The aim of this study was to apply a generated Δtku70 strain with increased homologous recombination efficiency from the mycoparasitic fungus Trichoderma virens for studying the involvement of laccases in the degradation of sclerotia of plant pathogenic fungi. Inactivation of the non-homologous end-joining pathway has become a successful tool in filamentous fungi to overcome poor targeting efficiencies for genetic engineering. Here, we applied this principle to the biocontrol fungus T. virens, strain I10, by deleting its tku70 gene. This strain was subsequently used to delete the laccase gene lcc1, which we found to be expressed after interaction of T. virens with sclerotia of the plant pathogenic fungi Botrytis cinerea and Sclerotinia sclerotiorum. Lcc1 was strongly upregulated at early colonization of B. cinerea sclerotia and steadily induced during colonization of S. sclerotiorum sclerotia. The Δtku70Δlcc1 mutant was altered in its ability to degrade the sclerotia of B. cinerea and S. sclerotiorum. Interestingly, while the decaying ability for B. cinerea sclerotia was significantly decreased, that to degrade S. sclerotiorum sclerotia was even enhanced, suggesting the operation of different mechanisms in the mycoparasitism of these two types of sclerotia by the laccase LCC1.

The chitinolytic enzyme machinery of fungi consists of chitinases and β‐N‐acetylglucosaminidases. These enzymes are important during the fungal life cycle for degradation of exogenous chitin, which is the second most abundant biopolymer, as well as fungal cell‐wall remodelling. In addition, involvement of chitinolytic enzymes in the lysis of the host cell wall in mycoparasitic Trichoderma spp. has been reported. In view of the fact that fungi have on average 15–20 chitinases, but only two β‐N‐acetylglucosaminidases, the question arises how important the latter enzymes actually are for various aspects of chitin degradation. In this study, the role of two β‐N‐acetylglucosaminidases, NAG1 and NAG2, was analysed in the mycoparasitic fungus Trichoderma atroviride. No β‐N‐acetylglucosaminidase activity was detected in T. atrovirideΔnag1Δnag2 strains, suggesting that NAG1 and NAG2 are the only enzymes in T. atroviride that possess this activity. Δnag1Δnag2 strains were not able to grow on chitin and chitobiose, but the presence of either NAG1 or NAG2 was sufficient to restore growth on chitinous carbon sources in solid media. Our results demonstrated that T. atroviride cannot metabolize chitobiose but only the monomer N‐acetylglucosamine, and that N‐acetylglucosaminidases are therefore essential for the use of chitin as a nutrient source. NAG1 is predominantly secreted into the medium, whereas NAG2 mainly remains attached to the cell wall. No physiological changes or reduction of the mycoparasitic potential of T. atroviride was detected in the double knockout strains, suggesting that the use of chitin as carbon source is only of minor importance for these processes.

A reliable and species-specific real-time quantitative polymerase chain reaction (qPCR) assay was developed for detection of the complex soilborne anamorphic fungus Fusarium oxysporum. The new primer pair, designed on the translation elongation factor 1-α gene with an amplicon of 142 bp, was highly specific to F. oxysporum without cross reactions with other Fusarium spp. The protocol was applied to grafted melon plants for the detection and quantification of F. oxysporum f. sp. melonis, a devastating pathogen of this cucurbit. Grafting technologies are widely used in melon to confer resistance against new virulent races of F. oxysporum f. sp. melonis, while maintaining the properties of valuable commercial varieties. However, the effects on the vascular pathogen colonization have not been fully investigated. Analyses were performed on 'Charentais-T' (susceptible) and 'Nad-1' (resistant) melon cultivars, both used either as rootstock and scion, and inoculated with F. oxysporum f. sp. melonis race 1 and race 1,2. Pathogen development was compared using qPCR and isolations from stem tissues. Early asymptomatic melon infections were detected with a quantification limit of 1 pg of fungal DNA. The qPCR protocol clearly showed that fungal development was highly affected by host-pathogen interaction (compatible or incompatible) and time (days postinoculation). The principal significant effect (P ≤ 0.01) on fungal development was due to the melon genotype used as rootstock, and this effect had a significant interaction with time and F. oxysporum f. sp. melonis race. In particular, the amount of race 1,2 DNA was significantly higher compared with that estimated for race 1 in the incompatible interaction at 18 days postinoculation. The two fungal races were always present in both the rootstock and scion of grafted plants in either the compatible or incompatible interaction.

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