Improvement of a dry formulation of Pseudomonas fluorescens EPS62e for fire blight disease biocontrol by combination of culture osmoadaptation with a freeze-drying lyoprotectant.

AIMS: To study the effect of lyoprotectants and osmoadaptation on viability of Pseudomonas fluorescens EPS62e during freeze-drying and storage and to evaluate the formulation in terms of efficacy in biocontrol and fitness on pear flowers. METHODS AND RESULTS: A wettable powder formulation of a biocontrol agent of fire blight was optimized by means of lyoprotectants and culture osmoadaptation. Freeze-drying was used to obtain dehydrated cells, and the best viability (70% of survival) was obtained using lactose as lyoprotectant. Survival during lyophilization was additionally improved using physiological adaptation of cells during cultivation under salt-amended medium (osmoadaptation). The procedure increased the survival of cells after freeze-drying attaining viability values close to a 100% in the lactose-formulated product (3 × 10(11) CFU g(-1) ), and through the storage period of 1 year at 4°C. The dry formulation showed also an improved biocontrol efficacy and survival of EPS62e on pear flowers under low relative humidity conditions. CONCLUSIONS: Cell viability after freeze-drying was improved using lactose as lyoprotectant combined with a procedure of osmoadaptation during cultivation. The powder-formulated product remained active for 12 months and retained biocontrol levels similar to that of fresh cells. The formulation showed an improved survival of EPS62e on flowers and an increase of the efficacy of biocontrol of fire blight at low relative humidity. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have a potential value for commercial application in biocontrol agents not only of fire blight but also of other plant diseases.

Prospects and limitations of microbial pesticides for control of bacterial and fungal pomefruit tree diseases.

The tree constitutes an ecosystem in which microorganisms play an essential role in its functionality. Interactions that microorganisms establish with plants may be beneficial or detrimental and are of extreme importance in the exploitation of trees in agriculture as crop production systems. Fruit trees, especially pomefruit trees including apple, pear and several ornamentals are of great economic importance but its production is affected by several diseases. Fungal and bacterial fruit tree diseases are mainly controlled with chemical fungicides and bactericides, but health and environmental concerns about the use of chemical pesticides have result in strong regulatory actions and have stimulated the development of beneficial microorganisms as microbial pesticides. Up to now, several microorganisms have been registered in different countries and in the EU as biocontrol agents (BCA) covering mainly fire blight, soil-borne fungal diseases and postharvest fruit fungal rot. The key aspects in the success of this technology for disease control are related to biosafety and environmental impact of biocontrol agents, the traceability and fate in the environment and food chain, the improvement by physiological, genetic engineering or the use of mixtures or formulations as well as the industrial production and development of delivery systems for treatment application to trees.

Improvement of fitness and efficacy of a fire blight biocontrol agent via nutritional enhancement combined with osmoadaptation.

The efficacy of Pseudomonas fluorescens EPS62e in the biocontrol of Erwinia amylovora was improved by a procedure of physiological adaptation to increase colonization and survival in the phytosphere of rosaceous plants. The procedure consisted of osmoadaptation (OA) and nutritional enhancement (NE). OA was based on saline stress and osmolyte amendment of the growth medium during inoculum preparation. NE consisted of addition of glycine and Tween 80 to the formulation. NE and OA increased the growth rate and carrying capacity of EPS62e under high-relative-humidity (RH) conditions and improved survival at low RH on flowers under controlled environmental conditions. NE did not promote growth or affect infection capacity of E. amylovora. The effect of both methods was tested in the field by following the population of EPS62e using quantitative PCR (Q-PCR) (total population) and CFU counting (culturable population) methods. Following field application, EPS62e colonized blossoms, but it was stressed, as indicated by a sharp decrease in culturable compared to total population levels. However, once established in flowers and at the end of bloom, almost all the total population was culturable. The physiological adaptation treatments increased population levels of EPS62e over those of nonadapted cells during the late stage of the flowering period. Control of fire blight infections in flowers and immature fruits was tested by field application of EPS62e and subsequent inoculation with E. amylovora under controlled-environment conditions. The efficacy of fire blight control increased significantly with the combination of nutritional enhancement and osmoadaptation, in comparison with the absence of physiological adaptation.