Development of SCAR markers and a semi-selective medium for the quantification of strains Ach 1-1 and 1113-5, two Aureobasidium pullulans potential biocontrol agents

Abstract

Aureobasidium pullulans strains Ach 1-1 and 1113-5 are two effective biocontrol agents against Botrytis cinerea and Penicillium expansum on stored apples. In the present work, a monitoring system allowing their identification and quantification was developed. The methodology used consisted of the development of both molecular markers and a semi-selective medium. The random amplified polymorphic DNA (RAPD) technique was applied to a collection of 15 strains of A. pullulans, including Ach 1-1 and 1113-5. Five specific RAPD fragments were 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) were selected, cloned, sequenced, and used to design sequence-characterized amplified region (SCAR) primers. Three different SCAR markers were amplified: two specific to strain Ach 1-1 (189 bp and 387 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 apple fruit surfaces. Their selectivity was also tested using DNA extracted from epiphytic microflora of the apple surface. As a semi-selective medium, PDA medium supplemented with 0.5 mg L −1 euparen, 1 mg L −1 sumico, 2.5 mg L −1 hygromycin B, 30 mg L −1 streptomycin sulphate, and 1 mg L −1 cycloheximide was selected. It 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 semi-selective medium and SCAR markers provides a valuable monitoring tool to specifically identify and quantify A. pullulans strain Ach 1-1 and strain 1113-5 and could be used in future studies to evaluate their population dynamics under various laboratory and practical conditions.