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 |
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| Institution: | 1. Unité de Phytopathologie, Faculté des Sciences Agronomiques de Gembloux, Passage des Déportés 2, B-5030 Gembloux, Belgium;2. Laboratoire de Biotechnologie et Amélioration des Plantes, Faculté des Sciences, Université Moulay Ismail, BP 4010, 50000 Meknès, Morocco;3. Laboratoire de Phytobactériologie, Institut National de la Recherche Agronomique, BP 579, 50000 Meknès, Morocco;4. Unité de Biotechnologie, Laboratoire de Microbiologie, Faculté des Sciences, Université Libre de Bruxelles, CP 700, Avenue Emile Gryson 1, B-1070 Bruxelles, Belgium;1. Center for Food Safety and Department of Food Science, University of Arkansas, Fayetteville, AR 72704, USA;2. Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72704, USA;1. Department of Biological Sciences, Sungkyunkwan University (SKKU), Suwon 440-746, South Korea;2. College of Pharmacy, Korea University, Sejong City 339-700, South Korea;3. Department of Biological Sciences, Sookmyung Women''s University, Seoul 04310, South Korea |
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| 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. |
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