Ability of nonpathogenic Fusarium oxysporum strain Fo47 to induce resistance against Pythium ultimum infection in cucumber

Abstract

The influence exerted by nonpathogenic Fusarium oxysporum strain Fo47 in triggering cucumber protection against infection by Pythium ultimum was investigated ultrastructurally. Macroscopic and microscopic observations of the pathogen colony in dual cultures revealed that reduction of Pythium growth was associated with marked disorders, including generalized disorganization of the host cytoplasm, retraction of the plasmalemma, and complete loss of the protoplasm. Cytochemical labeling of cellulose with an exoglucanase-gold complex showed that the cellulose component of the host cell walls was structurally preserved at a time when the host cytoplasm had undergone complete disorganization. A similar antagonistic process was observed at the root cell surface. Most striking and interesting was the finding that mycoparasitism, as evidenced by the frequent occurrence of Fo47 hyphae within nearly empty cells of the pathogen, occurred not only at the root surface but also within the invaded root tissues. The specific labeling pattern obtained with the exoglucanase-gold complex confirmed that Fo47 successfully penetrated cells of the pathogen, both in the rhizosphere and inside the root tissues. Pythium cells that could evade the first defensive line in the rhizosphere could penetrate the root epidermis, but their growth was restricted to the outermost tissues. Positive correlations between Fo47 treatment and induced resistance to infection by P. ultimum in cucumber were confirmed by (i) the reduction of pathogen viability; (ii) the elaboration of newly formed barriers, a phenomenon which was not seen in Fo47-free plants, where the pathogen proliferated in all root tissues within a few days; and (iii) the occlusion of intercellular spaces with a dense material likely enriched in phenolics. Taken together, our observations provide the first convincing evidence that Fo47 exerts a direct inhibitory effect on P. ultimum through a combination of antibiosis and mycoparasitism, in addition to being a strong inducer of plant defense reactions.

FIG. 1.

In vitro interactions between F. oxysporum strain Fo47 and P. ultimum 2 days after inoculation of the fungi on PDA medium. (A) P. ultimum (P) grown in single culture. (B) P. ultimum (P) grown in the presence of Fo47 (F). Growth of the pathogen is delayed, and changes in hyphal density at the edge of the colony are evident (arrow).

FIG. 2.

Transmission electron micrographs of mycelial samples collected 2 days after inoculation in the region of interaction between Fo47 and P. ultimum. (A) Cell of P. ultimum (P) grown in single culture. Upon labeling with gold-complexed exoglucanase, regular deposition of gold particles occurs over the cell wall whereas the cytoplasm and the organelles, such as mitochondria (M), are unlabeled. (B) In dual-culture tests, hyphae of P. ultimum (P) are severely altered at a time when the P. ultimum cell wall (PCW) is regularly labeled by the exoglucanase-gold complex. Cells of Fo47 (F) are not specifically labeled. (C) Labeling with the WGA-ovomucoid-gold complex results in labeling of Fo47 cell walls (FCW). Altered Pythium (P) cells are unlabeled. S, septum. Bars, 0.5 μm.

FIG. 3.

Transmission electron micrographs of mycelial samples collected 3 to 4 days after inoculation in the region of interaction between Fo47 and P. ultimum. (A and B). Local splitting of Pythium (P) cell wall (PCW) layers, accompanied by the deposition of vesicular material between the split layers (arrows), is evident. The split wall layers are labeled with the exoglucanase-gold complex (A) but not with the WGA-ovomucoid-gold complex (B). F, F. oxysporum strain Fo47. Bars, 0.5 μm.

FIG. 4.

Effect of F. oxysporum strain Fo47 on incidence of disease caused by P. ultimum. (A) In the absence of Fo47 preinoculation, cucumber seedlings infected with P. ultimum exhibit typical root symptoms, as well as leaf wilting. (B) Plants treated with Fo47 prior to Pythium inoculation have a more vigorous root system, on which only a few tiny lesions (arrow) are detectable.

FIG. 5.

Cross sections of pea roots stained with toluidine blue. (A and B) Roots inoculated with P. ultimum (controls). Hyphae of P. ultimum (P) propagate through much of the root tissues, including the epidermis (E) and the cortex (Co) (panel A). Fungal growth occurs in intercellular spaces (IS), as well as intracellularly (panel B). Direct host cell wall penetration is visible (panel B, arrow). Bars: A, 25 μm; B, 10 μm. (C through G) Roots inoculated with F. oxysporum strain Fo47. The fungus (F) develops actively at the root surface and penetrates the epidermis (E) and the outer cortex (Co) (panel C). Colonization of the outermost root tissues correlates with marked thickening of the host cell walls (panel D), formation of wall appositions (panel E), deposition of a fibrillar matrix in which fungal cells (F) are trapped (panel F, arrow), and occlusion of intercellular spaces with amorphous material in which fungal cells are also captured (panel G, arrow). Bars: C, 25 μm; D through G, 10 μm.

