Detection of Pseudomonas savastanoi pv. savastanoi in Olive Plants by Enrichment and PCR

Abstract

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 Contains Two iaaL Paralogs, One of Which Exhibits a Variable Number of a Trinucleotide (TAC) Tandem Repeat

“…Amplicons obtained using both primer pairs from eight different P. savastanoi pv. savastanoi isolates (Table 1) were sequenced using primers IAAL-F and IAAL-R (29). The amplified DNA fragments were sequenced on both strands by the dideoxy sequencing termination method using an ABI Prism 310 (Applied Biosystem) sequencer.…”

“…savastanoi in olive plants is usually performed by an enrichment-PCR assay based on amplification of an internal 454-bp fragment of the iaaL gene followed by HaeIII digestion (29) or by nested PCR followed by dot blot hybridization (2). Although iaaL is widespread among plant-associated bacteria (10,16), this assay has been designed using the published sequence of iaaL from an oleander isolate (37) and appears to be specific for P. savastanoi (29).…”

The Mangotoxin Biosynthetic Operon ( mbo ) Is Specifically Distributed within Pseudomonas syringae Genomospecies 1 and Was Acquired Only Once during Evolution

“…Another example is the amplification of the gene iaaL, yielding a 454-bp fragment that allows the identification and detection of P. syringae pv. savastanoi strains (18). In the current study, we have developed a sensitive and specific method for the detection of mangotoxin-producing strains by a PCR that is based on the amplification of an mbo operon region in P. syringae.…”

Detecção de Erwinia psidii via enriquecimento em extrato de folhas de goiabeira e imunodifusão radial dupla

“…savastanoi (ex Smith 1908 Gardan et al 1992 e Acidovorax avenae subsp. citrulli (Schaad et al 1978) Willems et al 1992, assim como no estudo de bactérias não fitopatogênicas (Bultreys & Gheysen, 1999;Penyalver et al, 2000;Ghosh et al, 2004).…”