Endophytic Microorganisms Associated with Reversion of Silverleaf Disease Symptoms in Apple.

Silverleaf is caused by the fungus Chondrostereum purpureum, which produces wood necrosis and foliar silvering in woody plants. Field observations and studies in apple have shown the reversion of foliar symptoms. Because plants were clones and received identical agronomical management, it was hypothesized that reversion is driven by endophytic microbiota. Thus, the objectives of this study were to compare healthy, diseased, and reverted plants with respect to their physiology, endophytic microbial communities, antagonistic ability of their endophytes against C. purpureum, and defense genes expression. Water potential, stomatal conductance, chlorophyll content, and fluorescence were measured. Endophytic bacterial and fungal DNA were analyzed by denaturing gradient gel electrophoresis, and community richness and similarity were calculated. Wood cores were collected and bacterial and fungal endophytes were isolated and confronted with C. purpureum-virulent strains in dual-culture assays. Defense genes expression was measured by quantitative PCR. Results indicated that there were no differences in physiological parameters between healthy and reverted plants, except for fluorescence, and both type of plants differed from diseased ones. Bacterial and fungal community richness was similar in healthy and reverted plants and higher than in diseased ones. Endophytes from reverted and healthy plants showed high antagonism to C. purpureum. Furthermore, nonexpressor of pathogenesis-related gene 1 expression was upregulated in reverted plants, whereas phenylalanine ammonia lyase and polygalacturonase-inhibiting protein genes showed higher values in diseased plants. Overall, physiological, molecular, and microbial characteristics were similar between healthy and reverted plants, and both differed from diseased ones. Therefore, reversion of symptoms is associated with changes in the endophytic microbiota, which seems to be a promising source of biological control agents against C. purpureum.

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

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.