Biological control of Sclerotinia sclerotiorum by yeast and Bacillus spp. in Allium sativum L. and its effect on the bioactive compounds content and antioxidant activity

Abstract

Phytopathogen attack may influence the production of secondary metabolites related to plant defense, as well as biocontrol agents application, since they can act through induction of resistance, promoting an increase in the production of secondary metabolites related to defense response, such as thiosulfinates and phenolic compounds in garlic. These act as antioxidants and antifungals, protecting the garlic from the phytopathogen and the oxidative stress generated during the infection. The increase of these metabolites is desirable, even as the use of efficient extractive methods to obtain them. In this context, the objective was (1) to evaluate the control of Sclerotinia sclerotiorum by yeasts and Bacillus spp. in vitro and in vivo in garlic; (2) to optimize the extraction process of total phenolic compounds (TPC) and thiosulfinates (TS) and verify th e total antioxidant capacity (TAC) using mixture design; (3) to evaluate the effect of plant x phytopathogen and antagonistic microorganisms interaction on the content of TPC and TS and antioxidant activity. The effect of volatile and diffusible antifungal metabolites on mycelial growth of S. sclerotiorum was evaluated in vitro and the lesion diameter in garlic cloves treated with suspensions of each yeast and bacteria (10 8 cells/ml), inoculated with the phytopathogen and kept in a moist chamber for 15 days. Extractions were performed by sonication using dry and pulverized garlic. For extractive optimization it was used the statistical mixture design of simpleaxial design (SAD) and the central composite desing (CCD), to evaluate the effect of solvents water, ethanol and acetone, as well as mixtures of these solvents, in three extraction times (15, 30 and 45 min) in TPC and TS content and TAC. From the optimized extraction the extracts were carried out with the garlic cloves of the biological control experiment to verify the effects of the plant x microorganisms interactions on the content of TPC and TS and on the antioxidant activity (TAC, free radical scavenging activity DPPH, ability to absorb oxygen radical - ORAC, reducing power and chelating power). A higher inhibition was observed in the growth of S. sclerotiorum by the volatile metabolites produced by Bacillus pumilus, B. amyloliquefaciens, B. macauenses and Candida labiduridarum, ranging from 74.61-87.61%, and by the diffusible metabolites produced by B. amyloliquefaciens (84%). The reduction in the area affected by the disease was more significant in garlic cloves treated with B. pumilus (86.74%) and C. labiduridarum (61.47%). Electromicrographs showed surface colonization of garlic cloves by all microorganisms tested and the colonization of the hyphae of the pathogen by Pichia kudriavzevii, B. acidiceler and B. amyloliquefaciens. In the optimization, the extraction for 30 min was sufficient to extract TPC from the garlic by sonication and the highest content of TPC and TS and CAT were obtained using water / acetone (80:20). The highest amount of TPC was observed in garlic treated with Saccharomyces cerevisiae and TS with Bacillus amyloliquefaciens and Acidithiobacillus ferrooxidans. Bacillus macauenses showed the best chelation power and antioxidant activity measured by DPPH and TAC, while Bacillus pumilus and Bacillus acidiceler presented the best ORAC.