers defines the sequence type (ST). The MLST-*Xf* data are stored in a public database (https://pubmlst.org/xfastidiosa/) that can be used to automatically identify and assign new allele variants, and provide tools to analyze the potential origin of the strains. The association of the different subspecies with their host plants is useful to better understand *Xf* epidemiology.

A reliable and enough informative typing method is particularly relevant in cases of new outbreaks or for the description of new host. Due to the large number of host plants to be analyzed, various types of inhibitors can interfere with reagents of PCR and low bacterial loads compromising PCR efficiency and hence typing. Improving DNA extraction methods can, at least partly, solve the problem of PCR inhibitors, and nested PCR appears a solution to allow the detection of low bacterial population sizes. A nested-MLST was already successfully developed to detect and type *Xf* in vectors [24]. Primers were designed inside the gene fragments used in the conventional-MLST scheme and hence some informative sites are lost. MLST with nested PCRs has also been developed in medical field to enable the direct typing of samples infected by *Leptospira* or *Trichomonas*, for example [25,26].

The objective of this study was to develop a *Xf* detection assay based on the largely used MLST scheme [22] that lowers the limit of detection (LoD) to enable at least the identification of *Xf* subspecies and, if possible, provide larger sets of typing data directly from plant samples. We used genomic sequences to improve each PCR efficiency and showed a drastic increase in the sensitivity as compared to that of the conventional-MLST approach.

## 2. Materials and Methods

### 2.1. Strains and Media

A collection of target and non-target bacterial strains was used to test in vitro the specificity of the newly designed primers and the nested PCR assays. This set was made of five *X. fastidiosa* strains from different subspecies and 34 strains representing bacteria phylogenetically close to *Xf*, i.e., various *Xanthomonas*, as well as strains of other plant pathogenic bacteria and endosymbionts potentially
inhabiting the same niches as *Xf* (Table 1), available at the French Collection of Plant-Associated Bacteria (CIRM-CFBP; https://www6.inra.fr/cirm_eng/CFBP-Plant-Associated-Bacteria).

The *Xf* strains were grown on modified PWG media (agar 12 g.L⁻¹; soytone 4 g.L⁻¹; bacto tryptone 1 g.L⁻¹; MgSO₄·7H₂O 0.4 g.L⁻¹; K₂HPO₄ 1.2 g.L⁻¹; KH₂PO₄ 1 g.L⁻¹; hemin chloride (0.1% in NaOH 0.05 M) 10 mL.L⁻¹; BSA (7.5%) 24 mL.L⁻¹; L-glutamine 4 g.L⁻¹) at 28 °C for one week. *Agrobacterium* and *Rhizobium* were grown at 25 °C for one to two days on MG medium [27]; TSA was used (tryptone soybroth 30 g.L⁻¹; agar 15 g.L⁻¹) for *Clavibacter*, *Ensifer*, *Stenotrophomonas*, *Xanthomonas* and *Xylophilus*; and King's medium B [28] for *Dickeya*, *Erwinia*, *Pantoea* and *Pseudomonas*. For PCRs, bacterial suspensions were prepared from fresh cultures in sterile distilled water, adjusted at OD₆₀₀ nm = 0.1 and used as templates for amplification after boiling for 20 min, thermal shock on ice and centrifugation 10 000 g, 10 min.

## 2.2. DNA Extraction

Genomic DNA from *Xf* strain CFBP 8070 was extracted with the Wizard genomic DNA Purification Kit (Promega, France) and used to prepare a 10-fold serial dilutions from 220 ng.mL⁻¹ (corresponding to 0.8 × 10⁸ copies.mL⁻¹ of genomic DNA) to 22 fg.mL⁻¹ (8 copies.mL⁻¹) to evaluate the LoD of the nested-MLST. Copies number were calculated using an estimated genome size of 2 903 976 bp, knowing that 1 pg = 9.78 × 10⁸ bp [29]. A total of 268 plant samples were collected in Corsica, France, based on symptoms compatible with those caused by *X. fastidiosa*; 106 samples were collected in June 2017 and 162 in September 2018. For each French sample, DNA was extracted as described in [10] using two methods in order to optimize the chances of detection. CTAB-based (Cetyl TrimethylAmmonium Bromide) extraction and robotic QuickpickTM SML kit from Bio-Nobile were used with the following modification: a sonication step (1 min, 42 KHz) was added after the samples (petioles, twigs) were finely cut, and was followed by a 15-min incubation period at room temperature. For initial laboratory diagnosis MLST results were compared with the Harper's qPCR test [12] as in [10] with following modifications: primers XF-F and XF-R, and probe XF-P [12] were used at a final concentration of 0.6 µM and 0.2 µM respectively, non-acetylated BSA (Bovine serum Albumine) was used at final concentration of 1.5 µg.µL⁻¹, and 2 µL of DNA were used in 10 µL reaction volume. The target of this PCR is located in the gene coding for the 16S rRNA-processing RimM protein. Each DNA sample was tested in triplicates. To validate the nested PCR, DNA samples were provided by the National Reference Laboratory for Phytopathogenic Bacteria, Valencia, Spain, and from the Official Phytosanitary Laboratory of the Balearic Islands for determining *Xf* subspecies. Those DNA samples correspond to DNA extractions made from symptomatic plants sampled during official monitoring surveys. A total of 70 *Xf*-infected samples were analyzed from Balearic Islands and mainland Spain during 2018 (40 samples) and 2019 (30 samples), as well as 26 insect samples from both regions. DNA was extracted from petioles of symptomatic leaves as described in [10] using a CTAB-based extraction method for plant samples from Alicante and insect samples from Alicante and Balearic Islands. A Mericon DNeasy Food kit from Qiagen was used for plant samples from Balearic Islands. All DNA extraction methods have been validated; validation data is available in the EPPO (European and Mediterranean Plant Protection Organization) Database on Diagnostic Expertise [10].

