Efficient Detection of Erwinia Amylovora by PCR-Analysis

Bereswill, Stefan; Pahl, Armin; Bellemann, Peter; Berger, F.; Zeller, W.; Geider, Klaus

doi:10.17660/actahortic.1993.338.6

Cited by 20 publications

(29 citation statements)

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“…PCR analyses from this study and from a previous study (Roselló et al, 2006) showed negative results for the novel isolates with the primers targeting plasmid pEA29 of E. amylovora (Bereswill et al, 1992;Llop et al, 2000;McManus & Jones, 1995) and with some primers based on chromosomal sequences of E. amylovora, such as primers Ea71/72 (Guilford et al, 1996). Nevertheless, with primers such as amsBL and amsBR (Bereswill et al, 1995) and EaF and EaR (Maes et al, 1996) targeting other chromosomal sequences, the amplification was positive, indicating similarities in some chromosomal sequences.…”

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confidence: 47%

“…Amplification is observed when using the primers EaF-EaR (Maes et al, 1996), whose targets are 23S rRNA gene sequences, and primers from other chromosomal sequences such as amsBL-amsBR (Bereswill et al, 1995). PCR analyses give negative results using the E. amylovora pEA29 primers (Bereswill et al, 1992;Llop et al, 2000;McManus & Jones, 1995) and using other primers based on E. amylovora chromosomal sequences, such as the Ea71 primers (Guilford et al, 1996). The isolates contain a plasmid of similar size to the pEA29 of E. amylovora.…”

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confidence: 99%

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Erwinia piriflorinigrans sp. nov., a novel pathogen that causes necrosis of pear blossoms

López

Roselló

Llop

et al. 2011

International Journal of Systematic and Evolutionary Microbiology

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Eight Erwinia strains, isolated from necrotic pear blossoms in Valè ncia, Spain, were compared with reference strains of Erwinia amylovora and Erwinia pyrifoliae, both of which are pathogenic to species of pear tree, and to other species of the family Enterobacteriaceae using a polyphasic approach. Phenotypic analyses clustered the novel isolates into one phenon, distinct from other species of the genus Erwinia, showing that the novel isolates constituted a homogeneous phenotypic group. Rep-PCR profiles, PCR products obtained with different pairs of primers and plasmid contents determined by restriction analysis showed differences between the novel strains and reference strains of E. amylovora and E. pyrifoliae. Phylogenetic analysis of 16S rRNA, gpd and recA gene sequences showed that the eight novel strains could not be assigned to any recognized species. On the basis of DNA-DNA hybridization studies, the novel isolates constituted a single group with relatedness values of 87-100 % to the designated type strain of the group, CFBP 5888 T . Depending on the method used, strain CFBP 5888 T showed DNA-DNA relatedness values of between 22.7 and 50 % to strains of the closely related species E. amylovora and E. tasmaniensis. The DNA G+C contents of two of the novel strains, CFBP 5888 T and CFBP 5883, were 51.1 and 50.5 mol%, respectively. On the basis of these and previous results, the novel isolates represent a novel species of the genus Erwinia, for which the name Erwinia piriflorinigrans sp. nov. is proposed. The type strain is CFBP 5888 T (5CECT 7348 T ).Necrotic blossoms were observed in Ercolini (Coscia) and Tendral pear trees growing in Valencia, Spain, from which an unknown bacterium was consistently isolated in 1999 and during the following two years. Infected blossoms were similar in appearance to those affected by the disease fire blight, caused by Erwinia amylovora, but only occurred in spring and the blossoms were the only part of the trees that were affected, unlike the disease caused by E. amylovora (Roselló et al., 2002(Roselló et al., , 2006. The isolated bacterium was subjected to phenotypic and molecular characterization and, although it was recognized as belonging to the genus Erwinia, many of its characteristics differed from those of other pathogenic and non-pathogenic species of the genus Erwinia that have been isolated from pear trees, including E. amylovora (Hauben et al., 1998), Erwinia billingiae (Mergaert et al., 1999), Erwinia pyrifoliae (Kim et al., 1999) and Erwinia tasmaniensis (Geider et al., 2006). The novel isolates provoked a hypersensitivity response when they were allowed to infiltrate tobacco and tomato leaves. Their 3These authors contributed equally to this work.Abbreviations: ERIC, enterobacterial repetitive intergenic consensus; ML, maximum-likelihood; MP, maximum-parsimony; NJ, neighbourjoining; REP, repetitive extragenic palindromic; UPGMA, unweighted pair group method with arithmetic mean.

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confidence: 47%

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confidence: 99%

Erwinia piriflorinigrans sp. nov., a novel pathogen that causes necrosis of pear blossoms

López

Roselló

Llop

et al. 2011

International Journal of Systematic and Evolutionary Microbiology

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“…The mixture was centrifuged, the supernatant was discarded, and the dried pellet was resuspended in 200 l of sterile water. Five microliters of DNA extract was used for standard PCRs (2,11,23,27) and for the first round of the two-tube nested-PCR assay (27), while 1 l was used for the nested PCR in a single closed tube. All the analyses were performed twice.…”

Section: Methodsmentioning

confidence: 99%

“…The criteria we used for selecting the external and internal primer pairs were (i) the external primer pair should amplify a fragment large enough to permit the design of an appropriate internal couple, (ii) annealing temperatures of the primer pairs should allow for the separation of both PCRs only by this parameter, and (iii) high sensitivity of the primers, to increase as much as possible the detection threshold of the nested PCR in one tube. The standard PCRs were performed as described by Bereswill et al (2), using primers A and B; by McManus and Jones (27), using primers AJ75-AJ76; by Maes et al (23), using primers EAF-EAR; and by Guilford et al (11), using primers EA71-EA72. After some sensitivity assays, we choose as external primers those designed by McManus and Jones (27), which were used at an annealing temperature of 72°C.…”

Section: Methodsmentioning

confidence: 99%

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Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material

Llop

Bonaterra

Peñalver

et al. 2000

Appl Environ Microbiol

127

109

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A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 l of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.

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Expression of ﬁreﬂy luciferase gene in Erwinia amylovora

et al. 2003

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In this study we describe an efficient stable genetic transformation of the phytopathogenic bacterium Erwinia amylovora using a recombinant expression vector encoding the firefly luciferase gene of Photinus pyralis, which is further controlled by IPTG-inducible promoter. Stably transformed E. amylovora cells maintain the same infectivity as the wild-type strain and, after induction with IPTG, produce luciferase. Luminescence produced by the action of luciferase on an exogenous substrate was easily detectable by a simple and rapid bioluminescent assay (BL). The transformed E. amylovora strain maintains the same high emission level, even after passage in pears, until about 15 days post-infection. Our findings therefore show that the luciferase assay can be conveniently used to follow the bacterial movement in plant tissue and its dissemination in controlled environments.

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Efficient Detection of Erwinia Amylovora by PCR-Analysis

Cited by 20 publications

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Erwinia piriflorinigrans sp. nov., a novel pathogen that causes necrosis of pear blossoms

Erwinia piriflorinigrans sp. nov., a novel pathogen that causes necrosis of pear blossoms

Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material

Expression of ﬁreﬂy luciferase gene in Erwinia amylovora

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