Metschnikowia strains isolated from botrytized grapes antagonize fungal and bacterial growth by iron depletion

Abstract

Noble-rotted grapes are colonized by complex microbial populations. I isolated pigment-producing Metschnikowia strains from noble-rotted grapes that had antagonistic activity against filamentous fungi, yeasts, and bacteria. A red-maroon pigment was formed from a diffusible colorless precursor released by the cells into the medium. The conversion of the precursor required iron and could occur both in the cells (red colonies) and in the medium (red halos around colonies). The intensity of pigmentation was correlated with the intensity of the antimicrobial activity. Mutants that did not form pigment also lacked antifungal activity. Within the pigmented halos, conidia of the sensitive fungi did not germinate, and their hyphae did not grow and frequently lysed at the tips. Supplementation of the medium with iron reduced the size of the halos and the inhibition zones, while it increased the pigment accumulation by the colonies. The iron-binding agent tropolone had a similar effect, so I hypothesize that pigmented Metschnikowia isolates inhibit the growth of the sensitive microorganisms by pigment formation, which depletes the free iron in the medium. As the pigment is a large nondiffusible complex produced in the presence of both low and high concentrations of ferric ions, the proposed mechanism is different from the mechanisms operating in microbes that release siderophores into the environment for iron acquisition.

FIG. 1.

Halo formation and inhibition of germination of B. cinerea conidia by Metschnikowia isolate 02.11.1.21. (A) Pigmented halo on YPD supplemented with 0.005 mg/ml FeCl₃. (B) Inhibition zone on YPD. (C to F) Microscopic images of conidia on PDA. m, Metschnikowia colony; n, group of nongerminating conidia (within a colored halo); d, conidia with dying germination tubes (at the edge of a colored halo); g, group of germinating conidia developing mycelium (outside a colored halo). (C) Bar = 120 μm. (F) Bar = 50 μm.

FIG. 2.

Degeneration of hyphae at the edge of the inhibition zone around a colony of Metschnikowia isolate 02.11.1.21. (A) B. cinerea 3318. (B) A. pullulans 27/2.36. (C) M. piriformis. v, viable hypha; r, hypha ruptured near its tip; c, hypha with coagulated cytoplasm. Bar = 20 μm.

FIG. 3.

Effect of FeCl₃ on halo formation and inhibition of the growth of B. cinerea on PDA. (A) Without FeCl₃. (B) With 0.005 mg/ml FeCl₃. (C) With 0.02 mg/ml FeCl₃. m, Metschnikowia colony; b, Botrytis mycelium.

FIG. 4.

Inhibition of B. cinerea by tropolone. Fifty microliters of a solution containing 0.5 mg tropolone was placed into a hole in the middle of the plate. The asterisk indicates the ring of stimulated mycelial growth. The arrow indicates hyphae growing into the inhibition zone. b, Botrytis mycelium.

FIG. 5.

Effect of Metschnikowia isolate 02.11.1.21 on yeasts. (A) S. cerevisiae. (B) C. zemplinina. (C) A. pullulans (yeast phase). m, Metschnikowia colony. There is a zone (marked with an asterisk) in which there is increased melanin production in the A. pullulans lawn.