## 2.3. Nested-MLST Primers and Reactions

The seven HKG sequences (*cysG*, *gltT*, *holC*, *leuA*, *malF*, *nuoL*, *petC*) were extracted from 39 *Xf* genome sequences (Table S1) [2] to design the nested primers. Alignments were performed with BioEdit sequence alignment editor. The primers designed by [22] were destined to be used as inner primers (PCR2) (Table 2) in our nested assay.
<table><caption>**Table 1.** List of target and non-target strains used to verify the specificity of nested-MLST primers.</caption><thead><tr><th>CFBP Code</th><th>Bacterial Species</th><th>Host Plant</th><th>Origin</th></tr></thead><tbody><tr><td>6448</td><td>Agrobacterium rubi</td><td>Rubus ursinus var. loganobaccus</td><td>USA (1942)</td></tr><tr><td>2413</td><td>Agrobacterium tumefaciens</td><td>Malus sp.</td><td>NA (1935)</td></tr><tr><td>5523</td><td>Agrobacterium vitis</td><td>Vitis vinifera</td><td>Australia (1977)</td></tr><tr><td>2404</td><td>Clavibacter insidiosus</td><td>Medicago sativa</td><td>USA (1955)</td></tr><tr><td>4999</td><td>Clavibacter michiganensis</td><td>Lycopersicon esculentum</td><td>Hungary (1957)</td></tr><tr><td>3418</td><td>Curtobacterium flaccumfaciens pv. flaccumfaciens</td><td>Phaseolus vulgaris</td><td>Hungary (1957)</td></tr><tr><td>1200</td><td>Dickeya dianthicola</td><td>Dianthus caryophyllus</td><td>United Kingdom (1956)</td></tr><tr><td>5561</td><td>Ensifer meliloti</td><td>Medicago sativa</td><td>VA, USA (1984)</td></tr><tr><td>1232</td><td>Erwinia amylovora</td><td>Pyrus communis</td><td>United Kingdom (1959)</td></tr><tr><td>3845</td><td>Pantoea agglomerans</td><td>Knee laceration</td><td>Zimbabwe (1956)</td></tr><tr><td>3167</td><td>Pantoea stewartii pv. stewartii</td><td>Zea mays var. rugosa</td><td>USA (1970)</td></tr><tr><td>3205</td><td>Pseudomonas amygdali</td><td>Prunus amygdalus</td><td>Greece (1967)</td></tr><tr><td>8305</td><td>Pseudomonas cerasi</td><td>Prunus cerasus</td><td>Poland (2007)</td></tr><tr><td>7019</td><td>Pseudomonas congelans</td><td>na<fnref n="1" /></td><td>Germany (1994)</td></tr><tr><td>1573</td><td>Pseudomonas syringae pv. persicae</td><td>Prunus persica</td><td>France (1974)</td></tr><tr><td>1392</td><td>Pseudomonas syringae pv. syringae</td><td>Syringa vulgaris</td><td>United Kingdom (1950)</td></tr><tr><td>7436</td><td>Rhizobium nepotum</td><td>Prunus cerasifera myrobolan</td><td>Hungary (1989)</td></tr><tr><td>13100</td><td>Stenotrophomas maltophilia</td><td>Phaseolus vulgaris</td><td>Cameroon (2009)</td></tr><tr><td>3371</td><td>Xanthomonas euvesicatoria pv. citrumelonis</td><td>Citrus sp.</td><td>USA (1989)</td></tr><tr><td>2528</td><td>Xanthomonas arboricola pv. juglandis</td><td>Juglans regia</td><td>New Zealand (1956)</td></tr><tr><td>2535</td><td>Xanthomonas arboricola pv. pruni</td><td>Prunus salicina</td><td>New Zealand (1953)</td></tr><tr><td>4924</td><td>Xanthomonas axonopodis pv. axonopodis</td><td>Axonopus scoparius</td><td>Colombia (1949)</td></tr><tr><td>5241</td><td>Xanthomonas campestris pv. campestris</td><td>Brassica oleracea var. gemmifera</td><td>United Kingdom (1957)</td></tr><tr><td>2901</td><td>Xanthomonas citri pv. aurantifolii</td><td>Citrus limon</td><td>Argentina (1988)</td></tr><tr><td>2525</td><td>Xanthomonas citri pv. citri</td><td>Citrus limon</td><td>New Zealand (1956)</td></tr><tr><td>7660</td><td>Xanthomonas citri pv. viticola</td><td>Vitis vinifera</td><td>India (1969)</td></tr><tr><td>2625</td><td>Xanthomonas gardneri</td><td>Medicago sativa</td><td>Reunion Island (1986)</td></tr><tr><td>4925</td><td>Xanthomonas hortorum pv. hederae</td><td>Hedera helix</td><td>USA (1944)</td></tr><tr><td>2533</td><td>Xanthomonas hortorum pv. pelargonii</td><td>Pelargonium peltatum</td><td>New Zealand (1974)</td></tr><tr><td>1156</td><td>Xanthomonas hyacinthi</td><td>Hyacinthus orientalis</td><td>Netherlands (1958)</td></tr><tr><td>2532</td><td>Xanthomonas oryzae pv. oryzae</td><td>Oryza sativa</td><td>India (1965)</td></tr><tr><td>2054</td><td>Xanthomonas translucens</td><td>Hordeum vulgare</td><td>USA (1933)</td></tr><tr><td>2543</td><td>Xanthomonas vasicola pv. holcicola</td><td>Sorghum vulgare</td><td>New Zealand (1969)</td></tr><tr><td>7970</td><td>Xylella fastidiosa subsp. fastidiosa</td><td>Vitis vinifera</td><td>USA (1987)</td></tr><tr><td>8416</td><td>Xylella fastidiosa subsp. multiplex</td><td>Polygala myrtifolia</td><td>France (2015)</td></tr><tr><td>8084</td><td>Xylella fastidiosa subsp. morus</td><td>Morus alba</td><td>USA (na<fnref n="1" />)</td></tr><tr><td>8070</td><td>Xylella fastidiosa subsp. multiplex</td><td>Prunus spp.</td><td>USA (2004)</td></tr><tr><td>8402</td><td>Xylella fastidiosa subsp. pauca</td><td>Olea europea</td><td>Italy (2014)</td></tr><tr><td>1192</td><td>Xylophilus ampelinus</td><td>Vitis vinifera</td><td>Greece (1966)</td></tr></tbody></table>

