Fusarium graminearum forms mycotoxin producing infection structures on wheat

Abstract

Background: The mycotoxin producing fungal pathogen Fusarium graminearum is the causal agent of Fusarium head blight (FHB) of small grain cereals in fields worldwide. Although F. graminearum is highly investigated by means of molecular genetics, detailed studies about hyphal development during initial infection stages are rare. In addition, the role of mycotoxins during initial infection stages of FHB is still unknown. Therefore, we investigated the infection strategy of the fungus on different floral organs of wheat (Triticum aestivum L.) under real time conditions by constitutive expression of the dsRed reporter gene in a TRI5prom::GFP mutant. Additionally, trichothecene induction during infection was visualised with a green fluorescent protein (GFP) coupled TRI5 promoter. A tissue specific infection pattern and TRI5 induction were tested by using different floral organs of wheat. Through combination of bioimaging and electron microscopy infection structures were identified and characterised. In addition, the role of trichothecene production for initial infection was elucidated by a ΔTRI5-GFP reporter strain.

Results: The present investigation demonstrates the formation of foot structures and compound appressoria by F. graminearum. All infection structures developed from epiphytic runner hyphae. Compound appressoria including lobate appressoria and infection cushions were observed on inoculated caryopses, paleas, lemmas, and glumes of susceptible and resistant wheat cultivars. A specific trichothecene induction in infection structures was demonstrated by different imaging techniques. Interestingly, a ΔTRI5-GFP mutant formed the same infection structures and exhibited a similar symptom development compared to the wild type and the TRI5prom::GFP mutant.

Conclusions: The different specialised infection structures of F. graminearum on wheat florets, as described in this study, indicate that the penetration strategy of this fungus is far more complex than postulated to date. We show that trichothecene biosynthesis is specifically induced in infection structures, but is neither necessary for their development nor for formation of primary symptoms on wheat.

Figure 1

Infection structures of F. graminearum wild type isolate 8/1 on wheat cv Nandu. A-D Light microscopy of infected palea after trypan blue staining of mycelium at 10 dpi. A Abundance of infection cushions (blue) on the surface of palea under white light conditions using MZFLIII microscope. Different sizes of infection cushions and necroses of plant cells (white arrowhead) surrounding big infection cushions is remarkable, scale bar = 200 μm. B-D Higher magnification using Zeiss Axio Imager.Z1 reveals different infection structures. B Magnification of blue stained fungal structure (red arrowhead in A) showing a typical infection cushion, scale bar = 50 μm. C Typical cellular structure of a lobate appressorium and D foot structure arising from runner hyphae, scale bars = 10 μm. Abbreviations: FS Foot structures, IC infection cushions, LA lobate appressorium, RH runner hypha, S septum.

Figure 2

Infection structures and TRI5 induction of F. graminearum TRI5prom::GFP on wheat cv Nandu. A-C White light and fluorescence micrographs of infection cushions on palea at 8 dpi using MZFLIII microscope, scale bars = 100 μm. A Natural appearance of the inoculated surface of palea. B Infection cushions are visible by dsRed fluorescence. C GFP fluorescence demonstrates TRI5 induction in infection structures. D Laser scanning microscopy of GFP inductive fungal structures (white arrowhead in B and C). Overlay image of individually detected dsRed and GFP fluorescence of the fungus as well as blue plant autofluorescence. The image represents a maximum intensity projection of a z-stack, scale bar = 50 μm. E-G Scanning electron micrographs of different infection structures on glume at 8 dpi. E Infection cushion, scale bar = 50 μm. F Lobate appressorium, and G foot structures, scale bars = 2 μm. Abbreviations: FS Foot structures, IC infection cushion, IH infection hypha, LA lobate appressorium, PS papillae silica cell, RH runner hyphae.

Figure 3

Quantification of deoxynivalenol (DON) in glumes and caryopses infected with F. graminearum TRI5prom::GFP at 8 dpi. 28 infected glumes and 28 caryopses of the wheat cultivar Nandu were pooled for the estimation of DON after GFP-fluorescence of the fungus was visible by fluorescence microscope MZFLIII. The concentration of DON in ppb of infected fresh weight was estimated by the RIDASCREEN DON enzymatic immunoassay (R-Biopharm, Darmstadt, Germany). Three replicative measurements were done for statistical analyses. Mock inoculated glumes were used as a negative control.

Figure 4

Infection structures of a trichothecene deficient ΔTRI5-GFP mutant of F. graminearum on wheat cv Nandu. A-C White light and fluorescence micrographs of an infected glume at 13 dpi done with MZFLIII microscope, scale bars = 200 μm. A White light image of infection cushions formed by the ΔTRI5-GFP mutant. B GFP fluorescence of the mycelium due to constitutive GFP expression. C Brownish necroses around infection cushions visible by white light microscopy after critical point drying. D-F Scanning electron micrographs show different infection structures of a ΔTRI5-GFP mutant on glume at 8 dpi. D Infection cushions, E lobate appressoria, F foot structures and infection hyphae, scale bars: D and E = 4 μm, F = 1 μm. Abbreviations: FS Foot structure, IC infection cushion, IH infection hypha, LA lobate appressorium, RH runner hypha.