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. #
Three Gram-negative, yellow-pigmented strains were isolated from the rhizospheres of Spathiphyllum plants grown in a compost-amended potting mix. The strains showed biological control activity towards the root-rot plant pathogen Cylindrocladium spathiphylli, and were characterized to determine their taxonomic position. Cells of the strains were non-motile rods, and the strains were oxidase-and catalase-positive and unable to ferment most sugars tested. The three strains showed differences in growth temperature range, optimal growth temperature and some biochemical reactions. The majority of the fatty acids were branched, and large amounts of 15 : 0 iso and 17 : 1 iso v9c were present. The 16S rRNA gene sequence (1497 bp sequence similarity was observed for other members of the Gammaproteobacteria. The mean DNA-DNA reassociation value for the three strains was 100 % and was 25 % when the strains were compared with DNA from R. fulvus LMG 23003
A real-time PCR assay using a 3V -Minor Groove Binding (MGB) probe was developed for specific detection and monitoring of Candida oleophila (strain O), a biocontrol agent against Botrytis cinerea and Penicillium expansum, on harvested apples. The application of the RAPD technique on C. oleophila strains followed by reproducible sequence characterized amplified region (SCAR) amplifications allowed the identification of a semi-specific fragment of 244 bp, observed in the profiles of strain O and three other C. oleophila strains. After sequencing, polymorphisms (3%) were observed between the strain O sequence and the three other sequences. A 3V -Minor Groove Binding probe was designed to specifically match a region of the strain O sequence and was able to discriminate a single base mutation or a two-base difference in the corresponding sequences of the non-target strains. This specific detection method was applied to monitor strain O population, recovered by a washing buffer, from harvested apples. Population densities were calculated using an external standard curve consisting in a serial dilution of strain O cells in the washing buffer from untreated apples. Linearity in the standard curve was kept between 1.64Â10 2 and 1.64Â10 5 cfu cm À2 of apple surface. During a first practical experiment, the calculated population densities were similar to those obtained by plating on semi-selective media. This new real-time PCR method is a promising tool to monitor quickly and specifically strain O population on apple surface in middle-or large-scale experiments. D
The yeast Pichia anomala strain K was selected in Belgium from the apple surface for its antagonistic activity against post-harvest diseases of apples. The efficacy of this strain against P. expansum was evaluated in the laboratory in three scenarios designed to mimic practical conditions, with different periods of incubation between biological treatment, wounding of fruit surface, and pathogen inoculation. Higher protection levels and higher final yeast densities were obtained when the applied initial concentration was 1×10 8 cfu ml −1 than when it was only 1×10 5 cfu ml −1 . The protection level correlated positively with the yeast density determined in wounds and was influenced by apple surface wetness. In orchard trials spanning two successive years, biological treatment against P. expansum, based on a powder of P. anomala strain K (1×10 7 cfu ml −1 ), β-1,3-glucans (YGT 2 g l −1 ), and CaCl 2 .2H 2 0 (20 g l −1 ), was applied to apples pre-or post-harvest under practical conditions and its effect compared with standard chemical treatments. The first year, the highest reduction (95.2%) against blue decay was obtained by means of four successive fungicide treatments and the next-highest level (87.6%) with pre-harvest high-volume spraying of the threecomponent mixture 12 days before harvest. The second year, the best results were obtained with post-harvest Sumico (carbendazim 25% and diethofencarb 25%) treatment and post-harvest biological treatment, both by dipping the apples, 88.3 and 56.3% respectively. A density threshold of 1×10 4 cfu cm −2 of strain K on the apple surface seemed to be required just after harvest for high protective activity, whatever the method and time of application. In the case of pre-harvest biological treatments, variations in meteorological conditions between the 2 years may have considerably affected strain K population density and its efficacies.
Aims: Determination of the minimum requirements (time–temperature relationship and moisture content) that are needed for a sufficient eradication of an indicator organism. Methods and Results: To determine the hygienic safety of composting processes, the indicator organism Salmonella enterica ssp. enterica serotype Senftenberg strain W 775 (further abbreviated as W 775) was artificially inoculated on a meat carrier and monitored subsequently. Different types of composting processes, e.g. composting in enclosed facilities, in open‐air and in‐vessel composting, were investigated. The waste feedstocks used in this work were either biowastes (i.e. vegetable, fruit and garden wastes; also called source‐separated household wastes) or pure garden wastes. Beside these large‐scale trials, we also conducted some lab experiments in order to determine the impact of temperature, moisture content and the presence of an indigenous microflora on the eradication of W 775. We found the temperature to be the most important parameter to eradicate W 775 from compost. When the temperature of the compost heap is 60°C and the moisture content varies between 60–65%, W 775 (108 CFU g−1) will be inactivated within 10 h of composting. The moisture content is, beside temperature, a second parameter that influences the survival of W 775. When the water content of the composting materials or meat carriers is reduced, a higher survival rate of W 775 was observed (survival rate increases 0·5 log10 unit when there is a reduction of 5% in moisture content). In addition, other parameters (such as microbial antagonism, toxic compounds, etc.) have an influence on the survival of W 775 as well. Conclusions: Our study demonstrates that all types of composting processes tested in this work were sufficient to eradicate W 775 providing that they are well managed in terms of temperature and moisture content. Significance and Impact of the Study: To give a better view on the parameters of importance for the eradication of W 775 during composting.
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