Development of a SCAR marker and a semi-selective medium for specific quantification of Pichia anomala strain K on apple fruit surfaces

Abstract

Abstract A washing procedure for apple fruit surfaces, and a semi-selective medium, were developed to assess the population dynamics of Pichia anomala strain K, a biocontrol agent against Botrytis cinerea and Penicillium expansum on fruit. The application of this plating technique allowed more than 99% recovery of strain K population on treated apples (by dipping them in a suspension of strain K at 107 cfu/ml). A strain K population decline of 51% was observed after 14 days of cold storage. To overcome the lack of specificity of the plating method, the RAPD technique was applied to a collection of 11 strains of P. anomala, including strain K. RAPD amplification with primer OPN13 produced a reproducible fragment of about 2000 bp, which was specific for strain K. Based on this DNA fragment, a SCAR marker of 262 bp was amplified with K1 and K2 primers for strain K as confirmed by Southern blot analysis, and was negative for a collection of 30 yeast strains including 21 P. anomala strains. A mixed monitoring method was developed and consisted of a combined plating technique on a semi-selective medium followed by a direct strain K-SCAR amplification without DNA extraction, on released DNA from resuspended white yeast colonies. This method was used on apples treated with strain K (107 cfu/ml) produced in Petri dishes, or in a bio-reactor (as a dry powder) with or without additives (2% CaCl2 and 0.2% beta-1,3-glucan) previously identified to enhance the strain K efficacy against B. cinerea. The percentages of recovered white colonies identified as strain K with the use of the specific SCAR marker ranged between 91 and 100%. The population densities reached similar levels of 1.1×104 and 0.7×104 cfu/cm2 on apples, 24 h after treatment with the powder formulation, without and with the stimulating agents, respectively. In contrast, a stimulating effect of glucan and CaCl2 on strain K population density was observed in apples treated with fresh cells produced in Petri dishes. Whatever the treatment, population densities diminished 1 week after application in cold storage conditions.