<footnote n="1">¹: not available.</footnote>
<table><caption>**Table 2.** Primer sequences used in the *X. fastidiosa* nested-MLST scheme.</caption><thead><tr><th>Locus</th><th>PCR Round</th><th>5'-Forward Primer-3'</th><th>5'-Reverse Primer-3'</th><th>Position on Xf M12 Genome (CP000941.1)</th><th>Annealing Temperature (°C)</th><th>Size (pb) of Reaction Product</th></tr></thead><tbody><tr><td rowspan="2">cysG</td><td>1</td><td>CCAAACATAGAAGCACGCCG</td><td>GCGAGTGTTTTCAGCGTTCC</td><td>2111116-2111891</td><td>64</td><td>776</td></tr><tr><td>2</td><td>GCCGAAGCAGTGCTGGAAG<sup>1</sup></td><td>GCCATTTTCGATCAGTGCAAAAG<sup>1</sup></td><td>2111203-2111844</td><td>56</td><td>642</td></tr><tr><td rowspan="2">gltT</td><td>1</td><td>GGTGCCATCCAATCCGTTTT</td><td>TCAGGATGTCCCAATTCCAACG</td><td>1731589-1732504</td><td>60</td><td>916</td></tr><tr><td>2</td><td>TCATGATCCAAATCACTCGCTT<sup>1</sup></td><td>TTACTGGACGCTGCCTCG</td><td>1731783-1732482</td><td>56</td><td>700</td></tr><tr><td rowspan="2">holC</td><td>1</td><td>CCGATGGTGAAGAACAGTAGACA</td><td>GCTCGAGAAACTSGATTAATGG</td><td>133166-133714</td><td>62</td><td>549</td></tr><tr><td>2</td><td>GGTCACATGTCGTGTTTGTC</td><td>CACGCGCCGACTTCTATTT</td><td>133269-133692</td><td>59</td><td>424</td></tr><tr><td rowspan="2">leuA</td><td>1</td><td>CGAAGGTGCAAACAAAGTGA</td><td>CGCACTGGCTTCGATAATGTCT</td><td>1271664-1272549</td><td>58</td><td>886</td></tr><tr><td>2</td><td>GGTGCACGCCAAATCGAATG<sup>1</sup></td><td>ACTGGTCCCTGTACCTTCGT</td><td>1271752-1272525</td><td>60</td><td>774</td></tr><tr><td rowspan="2">malF</td><td>1</td><td>AACGTCGTCACCCCAAGAA</td><td>ATGAGGCGGGCTTCTTTGG</td><td>1680264-1681108</td><td>56</td><td>845</td></tr><tr><td>2</td><td>AGCAGAAGCACGTCCCAGAT</td><td>CTGGTCCTGCGGTGTTGG</td><td>1680308-1681074</td><td>60</td><td>767</td></tr><tr><td rowspan="2">nuoL</td><td>1</td><td>TTGGTACGTTGGCTTTGGTG</td><td>GACAAAACCAGATTGCGTGC</td><td>325347-326191</td><td>60</td><td>845</td></tr><tr><td>2</td><td>GCGACTTACGGTTACTGGGC</td><td>ACCACCGATCCACAACGCAT<sup>1</sup></td><td>325454-326050</td><td>54</td><td>597</td></tr><tr><td rowspan="2">petC</td><td>1</td><td>TCAATGCACGTCCTCCCAAT</td><td>GGCTGCCATTCGTTGAAGTA</td><td>2020498-2021079</td><td>60</td><td>582</td></tr><tr><td>2</td><td>ACGTCCTCCCAATAAGCCT</td><td>CGTTATTCACGTATCGCTGC</td><td>2020505-2021055</td><td>56</td><td>551</td></tr></tbody></table>

<footnote n="1">¹: primers from [22].</footnote>
We checked their characteristics with Primer3 V4.1.0 software (http://primer3.ut.ee/). Because of high Tm differences between forward and reverse primers for some primer pairs (*gltT*) (Table S2), or high hairpin Tm values (*holC* forward primer), some primers from [22] were redesigned nearly at the same positions to improve their efficiency. Moreover, as primer sequences were already near the locus sequence ends, we also had to relocate some of them to design nested primers inside the sequence alignments without loss of informative sites. Outer primers (PCR1) were designed with Primer3 V4.1.0 software (http://primer3.ut.ee/) in flanking regions targeted by the inner primers. Outer and inner primers were tested in silico using a primer search tool available in the galaxy toolbox of CIRM-CFBP (https://iris.angers.inra.fr/galaxypub-cfbp) on 194,438 bacterial Whole Genome Shotgun (WGS) sequences available in the NCBI database (as on March, 2019) including 58 *Xylella* and 1292 *Xanthomonas* (Table S3), and in vitro on target and non-target bacterial strains (Table 1).

PCRs were performed in 25 µL reaction buffer (Promega) with MgCl₂ at 1.5 mM final, 200 µM dNTP, 300 µM each of the forward and reverse primers, 0.6 U GoTaq G2 (Promega) and 2 µL of sample DNA. The first-round PCR program consisted of an initial denaturation step of 3 min at 95 °C followed by 35 cycles of 30 s denaturation at 95 °C, 30 s annealing at the relevant temperature according to each gene (determined by gradient PCR) and 60s elongation at 72 °C followed by a final extension step of 10 min at 72 °C (Table 2). The second round was performed with 30 cycles under same conditions and same concentrations but with a final volume of 50 µL for sequencing purposes and with 4 µL of first-round PCR product. The primer pairs of the second round of each nested PCR were used for sequencing (by Genoscreen, Lille, France for French samples and by Stabvida, Caparica, Portugal, for Spanish samples) the corresponding PCR products after 1.8% agarose gel visualization. To avoid contamination, one sample was opened at a time and stringent cleaning measures were applied after each experiment.

## 2.4. Statistical Analysis

The sensitivity of detection by conventional- and nested-MLST PCRs were compared in plant and vector samples for the seven HKGs that were analyzed by both approaches, by using a Chi square test using SAS (version 9.4, SAS Institute, Cary, NC, USA). Analysis was performed for the Spanish samples only, as HKG-PCRs were not systematically carried out on the French samples. Results were considered significantly different when *p* ≤ 0.05.

