A PCR-Based Method for the Rapid Detection of Chondrostereum purpureum in Apple

Abstract

Silverleaf caused by the basidiomycete Chondrostereum purpureum affects numerous woody species, including fruit tree crops like apple, resulting in wood necrosis and foliar silvering. There are no curative alternatives for this disease, and its management is by prevention methods. Therefore, the aim of this study was to develop a rapid diagnostic tool for the detection and identification of C. purpureum directly from woody tissues to help distinguish the pathogen from other basidiomycetes that are commonly found on apple. The silverleaf pathogen was isolated from different hosts and locations, and Koch’s postulates were performed by inoculating the isolates on apple cuttings and measuring internal necrosis. A previously described APN 1 pair of primers specificity was also tested against 25 C. purpureum isolates in this study, using other wood rotting species as negative controls. Seven virulent isolates were inoculated on apple cuttings, and DNA was extracted from the cuttings’ sawdust and amplified using APN 1, after 22 days of incubation. To prove the efficiency of the method in the field, DNA from healthy nursery plants inoculated with two virulent isolates, and naturally infected plants showing different levels of foliar symptoms, were tested. Presence of the fungus was verified by reisolation on APDA in all assays. Koch’s postulates indicated that all C. purpureum isolates were pathogenic, showing different virulence levels, and APN 1 primers were able to discriminate them from other basidiomycetes. The method was also able to detect C. purpureum from artificially inoculated plants as well as naturally infected ones, demonstrating that the protocol may become a rapid minimally destructive diagnostic tool to detect the pathogen without the need to isolate it from tissues, and thus taking measures to prevent its dissemination.