Abstract
For rapid and sensitive detection of Xanthomonas fragariae, the causal agent of angular leaf spot of strawberry, a nested polymerase chain reaction (Pcr) was developed. A specific fragment was amplified from X fragariae Dna using primers 245A and 245B, described previously (Pooler et al. 1996). The fragment was sequenced and internal primers suitable for nested Pcr were selected. Using this internal pair of primers, a specific fragment was amplified from all of the 14 X. fragariae isolates tested but no fragment was amplified from X. campestris isolates or from unidentified bacteria, which were isolated from strawberry plants. Whereas detection limit of simple Pcr was 20 pg Dna per reaction, nested Pcr was up to a hundred times more sensitive and even from 200 fg Dna per reaction, a fragment was amplified. In addition, fragments amplified by nested Pcr were always clearly detectable on agarose gels, whereas fragments amplified by simple Pcr were only visible as faint bands when template concentrations decreased. Applicability of nested Pcr was tested on samples from naturally infected fields and from nursery plants showing no symptoms by visible inspection. In symptomatic plants, X. fragariae was regularly detected in leaves, especially in old leaves. In the crown, it was only occasionally detected. Simple Pcr was sufficient for confirmation of symptomatic infection and sometimes for detection of X. fragariae in symptomless parts of symptom-bearing plants and in latently infected nursery plants, respectively. However, using more sensitive nested Pcr, latent infections were more frequently detected and Pcr fragments were more clearly visible on agarose gels.
Zusammenfassung
Fur einen möglichst schnellen und sensitiven Nachweis von Xanthomonas fragariae, dem Erreger der Eckigen Blattfleckenkrankheit an Erdbeere, wurde eine verschachtelte Polymerase-Kettenreaktion (nested Pcr) entwickelt. Mit den Primern 245A und 245B (Pooler et al. 1996) wurde ein spezifisches Fragment von X. fragariae Dna amplifiziert. Das Fragment wurde sequenziert und innere Primer, die sich fur eine nested Pcr eignen, wurden ausgewahlt. Mit diesem inneren Primer-Paar wurde an allen 14 getesteten X. fragariae Isolaten ein spezifisches Fragment amplifiziert, an X. campestris Pathovaren und an weiteren nicht identifizierten Bakterien, die von Erdbeerpflanzen isoliert worden waren, wurde dagegen kein Fragment amplifiziert. Während die Nachweisgrenze der einfachen Pcr bei 20 pg Dna pro Pcr-Ansatz lag, wurde mit der neu entwickelten nested Pcr eine bis zu 100 x höhere Sensitivität erzielt und auch mit 200 fg Dna/Ansatz ein nachweisbares Fragment amplifiziert. Darüber hinaus war das Fragment, das mit der nested Pcr amplifiziert wurde stets deutlich auf dem Agarosegel sichtbar, während bei der einfachen Pcr die Bandenstärke des amplifizierten Fragments mit abnehmender Matritzen-Konzentration abnahm. Die Praxistauglichkeit der nested Pcr wurde an Proben aus natürlich befallenen Feldbeständen und an äußerlich gesund aussehenden Jungpflanzen geprüft. X. fragariae wurde regelmäßig in Blättern, insbesondere in älteren Blättern, nachgewiesen. Im Rhizom wurde das Bakterium nur gelegentlich nachgewiesen. Die einfache Pcr war für den Nachweis von Befallssymptomen ausreichend und teilweise wurde auch an symptomlosen Pflanzenteilen von Symptome aufweisenden Pflanzen bzw. an latent infizierten Jungpflanzen X. fragariae nachgewiesen. Mit der sensitiveren nested Pcr wurde latenter Befall häufiger nachgewiesen und die PCR Fragmente waren auf dem Agarosegel deutlicher sichtbar.
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Zimmermann, C., Hinrichs-Berger, J., Moltmann, E. et al. Nested Pcr (polymerase chain reaction) for detection of Xanthomonas fragariae in symptomless strawberry plants. J Plant Dis Prot 111, 39–51 (2004). https://doi.org/10.1007/BF03356131
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DOI: https://doi.org/10.1007/BF03356131