## 2.5. Sequence Acquisition, Alignment and Analyses

Forward and reverse nucleotide sequences were assembled, and aligned using Geneious 9.1.8 software (French samples) or Bionumerics V7.6.3 software (Spanish samples) to obtain high quality sequences. ST or loci assignation was performed according to http://pubmlst.org/xfastidiosa/. To reduce the costs of sequencing for French samples, only PCR products obtained for samples showing the highest rate of successful HKG amplifications were sequenced. On the other hand, all positive *holC* amplifications were sequenced to obtain a larger view of alleles present in Corsica.

A flow chart summarizing the different steps of the nested-MLST method is presented in Figure 1.
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Flow chart summarizing the different steps of the nested-MLST method.</figcaption></figure>

# 3. Results

## 3.1. Nested-MLST Proved to be Specific

The specificity of the outer and inner primer pairs was tested in silico and in vitro. In silico, all primers pairs showed the best scores of alignment with *Xf* genomic sequences. Some non-target organisms showed sequences nearly identical at outer primer locations with only one mismatch and a similar expected fragment size, but sequences of inner primers were more different indicating that there will be no amplification. This was the case for various *Xanthomonas* strains that contained one mismatch at position 15 of the *petC* forward outer primer and an identical sequence for the outer reverse primer. *X. taiwanensis* *holC* sequence corresponding to inner primers contained also only one mismatch. The fragment size predicted was as expected for *Xf*. Other predictions with one mismatch located in primers did not end in fragment amplifications of the same expected size. Then, the specificity of the outer and inner primer pairs (Table 2) was validated in vitro on five target strains and 34 non-target strains (Table 1). Specificity of the nested-MLST assay could not been tested in vitro on *X. taiwanensis* as no strain was available. Amplifications were obtained for all *Xf* strains. No amplification was detected on the non-target strains except for strain CFBP 2532 (*Xanthomonas oryzae* pv *oryzae*) and CFBP 2533 (*Xanthomonas hortorum* pv. *pelargonii*) in the first round of the nested PCR for the *petC* outer primers, providing a product of the expected size. However, these products were not amplified in the second round of the nested PCR and no false positive signal was finally obtained.
## 3.2. Nested-MLST Limit of Detection is Comparable to That of qPCR

The sensitivity of each primer combination was evaluated on serial dilutions of a genomic DNA solution calibrated (Qubit fluorimeter, Invitrogen) at 220 ng.mL⁻¹ (Figure 2). First round PCRs gave a signal more or less intense for concentrations up to 2.2 ng.mL⁻¹ (0.8 × 10⁶ copies.mL⁻¹) for all HKG except *malF* and *cysG* (220 pg.mL⁻¹). The second round of PCRs allowed a sufficiently strong signal for sequencing for concentrations up to 22 pg.mL⁻¹ (0.8 × 10⁴ copies.mL⁻¹) for *gltT*, *holC*, *petC*, *leuA*, *cysG*, and up to 2.2 pg.mL⁻¹ (0.8 × 10³ copies.mL⁻¹) for *nuoL* and *malF*.

<figure><img src="image_5.png" /><figcaption>**Figure 2.** Detection threshold of conventional-MLST (a) and nested-MLST (b) for seven HKGs using genomic DNA dilution range (1:220 ng.mL⁻¹; 2:22 ng.mL⁻¹; 3:2.2 ng.mL⁻¹; 4:220 pg.mL⁻¹; 5:22 pg.mL⁻¹; 6:2.2 pg.mL⁻¹; 7:220 fg.mL⁻¹; 8:22 fg.mL⁻¹).</figcaption></figure>

The same range of genomic DNA solutions was tested with the Harper's qPCR test to compare sensitivity of these two tests (Table S4). The latest signal (LoD) for the Harper's qPCR test (Cq = 37.64) was obtained with the concentration of 0.8.10³ copies.mL⁻¹ and no amplification was obtained for lower concentrations.

Previously, we evaluated the LoD of the conventional PCRs for *cysG* and *malF* of the initial MLST scheme [22] on a range of dilutions of CFBP 8070 genomic DNA with the Platinum Taq polymerase (Invitrogen) and tested the effect of adding BSA (final concentration at 0.3µg. µL⁻¹) on the efficiency of the conventional PCRs. No improvement was obtained as all signals remained around 0.8 × 10⁶ bacteria.mL⁻¹ (Figure S1).

