Effective In Vitro Control of Two Phytopathogens of Agricultural Interest Using Cell-Free Extracts of Pseudomonas fluorescens and Chitosan

Abstract

A biofungicide is a natural product that can be derived from various sources such as, among others, microorganisms, higher plants, animal products, phytochemicals, semiochemicals, and antagonist microorganisms. One of the most important approaches for the production of biofungicides is the combination of biocontrol agents. This study showed the inhibition growth of Alternaria alternata and Fusarium solani treated with cell-free extracts of P. fluorescens. Using thin-layer chromatography and plate assays it was also demonstrated that the cell-free extracts of P. fluorescens contained siderophores and derivates of 4-diacetylphloroglucinol and phenazine. Moreover, the combination of cell-free extracts of P. fluorescens and chitosan [50-1.5% (v/v)] had a synergistic effect since they notably inhibited the mycelial growth of A. altenata and F. solani. Various morphological alterations to the mycelia and conidia of the treated fungi as a result of this combination were also observed. The present study could be a starting point to control other fungal phytopathogens using different cell-free extracts and chitosan as biocontrol agents.

Keywords: Alternaria altenata; Fusarium solani; antagonist; biofungicide; siderophores.

Figure 1

Growth kinetics of P. fluorescens and the antifungal effect of cell-free extracts of P. fluorescens on A. alternata and F. solani incubated for 168 h.

Figure 2

Readings of the TLC. Ph = Standard phenazine, CE = cell-free extracts of P. fluorescens and DAGP = standard of for 2,4-diacetylphloroglucinol. Readings are on UV 245 nm. Numbers 1, 2, 3, 4 indicate the possible secondary metabolites with antifungal effects.

Figure 3

Petri plate with sterile distilled water as a control (A) and cell-free extracts of P. fluorescens (B). The scale of color is as follows for siderophore production: blue = none, green (blue + pale yellow) = limited, yellow = very strong.

Figure 4

Mycelia of A. alternata without treatment (A) and treated (B,C) with a mixture of cell-free extracts of P. fluorescens and chitosan [50–1.5% (v/v)]. Mycelia of F. solani without treatment (D) and treated (E,F) with the mixture of cell-free extracts of P. fluorescens with chitosan [50–1.5% (v/v)].

Figure 5

Conidia of A. alternata without treatment (A) and treated (B,C) with a mixture of cell-free extracts of P. fluorescens and chitosan [50–1.5% (v/v)]. Conidia of F. solani without treatment (D) and treated (E) with a mixture of cell-free extracts of P. fluorescens and chitosan [50–1.5% (v/v)].