Deciphering the mechanisms involved in reduced sensitivity to azoles and fengycin lipopeptide in Venturia inaequalis.

Apple scab, caused by the hemibiotrophic fungus Venturia inaequalis, is currently the most common and damaging disease in apple orchards. Two strains of V. inaequalis (S755 and Rs552) with different sensitivities to azole fungicides and the bacterial metabolite fengycin were compared to determine the mechanisms responsible for these differences. Antifungal activity tests showed that Rs552 had reduced sensitivity to tebuconazole and tetraconazole, as well as to fengycin alone or in a binary mixture with other lipopeptides (iturin A, pumilacidin, lichenysin). S755 was highly sensitive to fengycin, whose activity was close to that of tebuconazole. Unlike fengycin, lipopeptides from the iturin family (mycosubtilin, iturin A) had similar activity on both strains, while those from the surfactin family (lichenysin, pumilacidin) were not active, except in binary mixtures with fengycin. The activity of lipopeptides varies according to their family and structure. Analyses to determine the difference in sensitivity to azoles (which target the CYP51 enzyme involved in the ergosterol biosynthesis pathway) showed that the reduced sensitivity in Rs552 is linked to (i) a constitutive increased expression of the Cyp51A gene caused by insertions in the upstream region and (ii) greater efflux by membrane pumps with the involvement of ABC transporters. Microscopic observations revealed that fengycin, known to interact with plasma membranes, induced morphological and cytological changes in cells from both strains. Sterol and phospholipid analyses showed a higher level of ergosta-7,22-dien-3-ol and a lower level of PI(C16:0/C18:1) in Rs552 compared with S755. These differences could therefore influence the composition of the plasma membrane and explain the differential sensitivity of the strains to fengycin. However, the similar antifungal activities of mycosubtilin and iturin A in the two strains indirectly indicate that sterols are probably not involved in the fengycin resistance mechanism. This leads to the conclusion that different mechanisms are responsible for the difference in susceptibility to azoles or fengycin in the strains studied.

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

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.