Metabolites derived from fungi and bacteria suppress in vitro growth of Gnomoniopsis smithogilvyi, a major threat to the global chestnut industry.

INTRODUCTION: Chestnut rot caused by the fungus Gnomoniopsis smithogilvyi is a disease present in the world's major chestnut growing regions. The disease is considered a significant threat to the global production of nuts from the sweet chestnut (Castanea sativa). Conventional fungicides provide some control, but little is known about the potential of biological control agents (BCAs) as alternatives to manage the disease. OBJECTIVE: Evaluate whether formulated BCAs and their secreted metabolites inhibit the in vitro growth of G. smithogilvyi. METHODS: The antifungal potential of BCAs was assessed against the pathogen through an inverted plate assay for volatile compounds (VOCs), a diffusion assay for non-volatile compounds (nVOCs) and in dual culture. Methanolic extracts of nVOCs from the solid medium were further evaluated for their effect on conidia germination and were screened through an LC-MS-based approach for antifungal metabolites. RESULTS: Isolates of Trichoderma spp., derived from the BCAs, significantly suppressed the pathogen through the production of VOCs and nVOCs. The BCA from which Bacillus subtilis was isolated was more effective in growth inhibition through the production of nVOCs. The LC-MS based metabolomics on the nVOCs derived from the BCAs showed the presence of several antifungal compounds. CONCLUSION: The results show that G. smithogilvyi can be effectively controlled by the BCAs tested and that their use may provide a more ecological alternative for managing chestnut rot. The in vitro analysis should now be expanded to the field to assess the effectiveness of these alternatives for chestnut rot management.

Rapid and Accurate Detection of Gnomoniopsis smithogilvyi the Causal Agent of Chestnut Rot, through an Internally Controlled Multiplex PCR Assay.

The fungus Gnomoniopsis smithogilvyi is a significant threat to the production of sweet chestnut (Castanea sativa) nuts in Australia and worldwide. The pathogen causes nut rot, which leads to substantial production losses. Early and accurate diagnosis of the disease is essential to delineate and implement control strategies. A specific and sensitive multiplex PCR was developed based on the amplification of three barcode sequences of G. smithogilvyi. The assay reliability was enhanced by including the amplification of a host gene as an internal control. Primers were thoroughly evaluated in silico before assessing them in vitro. Primer annealing temperature and concentration were optimised to enhance the assay sensitivity and specificity. The assay detection limit ranged between 0.1 and 1.0 pg (5 and 50 fg/µL) of genomic DNA per reaction. No cross-reactivity was observed with genomic DNA from closely and distantly related fungal species. We also characterised Australian G. smithogilvyi isolates phenotypically and genotypically and found significant differences in morphologic and virulence traits of the isolates. An understanding of the virulence of G. smithogilvyi and the availability of a reliable and accurate diagnostic technique will enable earlier detection of the pathogen, which will contribute to effective control strategies for the disease.