## 3.3. Analysis of Naturally Infected Samples

Using qPCR Harper's test, 22 samples from 2017 and eight samples from 2018 collected in France were positive (Cq values < 35) with one or both DNA extraction methods; 70 samples from 2017 and 36 samples from 2018 were equivocal (35 ≤ Cq ≤ 40), 14 samples from 2017 and 118 from 2018 were negative (Cq > 40) (Table 3 and Table S5). Positive and equivocal samples were tested using the first round of PCR of the MLST assay: five samples from 2017 (one *Spartium junceum*, three *Polygala myrtifolia*, and one *Genista corsica*) gave a signal for at least one gene, but no complete typing was obtained for any sample. No sample from 2018 gave a signal. Most of Spanish samples used to evaluate nested-MLST scheme were positive using Harper's qPCR (only two out of 40 plant samples were equivocal in 2018 and eight out of 26 vector samples).
<table><caption>**Table 3.** Number of samples, positive and equivocal in Harper's qPCR. Percentage of successful amplifications obtained for each locus in conventional and nested PCR.</caption><thead><tr><th rowspan="3">Sample Type</th><th rowspan="3">Country</th><th rowspan="3">Year</th><th rowspan="3">Number of Samples</th><th colspan="15">Percentage of Successful Amplifications Obtained for Each Locus in Conventional and Nested MLST-PCR</th></tr><tr><th colspan="2">qPCR Harper Number of Samples</th><th colspan="2">cysG</th><th colspan="2">gltT</th><th colspan="2">holC</th><th colspan="2">leuA</th><th colspan="2">malF</th><th colspan="2">nuoL</th><th colspan="2">petC</th><th colspan="2">Average per Year</th></tr><tr><th>Cq &lt; 35</th><th>Cq ≥ 35</th><th>conv</th><th>nest</th><th>conv</th><th>nest</th><th>conv</th><th>nest</th><th>conv</th><th>nest</th><th>conv</th><th>nest</th><th>conv</th><th>nest</th><th>conv</th><th>nest</th><th>conv</th><th>nest</th></tr></thead><tbody><tr><td>Plant</td><td>France</td><td>2017</td><td>106</td><td>22</td><td>70</td><td>1.1</td><td>28.3</td><td>2.2</td><td>26.1</td><td>4.3</td><td>55.4</td><td>4.3</td><td>34.8</td><td>1.1</td><td>35.9</td><td>0</td><td>26.1</td><td>1.1</td><td>46.7</td><td>2</td><td>36.2</td></tr><tr><td>Plant</td><td>France</td><td>2018</td><td>162</td><td>8</td><td>36</td><td>0</td><td>11.4</td><td>0</td><td>9.1</td><td>0</td><td>27.3</td><td>0</td><td>27.3</td><td>0</td><td>15.9</td><td>0</td><td>27.3</td><td>0</td><td>25</td><td>0</td><td>20.5</td></tr><tr><td>Plant</td><td>Spain</td><td>2018</td><td>40</td><td>38</td><td>2</td><td>55</td><td>90<sup>*</sup></td><td>10</td><td>77.5<sup>*</sup></td><td>15</td><td>80<sup>*</sup></td><td>12.5</td><td>75<sup>*</sup></td><td>30</td><td>75<sup>*</sup></td><td>40</td><td>85<sup>*</sup></td><td>15</td><td>85<sup>*</sup></td><td>25.4</td><td>81.1</td></tr><tr><td>Plant</td><td>Spain</td><td>2019</td><td>30</td><td>30</td><td>0</td><td>30</td><td>90<sup>*</sup></td><td>13.3</td><td>90<sup>*</sup></td><td>16.7</td><td>93.3<sup>*</sup></td><td>16.7</td><td>90<sup>*</sup></td><td>20</td><td>90<sup>*</sup></td><td>66.7</td><td>90<sup>*</sup></td><td>20</td><td>90<sup>*</sup></td><td>26.2</td><td>90.5</td></tr><tr><td>Insect</td><td>Spain</td><td>2018</td><td>26</td><td>18</td><td>8</td><td>65.4</td><td>80.8</td><td>7.7</td><td>73.1<sup>*</sup></td><td>19.2</td><td>69.2<sup>*</sup></td><td>11.5</td><td>57.7<sup>*</sup></td><td>7.7</td><td>53.8<sup>*</sup></td><td>26.9</td><td>57.7<sup>*</sup></td><td>15.4</td><td>73.1<sup>*</sup></td><td>22</td><td>66.5</td></tr></tbody></table>

<footnote>(*) Asterisk indicates a significant (p < 0.05) higher number of successful amplifications for nested-MLST as compared to conventional-MLST [22] according to a Chi-square test. The test was conducted only for the Spanish samples on the number of samples, even if frequencies are indicated in the table for MLST-PCR.</footnote>
### 3.4. Nested-MLST Improved Successful HKG Typing by Increasing Sensitivity Level

Using nested-MLST for French samples, full allelic profiles were obtained for five samples from 2017 and one from 2018 corresponding to the lowest Cq in Harper's qPCR test (Table 4 and Table S5). Among fully typed samples, four were *X. fastidiosa* subsp. *multiplex* ST7 (*Genista corsica*, *Polygala myrtifolia*, *Spartium junceum*), and two were *X. fastidiosa* subsp. *multiplex* ST6 (*Polygala myrtifolia*).

Our scheme was also evaluated on Spanish samples already proved infected by Xf. These samples from different outbreaks showed a wide range of Cq values ranging from 18.8 to 36.0 for plant samples and from 23.29 to 37.0 for insect samples (Table 3 and Table S5). Samples were first analyzed using the conventional-MLST assay [22]. Amplification efficiency was variable and ranged from 10% for *gltT* to 67% for *nuoL* with an average of 25% and 26% for the seven HKG in 2018 and 2019, respectively. The nested-MLST assay improved the amplification efficiency that increased to 75% for *leuA* and up to 93% for *holC* with an average of 81% and 91% in 2018 and 2019, respectively. In total, full allelic profiles were obtained in seven plant samples using the conventional-MLST assay, whereas a total of 55 samples were fully typed with the improved nested-MLST assay (Table 4). For the 70 plant DNA samples that were tested by both protocols, for all the seven HKGs, conventional-MLST showed a significant (*p* < 0.0005 for 2018 and *p* < 0.0283 for 2019) lower number of samples amplified as compared to nested-MLST. Among fully typed plant samples using the nested-MLST, we identified *X. fastidiosa* subsp. *fastidiosa* ST1 in *Ficus carica* and *Juglans regia*, *X. fastidiosa* subsp. *multiplex* ST6 in *Helichrysum italicum*, *Olea europaea*, *Phagnalon saxatile*, *Polygala myrtifolia*, *Prunus armeniaca*, *Prunus domestica*, *Prunus dulcis*, *Rhamnus alaternus*, and *Rosmarinus officinalis*, *X. fastidiosa* subsp. *multiplex* ST7 in *Prunus dulcis*, *X. fastidiosa* subsp. *multiplex* ST81 in *Lavandula angustifolia* and *Prunus dulcis*, and *X. fastidiosa* subsp. *pauca* ST80 in *Cistus albidus*, *Prunus dulcis*, and *Rosmarinus officinalis*.

