A new inclusive MLVA assay to investigate genetic variability of Xylella fastidiosa with a specific focus on the Apulian outbreak in Italy

Abstract

The Olive Quick Decline Syndrome by Xylella fastidiosa subspecies pauca is among the most severe phytopathological emergencies nowadays. In few years, the outbreak devastated olive groves in Apulia (Italy), potentially endangering the entire Mediterranean basin. This research aimed to develop a multiple locus VNTR analysis assay, a molecular tool to differentiate between populations of the pathogen. It has already been successfully applied to different X. fastidiosa subspecies from various plant hosts. The previously published TR loci, together with a set of new design, have been tested in silico on the genome of the Apulian De Donno strain. The resulting selection of 37 TR loci was amplified on the genomic DNAs of the Apulian strains AND from representatives of X. fastidiosa subspecies, and directly on DNA extracted from infected plants. The assay clearly discerned among subspecies or even sequence types (ST), but also pointed out variants within the same ST so as to provide more detailed information on the dynamics and pathogen diffusion pathways. Its effective application even on total DNAs extracted from infected tissues of different host plants makes it particularly useful for large-scale screening of infection and for the strengthening of containment measures.

Figure 1

Hierarchical clustering of 15 Xylella fastidiosa strains from CFBP based on MLVA results and obtained using Bruvo’s genetic distance, UPGMA as agglomerative algorithm, and 1,000 bootstrap replications. Backgroud colors refers to subspecies. Only bootstrap percentages above 80% are reported.

Figure 2

Hierarchical clustering of Xylella fastidiosa strains from both CFBP and from the Apulian oubreak, and of total DNAs from infected plants in Apulia, based on MLVA results and obtained using Bruvo’s genetic distance, UPGMA as agglomerative algorithm, and 1,000 bootstrap replications. Backgroud colors refers to subspecies. Only bootstrap percentages above 80% are reported.

Figure 3

Minimum spanning tree (MST) of Xylella fastidiosa strains. (a) the color of the haplotypes corresponds to the host plant species; (b) the color of the haplotypes corresponds to their geographic origin at locality scale. Numbers on lines connecting two haplotypes represents the number of variable loci out of 37.