Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum

Abstract

Fusarium head blight epidemics of wheat and barley cause heavy economic losses to farmers due to yield decreases and production of mycotoxin that renders the grain useless for flour and malt products. No highly resistant cultivars are available at present. Hyphae of germinating fungal spores use different paths of infection: After germination at the extruded tip of an ovary, the hyphae travel along the epicarp in the space between the lemma and palea. Infection of the developing kernel proceeds through the epicarp, successively destroying the layers of the fruit coat and finally the starch and protein accumulating endosperm. Hyphae reaching the rachis proceed to apically located developing kernels. Using a constitutively green fluorescence protein-expressing Fusarium wild-type strain, and its knockout mutant, preventing trichothecene synthesis, we demonstrate that trichothecenes are not a virulence factor during infection through the fruit coat. In the absence of trichothecenes, the fungus is blocked by the development of heavy cell wall thickenings in the rachis node of Nandu wheat, a defense inhibited by the mycotoxin. In barley hyphae of both wild-type and the trichothecene knockout mutant, are inhibited at the rachis node and rachilla, limiting infection of adjacent florets through the phloem and along the surface of the rachis. Effective resistance to Fusarium head blight requires expression of genes that combat these different pathways of infection.

Fig. 1.

Confocal laser microscopy images of the infection of epicarp (48 hai, A-F) and inner tissues of the caryopsis (72 hai, G-K) of wheat and barley by different GFP-expressing strains of F. graminearum. (A-C) Hyphae of WT-GFP in epicarp cells of wheat cv. Nandu. (A) Hyphae showing appressoria-like structures. (B) Constricted parts of hyphae traversing the cell wall through a pit. (C) Intercellular growth of hyphae. (D and E) Infection of wheat cv. Nandu with tri5-GFP. Contents of infected cells are disintegrated; arrow in E indicates infection pore. (F) Infection of barley cv. Ingrid with WT-GFP. Penetration of cell walls occurs preferentially at junction regions. (G) Cross section of barley caryopsis cv. Ingrid infected with tri5-GFP. Fungal growth is restricted to the hypodermis. (H) Cross section of wheat caryopsis cv. Nandu infected with WT-GFP. Arrowheads indicate fungal mycelium in the gap between hypodermis and cross cells. (I and J) Cross sections of barley caryopsis BCIngrid-mlo5 infected with WT-GFP showing disintegration of aleurone and endosperm cells (I). Extensive generation of macroconidia on the surface of necrotic caryopsis was observed (J). (K) Cross section of barley caryopsis BCPallas-mlo5 infected with tri5-GFP exhibiting destruction of cell walls and content of hypodermis cells. (Scale bars, 10 μm in A-G and 50 μm in H-L.) a, aleurone; c, cross cells; e, endosperm; h, hypodermis; t, testa.

Fig. 2.

Comparison of the progress of infection through the tissue layers from the epicarp to the endosperm of barley and wheat fruit coats by WT-GFP and tri5-GFP. The tissues were sampled at 72 hai (A) and 96 hai (B). Considerable differences are observed among the barley genotypes for the speed with which the pathogen travels through the tissues to reach the endosperm. With the exception of Chevron at 72 hai, the two F. graminearum strains progress with same speed through the tissue layers.

Fig. 3.

Micrographs of hand sections after infection of wheat cv. Nandu 6 dai (A-D) and barley cv. Chevron at 21 dai (E-H) with WT-GFP (A, B, E, and G) and tri5-GFP (C, D, F, and H). In wheat, WT-GFP infects the rachis (B), whereas tri5-GFP is contained in the rachis node (D). In barley, both fungal strains are inhibited at the rachis neck and rachilla, limiting infection of adjacent florets through the phloem and along the surface of the rachis (G and H). (A, C, E, and F) White light. (B, D, G, and H) GFP exciting blue light.

Fig. 4.

Fusarium resistance in the rachis node. (A) Confocal micrograph of rachis node showing the heavily increased thickness of the cell walls in wheat, preventing the trichothecene deficient mutant of F. graminearum tri5-GFP from entering the rachis (12 dai). (B) Heavy infection of the rachis node by wild-type F. graminearum (6 dai, WT-GFP). (Scale bars, 50 μm.)

Fig. 5.

Confocal micrographs of the infection in the rachis of wheat cv. Nandu by F. graminearum WT-GFP. (A and B) Intracellular movement of the fungus through vascular bundles of xylem and phloem 6 dai. (A) Cross section. (B) Longitudinal section. (C) Growth of F. graminearum in the apoplast of parenchymatic tissue, 8 dai. (D) F. graminearum released from the rachis through stomata, 23 dai. (Scale bars, 50 μm.)