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

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.