Nested Pcr (polymerase chain reaction) for detection of Xanthomonas fragariae in symptomless strawberry plants

Zimmermann, Christian; Hinrichs-Berger, J.; Moltmann, E.; Buchenauer, H.

doi:10.1007/bf03356131

Cited by 21 publications

(29 citation statements)

References 21 publications

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“…The sensitivity of this LAMP assay was also slightly lower when compared to the qPCR method for X . fragariae detection (Vandroemme et al ., ), but was in the same detection range as the previously designed nested PCR (Zimmermann et al ., ; Moltmann & Zimmermann, ). Although the qPCR‐based method (Vandroemme et al ., ) allowed the detection of very low concentrations of X. fragariae in strawberry leaf samples (300 CFU mg −1 of leaf tissue), the drawback is that the method involves a rigorous DNA extraction and so is not very user‐friendly.…”

Section: Discussionmentioning

confidence: 99%

A diagnostic tool for improved detection of Xanthomonas fragariae using a rapid and highly specific LAMP assay designed with comparative genomics

et al. 2017

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Molecular diagnostics of plant pathogens are crucial to prevent disease spread and to enhance food quality and security. A comparative genomics approach using genomes of different Xanthomonas species and pathovars was applied to identify highly specific targets in the genome of Xanthomonas fragariae, the causal agent of angular leaf spot of strawberry, listed under quarantine regulations in Europe. A reliable and sensitive loop‐mediated isothermal amplification (LAMP) assay was designed using a unique marker, providing a highly specific and rapid detection technique, convenient for on‐site detection. Specificity of the designed assay was tested on 37 strains from a culture collection of X. fragariae, 82 strains of other Xanthomonas species and pathovars and 11 strains of other bacterial genera isolated from strawberry leaves. A detection limit of 102 fg was achieved, approximating to 20 genome copies per reaction. When performing analyses with crude plant material, a consistent lower detection efficiency of 102 CFU mL−1 was achieved. The LAMP assay designed in this study was adapted to work on crude plant material without any prior extensive extraction steps or incubation period; moreover, it does not require advanced analytical knowledge or a fully equipped laboratory. Results were produced within 7–20 min, depending on the pathogen concentration, thus providing a high‐throughput and user‐friendly method for detection and screening of plant material in support of quarantine regulations.

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Section: Discussionmentioning

confidence: 99%

A diagnostic tool for improved detection of Xanthomonas fragariae using a rapid and highly specific LAMP assay designed with comparative genomics

et al. 2017

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“…Amplification products were obtained in a nested PCR with primer pairs 245A, 245B and 245.5, 245.267 as described in Zimmermann et al . () and were separated via agarose gel electrophoresis. Fragments of 286 bp indicate presence of X.f.…”

Section: Resultsmentioning

confidence: 55%

Resistance and systemic dispersal of Xanthomonas fragariae in strawberry germplasm (Fragaria L.)

et al. 2014

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The angular leaf spot disease caused by Xanthomonas fragariae is an important plant disease with major impact for the strawberry nursery industry. Currently there is no plant protection product available for controlling the disease effectively. Planting of resistant cultivars seems to be promising, but all commercially used cultivars are susceptible and no donor with a high level of resistance has yet been found. Therefore, a total of 145 genotypes from the Fruit Genebank Dresden (Germany) were evaluated for resistance to X. fragariae by artificial inoculation. Six genotypes were classified as partly resistant, out of which only two (US4808 and US4809) are octoploid. Fragaria vesca f. alba, Fragaria nilgerrensis ‘Yunnan’, F. vesca ‘Illa Martin’ and F. moschata ‘Bauwens’ were also classified as partially resistant, but they are only of limited use for breeding because of their variable ploidy level. Fully resistant genotypes could not be detected. The systemic dispersal of the bacteria in strawberry plants was investigated after inoculation of leaves with X. fragariae strain XF3.9.C and the GFP‐tagged strain XF3.9.C(pKAN). The systemic spread was evaluated after 3, 7, 14 and 28 days post‐inoculation (dpi) by nested PCR and fluorescence microscopy. After 3 dpi, X. fragariae could be found in all tissues tested including the inoculated leaf, its petiole, the rhizome, the heart bud up to the youngest fully expanded leaf and its petiole. The systemic spread was also detectable in partially resistant genotypes.

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“…The nested PCR assay, as described by Zimmermann et al . (2004), gave positive results for all Xf strains.…”

Section: Resultsmentioning

confidence: 99%

“…Normal PCR is inadequate because of too low sensitivity. Zimmermann et al . (2004) proposed a sensitive nested PCR assay.…”

Section: Discussionmentioning

confidence: 99%

Sensitive real‐time PCR detection of Xanthomonas fragariae in strawberry plants

Vandroemme

Baeyen²,

Vaerenbergh³

et al. 2008

Plant Pathology

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Xanthomonas fragariae , the causal agent of angular leaf spot on strawberry, is a quarantine organism in strawberry propagation material in the European Union. For the reliable screening of planting material for latent infections, a realtime PCR assay based on Taqman® chemistry for the detection of X. fragariae was developed. Primers and probe sequences were based on a DNA fragment amplified by a previously reported X. fragariae -specific technique. The sequence of this genomic fragment had no significant similarity with any published GenBank sequence. Specificity of the designed assay was tested with an extended range of X. fragariae collection strains and isolates, with other Xanthomonas spp. and with unidentified bacterial isolates from strawberry plants. A nested PCR, which until now was the reference method for sensitive detection in planta , cross-reacted with the reference strain of Xanthomonas campestris pv. campestris . In combination with an elaborated DNA extraction procedure, the Taqman® PCR enabled reliable detection down to 300 colony forming units in a 100 mg strawberry leaf sample. The assay offers a new tool for epidemiological research and for sanitary control of plant material with low level or latent infections of X. fragariae .

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Nested Pcr (polymerase chain reaction) for detection of Xanthomonas fragariae in symptomless strawberry plants

Cited by 21 publications

References 21 publications

A diagnostic tool for improved detection of Xanthomonas fragariae using a rapid and highly specific LAMP assay designed with comparative genomics

A diagnostic tool for improved detection of Xanthomonas fragariae using a rapid and highly specific LAMP assay designed with comparative genomics

Resistance and systemic dispersal of Xanthomonas fragariae in strawberry germplasm (Fragaria L.)

Sensitive real‐time PCR detection of Xanthomonas fragariae in strawberry plants

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