Not all insect samples could be tested by both protocols due to restrictions in DNA amount. In samples tested only by the original MLST assay [22], the percentages of successful amplifications ranged from 8% (*gltT* and *malF*) to 65% (*cysG*). With the nested-MLST assay, successful amplifications ranged from 54% (*malF*) to 81% (*cysG*), with an average efficiency for the seven HKG of 22% to 67% for conventional and nested approach, respectively (Table 3 and Table S5). Nine insect samples were fully typed using a combination of both protocols (Table 4). *X. fastidiosa* subsp. *fastidiosa* ST1 was identified in insects from Mallorca (Balearic Islands), *X. fastidiosa* subsp. *multiplex* ST6 in insects from Alicante (mainland Spain) and *X. fastidiosa* subsp. *multiplex* ST81 in insects from Balearic Islands. For the nine insect samples that were tested by both protocols, conventional-MLST showed a significant (*p* < 0.0247) lower number of samples amplified as compared to nested-MLST for six of the seven HKGs (excluding *cysG*). These results indicate that for insect samples it is also better to use directly the improved nested-MLST assay.

No nonspecific amplicons were observed in any of the samples. Negative controls (water) were run in the first and the second PCR and were always negative. The negative control coming from the first reaction always tested negative in the second one, confirming the absence of contamination during the entire process. Positive control was a suspension of strain CFBP 8084 (ST29) from the subspecies *morus* or strain CO33 (ST72) as this STs were not previously found in Corsica, France or Spain, respectively.
<table><caption>**Table 4.** Allele numbers and STs obtained for fully typed samples in France and Spain for plant and insect samples. The numbers correspond to the names of the samples.</caption><thead><tr><th>Country</th><th>Sample Names</th><th>cysG</th><th>gltT</th><th>holC</th><th>leuA</th><th>malF</th><th>nuoL</th><th>petC</th><th>Sequence Type (ST)</th></tr></thead><tbody><tr><td>France</td><td>Spartium junceum 2</td><td>7</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST7</td></tr><tr><td>France</td><td>Polygala myrtifolia 3, 4</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST6</td></tr><tr><td>France</td><td>Genista corsica 1</td><td>7</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST7</td></tr><tr><td>France</td><td>Polygala myrtifolia 5, 6</td><td>7</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST7</td></tr><tr><td>Spain</td><td>Cistus albidus 2</td><td>31</td><td>15</td><td>10</td><td>7</td><td>17</td><td>16</td><td>6</td><td>ST80</td></tr><tr><td>Spain</td><td>Ficus carica 1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>ST1</td></tr><tr><td>Spain</td><td>Helichrysum italicum 1</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST6</td></tr><tr><td>Spain</td><td>Juglans regia 1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>ST1</td></tr><tr><td>Spain</td><td>Lavandula angustifolia 1</td><td>32</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST81</td></tr><tr><td>Spain</td><td>Olea europaea 1</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST6</td></tr><tr><td>Spain</td><td>Phagnalon saxatile 1</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST6</td></tr><tr><td>Spain</td><td>Polygala myrtifolia 1</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST6</td></tr><tr><td>Spain</td><td>Prunus armeniaca 1</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST6</td></tr><tr><td>Spain</td><td>Prunus domestica 1</td><td>32</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST81</td></tr><tr><td>Spain</td><td>Prunus domestica 2</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST6</td></tr><tr><td>Spain</td><td>Prunus dulcis 4–8,10,11,15,18–26,30–47</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST6</td></tr><tr><td>Spain</td><td>Prunus dulcis 9</td><td>31</td><td>15</td><td>10</td><td>7</td><td>17</td><td>16</td><td>6</td><td>ST80</td></tr><tr><td>Spain</td><td>Prunus dulcis 1,2</td><td>32</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST81</td></tr><tr><td>Spain</td><td>Prunus dulcis 3</td><td>7</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST7</td></tr><tr><td>Spain</td><td>Rhamnus alaternus 1</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST6</td></tr><tr><td>Spain</td><td>Rosmarinus officinalis 4</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST6</td></tr><tr><td>Spain</td><td>Rosmarinus officinalis 1,3</td><td>31</td><td>15</td><td>10</td><td>7</td><td>17</td><td>16</td><td>6</td><td>ST80</td></tr><tr><td>Spain</td><td>Prunus domestica 3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST6</td></tr><tr><td>Spain</td><td>Philaenus spumarius 6,7,8,10,11</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>ST1</td></tr><tr><td>Spain</td><td>Philaenus spumarius 1</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST6</td></tr><tr><td>Spain</td><td>Philaenus spumarius 22</td><td>32</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST81</td></tr><tr><td>Spain</td><td>Neophilaenus campestris 1,2</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>ST6</td></tr></tbody></table>
### 3.5. Nested-MLST Allowed Identification of New Alleles Among French Samples

Incomplete profiles were obtained for various French samples due to variable amplification efficiencies varying according to the HKG. From 9% (with *gltT*) to 55% (with *holC*) of French samples gave a signal applying the nested-MLST assay. Alleles that were not yet described in plant samples in France were detected in 2017. This was the case for *holC*_1 and *holC*_2 alleles known to occur in ST from ST1 to ST5 and ST75 that cluster in the subspecies *fastidiosa* (https://pubmlst.org/xfastidiosa/). These alleles were sequenced in samples of *Asparagus acutifolius*, *Eleagnus*, *Cistus monspeliensis* and *C. creticus*, *Quercus ilex*, *Myrtus myrtifolia*, *Olea europaea*, *Platanus*, *Arbutus unedo* (Table S5). Other *holC* alleles already described in STs clustering in the subspecies *fastidiosa* (*holC*_24) were also sequenced from *Cistus monspeliensis* and *Pistaccia lentiscus*. *HolC*_10 alleles described in STs clustering in the subspecies *pauca* were sequenced from *Cistus monspeliensis* and *C. salicifolius*, *Cypressus*, *Metrosideros excelsa*, *Myrtus communis*, *Pistaccia lentiscus*, *Quercus ilex*, *Rubia peregrina*, *Smilax aspera* samples. Similarly, *holC*_3 (known in ST6, ST7, ST25, ST34, ST35, ST79, ST81 and ST87 clustering in the subspecies *multiplex*) were obtained from samples of *Acer monspeliensis*, *Arbutus unedo*, *Calicotome spinosa*, *Cistus monspeliensis*, *Genista corsica*, *Myrtus communis*, *Olea europaea*, *Phyllirea angustifolia*, *Polygala myrtifolia*, *Quercus ilex* and *Quercus pubescens*, *Spartium junceum*. Among samples from 2018, only *holC*_1 allele was detected in *Olea europaea*, *Quercus ilex*, and *Platanus* sp. samples, and *holC*_3 allele in *Cistus monspeliensis*, *Acer monspeliensis*, *Myrtus communis*, and *Polygala myrtifolia* samples.

