Mechanisms of biological control of Fusarium root and stem rot of greenhouse cucumber by Gliocladium catenulatum

Gliocladium catenulatum strain J1446 (formulated as Prestop WP, Verdera Oy) is a biological control agent of Fusarium root and stem rot caused by Fusarium oxysporum f. sp. radicis-cucumerinum on greenhouse cucumber plants. The mechanisms involved in biocontrol efficacy are currently unknown. Following transformation of G. catenulatum with the ß-glucuronidase (uidA) gene, blue-stained mycelia could be seen growing on the surface and within epidermal and cortical cells of roots, stems and shoots 3 weeks after treatment. Application of G. catenulatum preceding inoculation with Fusarium significantly reduced pathogen populations on roots compared to plants inoculated with Fusarium alone, while densities of the biocontrol agent increased in the presence of the pathogen. Factors influencing root population levels included nutrient solution pH, temperature and growing media type, while cucumber cultivar, root wounding and addition of nutrients did not appear to significantly affect colonization. In culture, G. catenulatum produced chitinase and ß-1,3-glucanase enzymes on chitin or laminarin as a sole carbon source, respectively, and caused localized degradation of Fusarium hyphae. Cucumber root extracts from G. catenulatum-colonized plants had significantly higher levels of glucanase at 7 days post-application compared to untreated controls. Reverse-transcription polymerase chain reaction using primers designed to amplify a ß-1,3-glucanase gene confirmed G. catenulatum glucanase expression on roots. In a split-root system, G. catenulatum applied to one-half of the roots prior to inoculation with Fusarium on the other half did not significantly reduce disease compared to plants treated with Fusarium only. There was no detectable increase in chitinase, peroxidase or polyphenol oxidase enzyme activity in roots and leaves following treatment with G. catenulatum. Competitive colonization of the rhizosphere by G. catenulatum, which is facilitated by its mycoparasitic ability, are the primary mechanisms by which pathogen development and disease incidence is reduced.