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.
Highlights
Xylella fastidiosa is a Gram-negative phytopathogenic bacterium belonging to the Xanthomonadaceae family, which is able to infect and cause diseases on 563 plant species[1]
Regarding the markers described by Lin et al.[33], it was not possible to find the forward primer of OSSR-12 marker; OSSR-19 marker has an SNP in the reverse primer; only a single TR was found for CSSR-4, CSSR-6, GSSR-14, GSSR-15, GSSR-19, GSSR-20 markers; TR is absent for CSSR-12 and CSSR-13 markers; the primers of CSSR-16 marker show multiple annealing sequences; a different TR sequence and one SNP were found in the reverse primer for ASSR-16 marker, one SNP was found in the forward and one in the reverse primer of both ASSR-19 and GSSR-4 markers
multilocus sequence typing (MLST) is the method of choice for Xylella fastidiosa genotyping at subspecies level and beyond 44, based on the detection of SNPs included in seven constitutive g enes[45]
Summary
Xylella fastidiosa is a Gram-negative phytopathogenic bacterium belonging to the Xanthomonadaceae family, which is able to infect and cause diseases on 563 plant species[1]. It has been demonstrated that the introduction of foreign X. fastidiosa strains in new geographical areas and subsequent recombination with endemic strains may be relevant in increasing the genetic variability, shifting the target host and inducing new crop d iseases[22] In this regard, multilocus sequence typing (MLST), based on the identification of nucleotide sequence differences in seven housekeeping genes, has been applied to study the evolution of X. fastidiosa and its subspecies[6, 23]. Despite massive efforts in containment measures, nowadays thousands of hectares of olive groves in Apulia are sevrely affected by the syndrome and the spread is still going on In this situation, a fine-tuned genotyping of the strains responsible for the outbreak is crucial to understand where the disease comes from, how it moves in the infected areas and to monitor if new variants would appear in this scenario or if the present type undergoes evolutionary forces that can lead to new variants. A further search for new VNTR loci was independently conducted on the same genome, aiming to obtain a final selection of markers to be used in a novel, inclusive MLVA assay capable to generate novel and deeper information about the genetic diversity of this subspecies, with specific reference to the Italian outbreak in Apulia
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