First Report of Knot Disease Caused by Pseudomonas savastanoi on Sweet Olive in Central Italy

Abstract

In April 2012 the presence of hyperplastic outgrowths on trunks, branches, and twigs of sweet olive plants, Osmanthus fragrans Lour (Fam. Oleaceae), was recorded in two ornamental hedges made up of five and four plants, respectively, in the city center of Montecatini (Pistoia-Italy). All sweet olive plants were seriously affected by the disease with outgrowths appearing either singly or close together, often forming a single mass that could extend up to 20 cm along the stems, occasionally surrounding the entire circumference. The symptoms observed on O. fragrans closely resembled those induced by the bacterium Pseudomonas savastanoi on Olea europea (common olive) and other plant species. Suspecting a bacterial origin of the disorder, young knots were collected from four diseased plants and used for bacterial isolation with standard techniques on nutrient sucrose agar medium (1). After 3 days of incubation at 26°C, non-levan forming colonies about 3 mm in diameter that were circular, convex, smooth, and cream colored with entire margins appeared on the surface of the agar medium. Purified isolates were gram negative, levan negative, oxidase negative, potato rot negative, arginine dihydrolase negative, showed a tobacco hypersensitive reaction, and tested positive to PCR screening for the presence of the iaaM (tryptophan-2-monooxygenase), iaaH (indoleacetamide hydrolase), ptz (isopentenyl transferase) (1) and iaaL (IAA-lysine synthethase) (3) genes. Three isolates were selected arbitrarily and further characterized by sequencing a fragment of the housekeeping genes rpoD (sigma factor 70) and pgi (phosphoglucose isomerase) (2). All sequenced gene fragments, of 620 bp and 552 bp for the rpoD and pgi genes, respectively, were identical to those of P. savastanoi pv. savastanoi strain NCPPB3335. The pathogenicity of the three isolates was verified on three O. fragrans plants and three Olea europea (cv. Frantoio) plants. Per each isolate, three 1-cm wounds were made on the branches of each plant using a sterile scalpel dipped in a bacterial suspension (1 × 108 CFU/ml). P. savastanoi pv. savastanoi PVFi-t2b isolated from olive was also inoculated as reference strain. After 30 days, all isolates including the reference strain induced typical knots on both plant species while no symptoms were observed on wounds inoculated with sterile water. Bacteria were reisolated from induced knots and Koch's postulates were confirmed. On the basis of biochemical tests, PCR screening, pathogenicity testing, and sequence analyses, the causal agent of knot disease on O. fragrans was identified as P. savastanoi. The potential susceptibility of O. aquifolium Sieb. to the causal agent of olive knot disease has been demonstrated in the past by means of artificial inoculations but interestingly, in the same trials, O. fragrans had tested negative (4). To the best of our knowledge, this is the world's first report of O. fragrans as natural host of P. savastanoi, which extends the growing list of cultivated and ornamental plant species affected by this phytopathogenic bacterium.