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.
Highlights
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
Among fully typed plant samples using the nested-MultiLocus Sequence Typing (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
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
Summary
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]. This member of the Xanthomonadaceae family inhabits the xylem of its host plants [9] and is naturally transmitted by insects from plants to plants
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