Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are available for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-operative-PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is micro-array technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of information for the future development of sensitive and specific detection techniques for these microorganisms.

SummaryPseudomonas savastanoi pv. savastanoi is a tumourinducing pathogen of Olea europaea L. causing olive knot disease. Bioinformatic analysis of the draft genome sequence of strain NCPPB 3335, which encodes 5232 predicted coding genes on a total length of 5856 998 bp and a 57.12% G + C, revealed a large degree of conservation with Pseudomonas syringae pv. phaseolicola 1448A and P. syringae pv. tabaci 11528. However, NCPPB 3335 contains twelve variable genomic regions, which are absent in all previously sequenced P. syringae strains. Various features that could contribute to the ability of this strain to survive in a woody host were identified, including broad catabolic and transport capabilities for degrading plant-derived aromatic compounds, the duplication of sequences related to the biosynthesis of the phytohormone indoleacetic acid (iaaM, iaaH) and its amino acid conjugate indoleacetic acid-lysine (iaaL gene), and the repertoire of strain-specific putative type III secretion system effectors. Access to this seventh genome sequence belonging to the 'P. syringae complex' allowed us to identify 73 predicted coding genes that are NCPPB 3335-specific. Results shown here provide the basis for detailed functional analysis of a tumour-inducing pathogen of woody hosts and for the study of specific adaptations of a P. savastanoi pathovar.

Pseudomonas savastanoi pv. savastanoi causes olive knot disease, which is present in most countries where olive trees are grown. Although the use of cultivars with low susceptibility may be one of the most appropriate methods of disease control, little information is available from inoculation assays, and cultivar susceptibility assessments have been limited to few cultivars. We have evaluated the effects of pathogen virulence, plant age, the dose/response relationship, and the induction of secondary tumors in olive inoculation assays. Most P. savastanoi pv. savastanoi strains evaluated were highly virulent to olive plants, but interactions between cultivars and strains were found. The severity of the disease in a given cultivar was strongly dependent of the pathogen dose applied at the wound sites. Secondary tumors developed in noninoculated wounds following inoculation at another position on the stem, suggesting the migration of the pathogen within olive plants. Proportion and weight of primary knots and the presence of secondary knots were evaluated in 29 olive cultivars inoculated with two pathogen strains at two inoculum doses, allowing us to rate most of the cultivars as having either high, medium, or low susceptibility to olive knot disease. None of the cultivars were immune to the disease.

The sequence of the gene iaaL of Pseudomonas savastanoi EW2009 was used to design primers for PCR amplification. The iaaL-derived primers directed the amplification of a 454-bp fragment from genomic DNA isolated from 70 strains of P. savastanoi, whereas genomic DNA from 93 non-P. savastanoi isolates did not yield this amplified product. A previous bacterial enrichment in the semiselective liquid medium PVF-1 improved the PCR sensitivity level, allowing detection of 10 to 100 CFU/ml of plant extract. P. savastanoi was detected by the developed enrichment-PCR method in knots from different varieties of inoculated and naturally infected olive trees. Moreover, P. savastanoi was detected in symptomless stem tissues from naturally infected olive plants. This enrichment-PCR method is more sensitive and less cumbersome than the conventional isolation methods for detection of P. savastanoi.Pseudomonas savastanoi and its pathovars savastanoi, fraxini, and nerii incite a disease of olive (Olea europaea L.), ash (Fraxinus excelsior L.), other Oleaceae plants and oleander (Nerium oleander L.) that is characterized by tumorous outgrowths (4,19). This development of knots is dependent on bacterial production of the phytohormone indoleacetic acid (IAA) and cytokinins (3,7,16,18). P. savastanoi can conjugate IAA with lysine to form 3-indoleacetyl-ε-L-lysine (IAA-lysine) (6). The two enzymes involved in IAA biosynthesis are tryptophan monooxygenase, which converts tryptophan to indoleacetamide, and indoleacetamide hydrolase, which catalyzes the conversion of indoleacetamide to IAA (12). The enzyme involved in the conversion of IAA to IAA-lysine is (indole-3-acetyl)-L-lysine synthethase (5). The genes for tryptophan monooxygenase (iaaM), indoleacetamide hydrolase (iaaH), and IAA-lysine synthethase (iaaL) reside on the 52-kb plasmid pIAA1 in the oleander P. savastanoi strain EW2009, and they have been sequenced (3,5,14,20). Sequence analysis revealed that iaaM and iaaH have significant similarity with homologous genes of other plant-associated bacteria (13,20). In contrast, to date, no nucleotide homologies have been found with the iaaL gene.Detection of P. savastanoi is currently based on bacterial isolation followed by pathogenicity tests and biochemical or serological techniques (2,8,17,21). These conventional methods are time-consuming and expensive, requiring bacterial isolation. We used the published sequence of iaaL (14) to design specific primers for amplification of this gene. We report here the development of a new sensitive and specific detection method for P. savastanoi based on amplification of iaaL after a bacterial enrichment. The developed enrichment-PCR assay can be applied to specifically detect low levels of P. savastanoi in inoculated and naturally infected plants.Specificity of the PCR assay. Seventy strains of P. savastanoi isolated from olive, oleander, ash, and jasmine (Jasminus officinalis L.) plants from different countries, 23 outgroup strains, and 70 saprophytic isolates from olive plants were used to te...

Pseudomonas savastanoi pv. savastanoi (Psv) is the causal agent of olive knot disease. The bacterium survives epiphytically and gains ingress through new wounds where infections and colonization result in knot formation. The natural spread of the bacterium and the subsequent appearance of the disease in olive orchards is poorly understood. The aim of this study was to monitor Psv epiphytic populations in inoculated plants with knots versus non-inoculated healthy trees within the same orchard over four years. Additionally, disease severity was measured in both inoculated and non-inoculated control trees. Epiphytic Psv populations moved from inoculated to non-inoculated trees, although average Psv populations were higher in inoculated trees. Olive knot severity increased over the course of the study in all treatments and cultivars, with all plants reaching a high level of disease by the end of the study. However, the delay in the onset of disease was longer in non-inoculated than in inoculated trees. Molecular typing of Psv isolates recovered from non-inoculated control trees confirmed that they were similar to the inoculated strain. These data demonstrate that Psv can move over short distances in olive orchards through dissemination of epiphytic bacteria and suggest a relationship between the presence of epiphytic Psv and the number of knots on trees.