No abstract

ATP-binding cassette (ABC) transporters mediate active efflux of natural and synthetic toxicants and are considered to be important for drug tolerance in microorganisms. In biological control agents (BCA), ABC transporters can play important roles in antagonism by providing protection against toxins derived from the fungal prey and by mediating the secretion of endogenous toxins. In the present study, we generated deletion and complementation strains of the ABC transporter abcG5 in the fungal BCA Clonostachys rosea to study its role in xenobiotic tolerance and antagonism. Gene expression analysis shows induced expression of abcG5 in the presence of the Fusarium mycotoxin zearalenone (ZEA), secreted metabolites of F. graminearum, and different classes of fungicides. Phenotypic analysis of abcG5 deletion and complementation strains showed that the deletion strains were more sensitive towards F. graminearum culture filtrates, ZEA, and iprodione- and mefenoxam-based fungicides, thus suggesting the involvement of abcG5 in cell protection. The ΔabcG5 strains displayed reduced antagonism towards F. graminearum in a plate confrontation assay. Furthermore, the ΔabcG5 strains failed to protect barley seedlings from F. graminearium foot rot disease. These data show that the abcG5 ABC transporter is important for xenobiotic tolerance and biocontrol traits in C. rosea.

The purpose of this study was to verify, under natural conditions, biological control effects obtained in a former screening programme against Fusarium culmorum. The most successful antagonists against seedborne Fusarium culmorum and Bipolaris sorokiniana were isolates of Chaetomium sp., Idriella bolleyi and Gliocladium roseum. These results were also obtained when the antagonists were applied to B. sorokiniana‐infected barley sown in field soil in pots. In field experiments, G. roseum gave the best control of F. culmorum in winter wheat. One month after sowing, germination increased by 170%, the disease index decreased by 73% and plant dry weight increased by 25%, compared to untreated plots. At harvest, the number of tillers per row was found to have increased by 53%, yield had increased by 160% and the 1000‐grain weight had increased by 4%. For all evaluated parameters the effect was significantly different from the untreated plots and not significantly different from the plots treated with the fungicide, Sibutol LS 280. I. bolleyi gave a lower but still significantly effective control, when evaluated for disease index and numbers of tillers per row, while Chaetomium sp. did not show any reduction in the disease. Furthermore, in field experiments using barley infected with B. sorokiniana, a significant effect of G. roseum was demonstrated as increased plant dry weight after 1 month and increased 1000‐grain weight at harvest. The disease‐controlling effect of G. roseum on F. culmorum was shown in a field experiment with spring barley.

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.

No abstract

For successful biocontrol interactions, biological control organisms must tolerate toxic metabolites produced by themselves or plant pathogens during mycoparasitic/antagonistic interactions, by host plant during colonization of the plant, and xenobiotics present in the environment. ATP-binding cassette (ABC) transporters can play a significant role in tolerance of toxic compounds by mediating active transport across the cellular membrane. This paper reports on functional characterization of an ABC transporter ABCG29 in the biocontrol fungus Clonostachys rosea strain IK726. Gene expression analysis showed induced expression of abcG29 during exposure to the Fusarium spp. mycotoxin zearalenone (ZEA) and the fungicides Cantus, Chipco Green and Apron. Expression of abcG29 in C. rosea was significantly higher during C. rosea-C. rosea (Cr-Cr) interaction or in exposure to C. rosea culture filtrate for 2 h, compared to interaction with Fusarium graminearum or 2 h exposure to F. graminearum culture filtrate. In contrast with gene expression data, ΔabcG29 strains did not display reduced tolerance towards ZEA, fungicides or chemical agents known for inducing oxidative, cell wall or osmotic stress, compared to C. rosea WT. The exception was a significant reduction in tolerance to H2O2 (10 mM) in ΔabcG29 strains when conidia were used as an inoculum. The antagonistic ability of ΔabcG29 strains towards F. graminearum, Fusarium oxysporum or Botrytis cinerea in dual plate assays were not different compared with WT. However, in biocontrol assays ΔabcG29 strains displayed reduced ability to protect Arabidopsis thaliana leaves from B. cinerea, and barley seedling from F. graminearum as measured by an A. thaliana detached leaf assay and a barley foot rot disease assay, respectively. These data show that the ABCG29 is dispensable for ZEA and fungicides tolerance, and antagonism but not H2O2 tolerance and biocontrol effects in C. rosea.

BackgroundFilamentous 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.ResultsBased 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 ΔHyd3 strain, but not for the ΔHyd1 strain. Furthermore, enhanced root colonization ability for the ΔHyd1ΔHyd3 strain was found in comparison to WT.ConclusionsThese 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.

The recently reported red fluorescent protein DsRed from the reef coral Discosoma sp. represents a new marker that has been codonoptimized for high expression in mammalian cells. To facilitate expression of DsRed in ascomycete fungi, we used the clone pDsRedExpress (Clontech) for constructing a plasmid vector, pPgpd-DsRed, containing the constitutive Aspergillus nidulans glyceraldehyde 3-phosphate (gpd) promoter. This vector was used for co-transformation of Penicillium paxilli, Trichoderma harzianum and Trichoderma virens (syn. Gliocladium virens) together with either pAN7-1 or gGFP, both containing a gene for hygromycin resistance for transformant selection. In addition, gGFP contains a green fluorescent protein (GFP) gene for expression in Ascomycetes. Expression of DsRedExpress was obtained in all three fungi, indicating that DsRed can be used as a highly effective vital marker in Ascomycetes. Dual marked transformants expressed both DsRed-Express and GFP in the same mycelium and were used for non-quantitative comparison of the intensity of the fluorescence using confocal laser scanning microscopy.