Highly sensitive detection of Pseudomonas savastanoi pv. savastanoi in asymptomatic olive plants by nested-PCR in a single closed tube

Bertolini, Edson; Penyalver, Ramón; García, Alberto Urbaneja; Olmos, Antonio; Quesada, José Miguel; Cambra, M.; López, Marı́a M.

doi:10.1016/s0167-7012(02)00163-x

Cited by 37 publications

(20 citation statements)

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“…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).…”

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

Matas

Pérez‐Martínez

Quesada

et al. 2009

Appl Environ Microbiol

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In this study, Pseudomonas savastanoi pv. savastanoi isolates were demonstrated to contain two iaaL paralogs, which are both chromosomally located in most strains. Comparative analysis of iaaL nucleotide sequences amplified from these two paralogs revealed that one paralog, iaaL Psn , is 100% identical to iaaL from P. savastanoi pv. nerii, while the other paralog, iaaL Psv , exhibited 93% identity to iaaL from Pseudomonas syringae pv. tomato (iaaL Pto ). A 3-nucleotide motif (TAC) comprised of 3 to 15 repeats, which remained stable after propagation of the strains in olive plants, was found in iaaL Psv . Based on the observed nucleotide sequence variations, a restriction fragment length polymorphism assay was developed that allowed differentiation among iaaL Psn , iaaL Psv , and iaaL Pto. In addition, reverse transcriptase PCR on total RNA from P. savastanoi pv. savastanoi strains demonstrated that both iaaL Psv and iaaL Psn containing 14 or fewer TAC repeats are transcribed. Capillary electrophoresis analysis of PCR-amplified DNA fragments containing the TAC repeats from iaaL Psv allowed the differentiation of P. savastanoi pv. savastanoi isolates.Infections with Pseudomonas savastanoi pv. savastanoi (46), pv. fraxini, and pv. nerii (13) result in knots and galls on members of the Oleaceae family, and P. savastanoi pv. nerii also causes galls on oleander. Symptoms of infected trees include hypertrophy formation on the stems and branches and occasionally on the leaves and fruits. In the olive, the disease is considered to reduce both olive yield and productivity (41, 42); however, quantitative data on the impact of the disease on crop yield or crop quality are not available (19). Nevertheless, losses can be caused directly by localized infections that inhibit flowering and affect fruit development, as well as taste, and can be caused indirectly by weakening immature main leader branches that result in later damage to the tree frame (48).Currently, the only molecular P. savastanoi determinants known to be involved in knot development are the phytohormones indoleacetic acid (IAA) and cytokinins (1,15,33,38,45), as well as biosynthesis of a functional type III secretion system, encoded by the hrp-hrc gene clusters (43,44). Recently, a global genomic analysis of P. savastanoi pv. savastanoi plasmids allowed the identification of several putative virulence factors in the olive pathogen, including several type III secretion system protein effectors and a variety of genes encoding known Pseudomonas syringae virulence factors (32).The genetic determinants of P. savastanoi that are involved in the conversion of tryptophan (Trp) to IAA are Trp monooxygenase (encoded by the iaaM gene), which converts Trp to indoleacetamide (IAM), and IAM hydrolase (encoded by the iaaH gene), which catalyzes transformation of IAM to IAA (28). IAA can be further metabolized in P. savastanoi to an amino acid conjugate, 3-indole-acetyl-ε-L-lysine (IAA-lysine), through the action of the iaaL gene (14). In P. savastanoi pv. savastanoi and pv. ne...

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

Matas

Pérez‐Martínez

Quesada

et al. 2009

Appl Environ Microbiol

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“…sepedonicus (Lee et al, 1997b), Erwinia amylovora (Llop et al, 2000) and Pseudomonas savastanoi pv. savastanoi (Bertolini, 2003b).…”

Section: Nested-pcrmentioning

confidence: 99%

Advancements in the diagnosis of bacterial plant pathogens: An overview

Mondal¹

2013

Biotechnol. Mol. Biol. Rev.

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The timely detection and appropriate identification of causal agents associated with disease of crop plants or seeds are considered to be the most important issue in formulating the management strategies for plant diseases. This is particularly important for plant diseases of a bacterial nature, where disease-free planting materials is the only effective way to restrict the disease. The detection of bacterial pathogens still largely depends on cultural, morphological and biochemical properties. The protocol requires skilled taxonomical expertise and is also time and labor intensive. Moreover, it cannot discriminate between closely related strains of same bacterial pathogens. With the advent of polymerase chain reaction (PCR), nucleic acid based techniques have made the diagnostic procedures for plant pathogens, including bacteria, easier than the conventional approaches. The wide acceptability of nucleic acid based techniques is due to them being more sensitive, more accurate, more specific, and much faster than conventional techniques. The serology-based diagnoses are very often preferred over nucleo-based techniques as they are more user-friendly and less cumbersome, besides being sensitive, accurate, specific, and much faster than conventional techniques. This review critically analyzes the recent developments and scope of various nucleic acid-and serology-based techniques for the diagnosis of bacterial plant pathogens.

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“…Lavermicocca & Surico (1987) simultaneously analyzed Psv populations on olive tree leaves and stems for the first time and during one year, reporting higher frequencies of Psv isolation in stems than in leaves with relatively high epiphytic Psv populations in July (about 10 5 cfu/cm 2 ) and only 10 cfu/cm 2 in September and March. However, in Spain no significant differences were found between either leaves or stems with respect to the number of analyzed samples where Psv was isolated, detected by PCR, or regarding the average Psv populations over several years (Bertolini et al, 2003a;Quesada et al, 2007;Quesada et al, 2010aQuesada et al, , 2010b. Given that in such studies the Psv number were evaluated on stems after they were cut into pieces, some endophytic Psv could be also counted (Penyalver et al, 2006).…”

Section: Epiphytic Populations Of Psvmentioning

confidence: 99%

“…Given that in such studies the Psv number were evaluated on stems after they were cut into pieces, some endophytic Psv could be also counted (Penyalver et al, 2006). Furthermore, both types of plant material (stems and leaves) should be analyzed from symptomless shoots to make the evaluation of Psv populations in the phyllosphere more accurate (Bertolini et al, 2003a;Quesada et al, 2007). Psv was also isolated from the surface of olive fruits, but at lower frequency than from leaves, reaching a high Psv population size in September (10 6 bacteria / g fresh weight) (Lavermicocca & Surico, 1987).…”

Section: Epiphytic Populations Of Psvmentioning

confidence: 99%

Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria: The Case of Olive Knot Disease Caused by Pseudomonas savastanoi pv. savastanoi

Quesada¹,

Penyalver²,

López³

2012

Plant Pathology

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Highly sensitive detection of Pseudomonas savastanoi pv. savastanoi in asymptomatic olive plants by nested-PCR in a single closed tube

Cited by 37 publications

References 18 publications

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

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

Advancements in the diagnosis of bacterial plant pathogens: An overview

Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria: The Case of Olive Knot Disease Caused by Pseudomonas savastanoi pv. savastanoi

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