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

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 10 7 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 (10 7 cfu/ml) produced in Petri dishes, or in a bio-reactor (as a dry powder) with or without additives (2% CaCl 2 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)/10 4 and 0.7 )/10 4 cfu/cm 2 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 CaCl 2 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. #

Development of real-time PCR using Minor Groove Binding probe to monitor the biological control agent Candida oleophila (strain O)

“…The RAPD, already successfully used to discriminate a specific yeast strain from other natural strains present in the environment (De Clercq et al, 2003;Psomas et al, 2001), failed to identify a specific DNA fragment for strain O. A cluster analysis based on RAPD results showed a close relatedness between Means and standard deviation (S.D.)…”

“…After three successive subcultures under the same conditions, strains were inoculated in YEPD (yeast extract 1% (w/v), peptone 2% (w/v), dextrose 2% (w/v); Merck) at 25 8C during 24 h before DNA extraction. For monitoring by plating, a semi-selective medium, composed of PDA supplemented with 12.5 mg l À1 hygromycine B (Duchefa, Haarlem, The Nederlands), 0.25 mg l À1 Thiram (UCB, Brussel, Belgium) and 5 mg l À1 Sumico (AgrEvo, Machelen, Belgium) was used (De Clercq et al, 2003).…”

“…Both repetitions from the same storage room were pooled. The realtime PCR method was applied and compared to the dilution plate method (De Clercq et al, 2003). For plating, serial dilution series were prepared from 1 ml of KPB buffer and plated in triplicate.…”

“…The methods based on conventional PCR allow the specific detection of the microorganisms but fail to quantify its population. Hence, they have already been combined with serial dilution plating (Abassi et al, 1999;De Clercq et al, 2003;Leibniger et al, 1997). The application of quantitative competitive PCR, like PCR-ELOSA, has enabled direct quantification of the microorganisms (Johnsen et al, 1999;Pujol et al, 2004).…”

Development and application of a SCAR marker to monitor and quantify populations of the postharvest biocontrol agent Pantoea agglomerans CPA-2

“…This has led to the development of more specific and more rapid monitoring tools based on DNA amplification (Olive and Bean, 1999). Several of these molecular tools have been developed to specifically detect and monitor a variety of biocontrol agents (De Clercq et al, 2003;Pujol et al, 2004Pujol et al, , 2006Massart et al, 2005;Cordier et al, 2007). In spite of their specificity, these tools are not able to assess a biocontrol population.…”