FIG. 6.

Effect of F. oxysporum strain Fo47 on the rate and extent of root cell colonization by P. ultimum. Staining was done with toluidine blue. (A through D) Both fungi, easily recognizable by their different electron densities and diameters, are restricted to the outermost root cell layers, including the epidermis (E) (panel A). Growth of P. ultimum (P) in planta is associated with formation of wall appositions (WA) at sites of potential fungal penetration (panel B), deposition of densely stained material along the host cell walls and the pathogen cell surface (panel C, arrowhead), and filling of intercellular spaces with fibrillar matrices (panel D, arrows). Features of potential mycoparasitism exerted by Fo47 (F) are visible (panel C, arrows). Bars: A, 25 μm; B to D, 10 μm.

FIG. 7.

Transmission electron micrographs of cross sections of cucumber roots inoculated with either P. ultimum alone (A) or F. oxysporum strain Fo47 (B through E). (A) Pythium hyphae (P) develop abundantly in all root tissues, causing some host cell wall (HCW) alterations. (B through E) Attempts of Fo47 hyphae to penetrate the epidermis are often halted by the formation of hemispheric wall appositions (WA) at sites of potential penetration (B and C). Elongated and stratified wall appositions are also seen in areas proximal to attempted fungal penetration (D, arrows). Electron-dense deposits (E, arrows) surround an Fo47 cell (F), which is distorted. Bars: A through C, 2 μm; D, 0.5 μm; E, 1 μm.

FIG. 8.

Transmission electron micrographs of cross sections of cucumber roots inoculated with F. oxysporum strain Fo47. (A) Osmiophilic material lining the primary walls of invaded intercellular spaces (arrow) extends toward the inside to coat Fo47 hyphae (F). The host cell wall (HCW) is labeled with gold-complexed exoglucanase. Bar, 0.5 μm. (B) Occlusion of an intercellular space (IS) with a fibrillar matrix (FM) in which fungal cells (F) are trapped. WA, wall apposition. Bar, 1 μm.

FIG. 9.

Cytological and cytochemical observations of the effect of F. oxysporum strain Fo47 on the rate and extent of root cell colonization by P. ultimum. (A through C) Colonization of the root surface by Fo47 and P. ultimum. Fusarium cells (F) develop abundantly at the root surface and interact with the pathogen either through intimate contact (A, arrows) or through mycoparasitism (B and C). The wall of penetrated Pythium hyphae is labeled with gold-complexed exoglucanase (C, arrow). (D and E) Colonization of intercellular spaces (IS) by Pythium hyphae (P) is associated with host defense reactions, including formation of wall appositions (WA) and deposition of osmiophilic material that forms a coating band around invading fungal cells (E, arrow). Bars: A, 2 μm; B, D, and E, 1 μm; C, 0.5 μm.

FIG. 10.

Cytological and cytochemical observations of effect of F. oxysporum strain Fo47 on the rate and extent of root cell colonization by P. ultimum. (A) Interactions between Fo47 (F) and P. ultimum (P) causing cytological damage similar to that observed at the root surface are evident in some intercellular spaces (IS). Incubation of sections with gold-complexed exoglucanase results in regular labeling of Pythium cell walls (PCW), except in areas adjacent to the F047 cell wall (arrows). (B) Feature of mycoparasitism observed in an intercellular space (IS). The penetrated Pythium cell wall (PCW) is labeled with gold-complexed exoglucanase. Labeling is absent over the wall of the invading Fusarium cell (F), in which a septum (S) and Woronin bodies (WB) are visible. Bars, 0.5 μm.

FIG. 11.

Cytological and cytochemical observations of effect of F. oxysporum strain Fo47 on the rate and extent of root cell colonization by P. ultimum. (A) Pythium hyphae (P) are altered even at a distance from Fusarium cells (F). (B and C) By 5 days after pathogen inoculation, Pythium hyphae (P) are morphologically and structurally altered. Labeling of cellulose with gold-complexed exoglucanase confirms their presence. F, Fo47 hypha; HCW, host cell wall. Bars: B, 1 μm; A and C, 0.5 μm.