Développement d’une méthode de traçage mixte pour Aureobasidium pullulans souche Ach 1-1 et souche 1113-5,deux agents antagonistes des maladies fongiques des pommes en conservation

摘要

Aureobasidium pullulans strains Ach 1-1 and 1113-5 are two efficient biocontrol agents against Botrytiscinerea and Penicillium expansum on stored apples. In the present work, a monitoring system allowing their identification and quantification has been developed. The used methodology consisted in the development of specific molecular markers for both strains and a semi-selective medium which facilitate the isolation and counting colonies of strains Ach 1-1 and 1113-5. The random amplified polymorphic DNA (RAPD) technique has been applied to a collection of 15 strains of A. pullulans, including the Ach 1-1 and 1113-5 strains. Five specific RAPD fragments have been amplified for strain Ach 1-1 and three others for strain 1113-5. Among them, a fragment of 528 bp specific to strain Ach 1-1 (generated with the OPR-13 RAPD primer) and another one of 431 bp specific to strain 1113-5 (amplified with the OPQ-03 RAPD primer) have been selected, cloned, sequenced, and used to design sequence-characterized amplified region (SCAR) primers. Four different SCAR markers have been amplified: three specific to strain Ach 1-1 (157 bp, 190 bp and 388 bp) and one specific to strain 1113-5 (431 bp). These SCAR primers can clearly identify strains Ach 1-1 and 1113-5 among 14 strains of A. pullulans and among eight yeast strains commonly present on the surface of apples. Their selectivity has been also tested using DNA extracted from epiphytic microflora of the apple surface. As a semi-selective medium, the PDA medium supplemented with 0.5 mg/L Euparen, 1mg/L Sumico, 2.5mg/L Hygromycin B, 30 mg/L Streptomycin sulphate, and 1 mg/L Cycloheximide has been selected. This medium inhibited the development of the air microflora and appeared highly toxic for the epiphytic microflora of apple surface without altering the growth of the targeted strains Ach 1-1 and 1113-5. The combination of the plating technique on semi-selective medium and the identification of the colonies by PCR amplification of SCAR markers of 190 bp and 431 bp, respectively specific to A. pullulans strain Ach 1-1 and strain 1113-5, provides a valuable monitoring tool to specifically identify and quantify our targets strains. This monitoring tool has been used to evaluate their population dynamics on apple surface under cold storage conditions. Using this mixed monitoring method, we have highlighted that the population density of strain Ach 1-1 and strain 1113-5 on apple surface has been respectively 1.43x104 ufc/cm2 and 1.47x104 ufc/cm², two hours after their application on apples at the concentration of 107 ufc/mL. After two months storage at 4°C, these two populations densities were reduced by 58% and 66% respectively for the strain Ach 1-1 and strain 1113-5. This technique allowed also to demonstrate that the natural population of A. pullulans on apples surface represented 1% of the number of A. pullulans recovered on apples treated by strain Ach 1-1 and 8% in the case of strain 1113-5.