Assessment of Lipopeptide Mixtures Produced by Bacillus subtilis as Biocontrol Products against Apple Scab (Venturia inaequalis)

Abstract

Apple scab is an important disease conventionally controlled by chemical fungicides, which should be replaced by more environmentally friendly alternatives. One of these alternatives could be the use of lipopeptides produced by Bacillus subtilis. The objective of this work is to study the action of the three families of lipopeptides and different mixtures of them in vitro and in vivo against Venturia inaequalis. Firstly, the antifungal activity of mycosubtilin/surfactin and fengycin/surfactin mixtures was determined in vitro by measuring the median inhibitory concentration. Then, the best lipopeptide mixture ratio was produced using Design of Experiment (DoE) to optimize the composition of the culture medium. Finally, the lipopeptides mixtures efficiency against V. inaequalis was assessed in orchards as well as the evaluation of the persistence of lipopeptides on apple. In vitro tests show that the use of fengycin or mycosubtilin alone is as effective as a mixture, with the 50-50% fengycin/surfactin mixture being the most effective. Optimization of culture medium for the production of fengycin/surfactin mixture shows that the best composition is glycerol coupled with glutamic acid. Finally, lipopeptides showed in vivo antifungal efficiency against V. inaequalis regardless of the mixture used with a 70% reduction in the incidence of scab for both mixtures (fengycin/surfactin or mycosubtilin/surfactin). The reproducibility of the results over the two trial campaigns was significantly better with the mycosubtilin/surfactin mixture. The use of B. subtilis lipopeptides to control this disease is very promising.

Keywords: Bacillus subtilis; Venturia inaequalis; apple scab; biopesticide; fengycin; lipopeptides; mycosubtilin; surfactin.

Figure 2

Effect of the lipopeptide ratios (w/w) on the IC₅₀ of fengycin/surfactin mixtures on V. inaequalis S755 strain. IC₅₀ in logarithmic scale.

Figure 3

Effect of the lipopeptide ratios (w/w) on the IC₅₀ of mycosubtilin/surfactin mixtures on V. inaequalis S755 strain. Logarithmic scale.

Figure 1

Schematic representation of scientific strategy developed in this work. Step 1: in vitro screening of the best lipopeptide mixture against V. inaequalis; Step 2: Optimization of the lipopeptide mixture production; Step 3: Production and purification of the different mixtures of lipopeptides in shacked flask; Step 4: Study of lipopeptide mixture efficiency against V. inaequalis in organic orchards; Step 5: Lipopeptide remanence evaluation using QuEChERS method.

Figure 4

Pareto diagram of normalized effect for fengycin concentration, surfactin concentration, specific production of fengycin and specific production of surfactin, biomass production and percentage of fengycin produced in relation to total lipopeptides produced. A: carbon source; B: Nitrogen source; C: Phosphate concentration; D: K_La.

Figure 5

Interaction diagrams for (A) fengcyin specific production, (B) surfactin specific production and for (C) Percentage of fengycin produced related to total lipopeptides. * indicates the interaction between the factors.

Figure 5

Interaction diagrams for (A) fengcyin specific production, (B) surfactin specific production and for (C) Percentage of fengycin produced related to total lipopeptides. * indicates the interaction between the factors.

Figure 6

Graphical representation of the levels of leaf scab contamination in the different modalities following the 2018 trials at the end of primary contamination. The different statistical groups are represented by the letters A and B. A rating of AB indicates that this modality belongs statistically to groups A and B.

Figure 7

Graphical representation of the levels of leaf scab contamination in the different modalities following the 2019 trials at the end of primary contamination. The different statistical groups are represented by the letters A and B. A rating of AB indicates that this modality belongs statistically to groups A and B.