Pro Ligno (Mar 2015)
CHARACTERIZATION OF APHYLLOPHORALES CAUSING BROWN AND WHITE ROT IN WOODS: ANALYSIS OF ITS RESTRICTION POLYMORPHISM (PCR/RFLP)
Abstract
Intraspecific polymorphism exists for many fungus species, making their macroscopic identification difficult. Easily usable molecular markers providing access to information about variability have boosted the development of fungus diversity analyses. For this study, on wood-decaying Aphyllophorales, the main problem to be solved consisted in detecting any existence of intraspecific polymorphism in these fungi, in order to complete and clarify their identification. In order to characterize 98 strains in the CIRAD collection, we developed a rapid discriminant analysis method based on analysing the diversity of partial ribosomal DNA sequences independently of the macro- and microscopic morphological traits of the fruiting bodies required for the identification of genera and species. Adaptation of the Qiagen kit for higher plants made it possible to extract fungal DNA from a few milligrams of mycelium; with most of the strains studied, these DNA extracts enabled us to obtain an ITS amplificate of a size varying from 550 to 895 base pairs. We also found a large margin of error, of around 12%, for the DNA molecular weight readings on gel, leading us to prefer direct sequencing for precise determination of amplificate lengths. PCR-RFLP showed a good ability to characterize a given strain by providing different profiles obtained using several restriction enzymes. We thus demonstrated that studying ITS diversity using PCR-RFLP is a discriminant method making it possible to distinguish between strains. However, this method calls for the compilation of a database whose use remains partly subjected to interpretation, which cannot be used to work on a material unknown to the database. This simple, rapid technique is efficient and perfectly suitable for monitoring known strains in controlled trials.