### 3.6. Recombinants or Mixed Infections Were Identified by Nested-MLST

Some French samples were further sequenced for several loci and these sequencing confirmed the presence of alleles occurring in the subspecies *fastidiosa*, *multiplex* and *pauca* (Table S5). All alleles were previously described but were detected in combinations that were not previously described, suggesting the presence of recombinants or of mix infections (Table S5). This is the case for *Cistus monspeliensis* 7 showing an unknown combination of *cysG*_2/*petC*_2/*nuoL*_2/*gltT*_2 (known in ST5) with *malF*_4 (known in ST2), both from subspecies *fastidiosa*; *Helichrysum italicum* 1 showing *leuA*_1 (known in subspecies *fastidiosa*) with *petC*_3/*holC*_3 known in subspecies *multiplex*; *Myrtus communis* 4 with *leuA*_3/*holC*_2 respectively known in subspecies *multiplex* and *fastidiosa*; *Myrtus communis* 8 and *Platanus* presenting form 1 alleles for five HKG mixed with *malF*_4 (all known in subspecies *fastidiosa*) and *Q. ilex* 10 presenting form 1 alleles for two HKG mixed with *malF*4); *Olea europaea* 2 with four *multiplex* alleles combined with *nuoL*_1 (subspecies *fastidiosa*); *Olea europaea* 5 with four *pauca* alleles combined with *malF*_15 (known in ST72 and ST76, subspecies *fastidiosa*). Two samples gave a double sequence for *holC* that were impossible to analyze (Table S5). Some sequences were ambiguous with superimposed peaks at some locations in otherwise good quality chromatograms revealing mixed infections. In those 12 samples, the number of potential combinations was too high to detect one probable allelic form, excepted for *Prunus dulcis* where the superimposed chromatograms corresponded to only two allelic forms (*holC*_3 or *holC*_6 which are found in subspecies *multiplex*). The *holC*_6 allelic form and the *leuA*_5 allele obtained for this sample are found in ST10, ST26, ST36, ST46, and ST63.

## 4. Discussion

A two-step nested procedure for MLST was developed to improve the typing of samples infected with low *Xf* population sizes that cannot be typed using the conventional protocol. In order not to affect the comparability of the results with the databases, the widely used MLST scheme developed for *Xf* that is supported by the pubMLST public website [22] was re-used.

The nested-MLST approach proved to be specific and efficient. No nonspecific amplifications were observed in any of the samples. Moreover, the sensitivities of the Harper's qPCR detection test and the nested-MLST were similar with a LoD ranging from 10³ bacteria.mL⁻¹ to 10⁴ bacteria.mL⁻¹ These LoDs are similar to other nested-MLST approaches such as those developed for *Burkholderia*
*cepacia* [30] but higher than for the one developed for *Neisseria meningitidis* (10 copies mL⁻¹) [31]. Consequently, in resource-limited settings where qPCR facilities are not available, the assay may be used as a useful diagnostic tool if applied with all necessary precautions to avoid cross-contamination between samples. The sequencing, which is costly, can be done as a consecutive but separate step to provide information on subspecies present in the sample. Higher bacterial loads (as indicated by lower Cq values) were observed in Spanish samples than in French samples, for which low amplification efficiency and partial profiles were observed. Full allelic profiles (ST6 and ST7 from *multiplex* subspecies) were obtained for *Polygala myrtifolia*, *Spartium junceum* and *Genista corsica* samples from France probably because they carried a higher bacterial load as shown by the low Cq obtained with the Harper's qPCR test: five of the six typed samples had a Cq value between 23.4 and 26.5. The use of the nested-MLST assay to type plant Spanish samples allowed a higher number of successful complete typing (55 samples versus seven samples with the conventional approach). Spanish samples generally showed higher *Xf* titer (i.e, lower Cq values in Harper's qPCR test) than the French samples but also concerned different plant species.

In our nested-MLST assay as well as in the original MLST assay, the amplification efficiencies were variable among genes, while all primers were designed using the same parameters from the software. For example, the *holC* gene for French samples tested with the nested-MLST assay was successfully amplified in 55% of samples collected in 2017 while the *gltT* and *nuoL* genes gave the lowest rates (around 26%). For samples collected in Spain tested with the original MLST assay, amplification rates among the seven HKGs ranged from 10 to 67%. Success rate variations were also observed in medical research using MLST between samples and between loci [25]. When conducted on strains, no differences about amplification rates are observed because of DNA excess. Robustness of a PCR reaction is determined by appropriate primers and it is not always obvious why some primer combinations do not amplify well, even if some parameters such as DNA folding can interfere in PCR efficiency [32]. In this study, even if primer annealing temperature was adjusted, design of primers was limited by their arbitrary localization.

Typing results of French samples were concordant with previously published results [23] but also revealed the presence of alleles not yet described in France. It should be noticed that no unknown sequence was obtained, refraining from evoking contaminations as the origin of these yet undescribed alleles in France. Thanks to the high rate of amplification of *holC* in nested PCR, it was also possible to obtain sequences for equivocal samples (Cq with the Harper's qPCR test above 35) to confirm the presence of the bacterium in these samples. Surprisingly, these amplifications led to alleles that correspond to subspecies other than the *multiplex* subspecies. Thereby, alleles from subspecies *pauca* (*holC* 10) and *fastidiosa* (*holC* 1, *holC* 2, *holC* 24) were sequenced. *holC* 10 was already reported in *Polygala myrtifolia* in the south of France in 2015 [23]. *holC* 1 finding is in agreement with [24], who also reported *holC* 1 in insects in Corsica. Up to now, no *holC* 2 was reported in France but it is known in the USA. *holC* 24 was also reported in *Polygala myrtifolia* in Corsica in 2015 [23]. Further plant sampling efforts are needed to confirm the establishment of those strains in the environment or to document further the dynamics of alleles revealing sporadic infections.

