Complete genome assembly of the levan-positive strain PVFi1 of Pseudomonas savastanoi pv. savastanoi isolated from olive knots in Central Italy.

Pseudomonas savastanoi pv. savastanoi, the causal agent of olive knot disease, is a fluorescent Gram-negative bacterium classified, according to the specific LOPAT profile, as Ib. However, during the 90s, a number of atypical non-fluorescent levan-positive strains of Pseudomonas savastanoi pv. savastanoi have been unexpectedly isolated from olive knots in Central Italy. Since its first report, several studies were conducted on this species variant, but its genome sequence has never been reported. The complete genome sequence and two additional plasmids of PVFi1, a representative strain, were here obtained using a hybrid sequencing approach with both Oxford Nanopore Technology and Illumina sequencing. A thorough genomic analysis unravelled several genetic features of this peculiar strain, showing a transposase insertion downstream a fragmented copy of the levansucrase gene. The same features were previously reported on levan-negative Pseudomonas savastanoi pv. savastanoi strains. In addition, a second copy of the levansucrase gene fully equipped for a gene expression and comparable to the levan-positive Pseudomonas savastanoi pv. glycinea, may explain the levan-positive test. This result provides a solid genetic demonstration that the bacterial species Pseudomonas savastanoi contains either levan-positive or levan-negative strains, providing insights for an update of the related LOPAT classification.

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

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.