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

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.

Investigating plant disease outbreaks with long-read metagenomics: sensitive detection and highly resolved phylogenetic reconstruction applied to Xylella fastidiosa

“…Methods of detection for X. fastidiosa include microscopy, immunological assays and molecular methods [12], none of which require culturing. Also, identification of X. fastidiosa to the subspecies level by quantitative PCR (qPCR) and to the strain level using multilocus sequence typing (MLST) can be performed using DNA directly extracted from plants [13,14]. However, to reconstruct transmission chains, whole-genome sequencing is necessary to identify a sufficient number of SNPs for phylogenetic analysis.…”

Use of traC Gene to Type the Incidence and Distribution of pXFAS_5235 Plasmid-Bearing Strains of Xylella fastidiosa subsp. fastidiosa ST1 in Spain

“…The sensitivity of the primer pair was evaluated in different plant DNA backgrounds calibrated at an initial concentration of 5 × 10 5 copies.µL −1 . The fact that there are no differences between hosts in the sensitivity of the technique may be due to the use of BSA, which in other studies has been shown to compensate for polymerase inhibitors, and thus favor PCR efficiency [28]. In the same way, the gene could be amplified in naturally-infected samples from various hosts, where the lack of amplification was mainly related to a low concentration of bacterial cells (i.e., it was associated with high threshold values of Harper's qPCR assay) [29].…”

“…These samples showed a Ct > 30 for Harper's qPCR test, which was over the detection limit of the ND116-pRIV5-F1 and ND117-pRIV5-R1 primer pair. Indeed, for those samples, it was necessary to perform the Nested-MLST analysis [28] to assign the Xf isolate infecting the sample at the subspecies and ST level. Table 1.…”

“…For the remaining Xf subsp. fastidiosa-infected samples, in which there was no amplification of the traC gene, the Ct values shown in Harper's qPCR ranged between 25 and 36. a Subspecies and sequence type (ST) were determined by MLST [8] or Nested-MLST [28] analysis. b All of the samples were obtained from fresh leaf petioles or stem portions of all analyzed hosts; except for samples of Prunus dulcis obtained from tree rings (TR) from a previous study [20].…”

Diseases Caused by Xylella fastidiosa in Prunus Genus: An Overview of the Research on an Increasingly Widespread Pathogen

“…In Majorca, both subspecies multiplex and fastidiosa have been detected in P. spumarius (Cesbron et al, 2020;Moralejo et al, 2020), and under experimental conditions, their role as a vector has been confirmed for bacterial transmission between almond and almond, vine to vine, and vine to almond tree (Cesbron et al, 2020;Moralejo et al, 2020). Other insects considered potential vectors in Majorca are N. campestris and N. lineatus (Olmo et al, 2021).…”