For French samples only, several samples could not be typed since the chromatograms showed an overlap of two peaks precisely on the polymorphic sites (mainly with *leuA* and *holC* genes). This has already been reported by [23], it suggests the simultaneous presence of several strains in the same sample since only one copy of these genes are known in *Xf* [22]. Moreover, the report of previously unknown combination of alleles belonging to different subspecies can also results from the presence of co-infection or of recombinants. Recombination events are reported in *Xf* [23,33–35] and could have led to host shift [36]. In this study, eight samples presented unknown combinations of alleles from the same or different subspecies which could be explained by intraspecies or interspecies recombination events. As reported in [37], such events may exist and occur but not with the same frequency. Moreover, natural competence can be variable among *Xf* strains [38]. These events could also reflect a mechanism of adaptation [39]. Five samples among these eight samples were collected
in 2017 and three in 2018, and were different between years. In 2018 the three cases were a similar combination of alleles and were found in three different plants. Future surveys will be necessary to know if some of these recombinants strains are indeed present in Corsica or are the consequence of mixed infections and if they have adapted and survived on different hosts.

The objective of this study was to improve the published MLST scheme supported by a public website (https://pubmlst.org/xfastidiosa/) by designing nested primers to lower the limit of detection and help in Xf diagnosis and typing. Thus, this improved MLST assay enables a higher sensitivity and specific typing of Xf directly from plant and insects samples without the need of isolating the strain and at an affordable cost.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4395/10/8/1099/s1, Figure S1: detection threshold of conventional PCR for *cysG* and *malF* (Yuan et al., 2010) with and without BSA (final concentration at 0.3µg. µL⁻¹) using genomic DNA dilution range (1: 220ng.mL⁻¹; 2: 22ng.mL⁻¹; 3: 2.2ng.mL⁻¹; 4: 220pg.mL⁻¹; 5: 22pg.mL⁻¹; 6: 2.2pg.mL⁻¹; 7: 220fg.mL⁻¹; 8: 22fg.mL⁻¹). (+): positive control; (-) negative control, Table S1: List of *X. fastidiosa* genome sequences used in this study for primer and probe design (Denancé et al. 2019), Table S2: Primers properties, Table S3 Primer sequences alignment using a primer search tool available in the galaxy toolbox of CIRM-CFBP (https://iris.angers.inra.fr/galaxypub-cfbp) on 194438 bacterial Whole Genome Shotgun (WGS) sequences available in the NCBI database (as on March, 2019) including 58 *Xylella* and 1292 *Xanthomonas*, Table S4: detection threshold for Harper's qPCR test using genomic DNA dilution range (1: 220ng.mL⁻¹; 2: 22ng.mL⁻¹; 3: 2.2ng.mL⁻¹; 4: 220pg.mL⁻¹; 5: 22pg.mL⁻¹; 6: 2.2pg.mL⁻¹; 7: 220fg.mL⁻¹; 8: 22fg.mL⁻¹), Table S5: results obtained with qPCR and nested-MLST. (+) means that a signal has been obtained in PCR but the PCR product has not been sequenced.

**Author Contributions:** Conceptualization, S.C.; methodology, S.C., E.D., M.B.; validation, S.C., Q.B.; formal analysis, B.B.L.; investigation, S.C., Q.B., M.M.-B., M.d.P.V.A.; resources, S.C., M.-A.J., M.M.-B., M.d.P.V.-A., B.B.L.; writing—Original draft preparation, S.C.; writing—Review and editing, S.C., E.D., M.-A.J., B.B.L.; visualization, S.C., B.B.L.; supervision, M.-A.J.; project administration, M.-A.J., B.B.L.; funding acquisition, M.-A.J., B.B.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** ED salary was funded by INRA SPE division and Anses. This work received support from the European Union's Horizon 2020 research and innovation program under grant agreement 727987 XF-ACTORS (*Xylella fastidiosa* Active Containment Through a multidisciplinary-Oriented Research Strategy), and from Projects E-RTA2017-00004-C06-02 from 'Programa Estatal de I+D Orientada a los Retos de la Sociedad' from Spanish State Research Agency and from the Organización Interprofesional del Aceite de Oliva Español, CSIC Intramural Project 2018 40E111, and from "Conselleria de Agricultura, Desarrollo Rural, Emergencia Climática y Transición Ecológica" from Valencia region, and the Ministry of Agriculture, Fisheries and Food of Spain. The present work reflects only the authors' view and the EU funding agency is not responsible for any use that may be made of the information it contains.

**Acknowledgments:** We thank Muriel Bahut (ANAN technical facility, SFR QUASAV, Angers, FR) for DNA extraction automatization, CIRM-CFBP (Beaucouzé, INRA, France; http://www6.inra.fr/cirm_eng/CFBP-Plant Associated-Bacteria) for strain preservation and supply. We thank Ester Marco-Noales from the National Reference Laboratory for Phytopathogenic Bacteria (IVIA), and Diego Olmo from the Official Phytosanitary Laboratory of the Balearic Islands for providing DNA samples for MLST typing.

**Conflicts of Interest:** The authors declare no conflict of interest. The present work reflects only the authors' view and no analysis has been made in the French Reference Lab; in particular ED is not authorized to perform any official tests at Anses.

Nomenclature

<table><tr><td>BLAST</td><td>Basic Local Alignment Search Tool</td></tr><tr><td>Cq</td><td>quantification cycle</td></tr><tr><td>HKG</td><td>housekeeping gene</td></tr><tr><td>INRA</td><td>French National Institute for Agricultural Research</td></tr><tr><td>IRHS</td><td>Research Institute of Horticulture and Seeds</td></tr><tr><td>LoD</td><td>Limit of Detection</td></tr><tr><td>MLST</td><td>Multilocus Sequence Typing</td></tr><tr><td>NCBI</td><td>National Center for Biotechnology Information</td></tr><tr><td>ST</td><td>Sequence Type</td></tr><tr><td>Xf</td><td>Xylella fastidiosa</td></tr><tr><td>WGS</td><td>Whole Genome Shotgun</td></tr></table>
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