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Background  Filamentous fungi produce small cysteine rich surface active amphiphilic hydrophobins on the outer surface of cell walls that mediate interactions between the fungus and the environment. The role of hydrophobins in surface hydrophobicity, sporulation, fruit body formation, recognition and adhesion to host surface and virulence have been reported. The aim of the present study was to characterize the biological function of hydrophobins in the fungal biocontrol agent  Clonostachys rosea  in order to understand their potential roles in biocontrol mechanisms.    Results  Based on the presence of hydrophobin domains, cysteine spacing patterns and hydropathy plots, we identified three class II hydrophobin genes in  C. rosea . Gene expression analysis showed basal expression of  Hyd1 ,  Hyd2  and  Hyd3  in all conditions tested with the exception of induced  Hyd1  expression in conidiating mycelium. Interestingly, up-regulation of  Hyd1 ,  Hyd2  and  Hyd3  was found during  C. rosea  self interaction compared to interactions with the fungal plant pathogens  Botrytis cinerea  or  Fusarium graminearum  in dual culture assays. Phenotypic analysis of  C. rosea  deletion and complementation strains showed that Hyd1 and Hyd3 are jointly required for conidial hydrophobicity, although no difference in mycelia hydrophobicity was found between wild type (WT) and mutant strains. Interestingly, mutant strains showed increased growth rates, conidiation and enhanced tolerances of conidia to abiotic stresses. Antagonism tests using  in vitro  dual culture and detached leaf assays showed that the mutant strains were more aggressive towards  B. cinerea ,  F. graminearum  or  Rhizoctonia solani , and that aggression was partly related to earlier conidial germination and enhanced tolerance of mutant strains to secreted fungal metabolites. Furthermore,  in vitro Arabidopsis thaliana  root colonization assays revealed reduced root colonization ability of the Δ Hyd3  strain, but not for the Δ Hyd1  strain. Furthermore, enhanced root colonization ability for the Δ Hyd1 Δ Hyd3  strain was found in comparison to WT.    Conclusions  These results show a role for hydrophobins in conidial hydrophobicity, control of conidial germination under stress conditions, and in root colonization in  C. rosea . However, functional studies of Hyd2 remains to be performed in order to fully assess the role of hydrophobins in  C. rosea .

Hydrophobins are required for conidial hydrophobicity and plant root colonization in the fungal biocontrol agent Clonostachys rosea