Improvement of biocontrol efficacy of Trichoderma harzianum vs. Fusarium oxysporum f. sp. lycopersici through UV-induced tolerance to fusaric acid

Abstract

Competition is one of the potential mechanisms of the antagonistic action of Trichoderma harzianum against Fusarium oxysporum. The competitive capability of the T. harzianum isolate ITEM 908 (Th908) vs. an isolate of F. oxysporum f. sp. lycopersici was improved via enhancement of the tolerance to growth-inhibitory metabolites produced by F. oxysporum. HPTLC and HPLC analyses led to the identification of fusaric acid (FA) as the major metabolite in culture filtrate of the phytopathogenic F. oxysporum strain ITEM 2797 (Fo2797). FA, a phytotoxin which has also been reported to be released in soil, totally inhibited the growth of Th908 on PDA containing 120μg of FA per gram in 3-day trials. Through UV-C irradiation and subsequent selection of mutants able to grow on PDA supplemented with culture extracts of Fo2797 or FA, one stable tolerant mutant (Th908-5) with unaltered physiological features and rhizosphere competence was isolated. The biocontrol capability of the UV-mutant Th908-5 was compared to that of the wild-type strain Th908 on tomato plants grown in a substrate heavily infested with Fo2797 in two separate trials. The reduction of the disease by Th908-5 was highly (P<0.01) to extremely (P<0.001) significant, while only marginally significant (P<0.05) and inconsistent biocontrol was achieved by Th908. In addition, in non-inoculated vermiculite, Th908-5 increased the emergence and growth of tomato plants compared to the control. Th908 and Th908-5 were investigated for the expression of five genes (MDR ProB, MDR BrefA, MDR Protein2, Hydro II, ThPTR2) encoding proteins putatively associated with T. harzianum biocontrol function and involved in the mechanisms of multidrug resistance (MDR) or competition for space and nutrients. When the mutant strain was exposed to FA, the expression of the gene MDR ProB, encoding a protein associated with MDR was suppressed, suggesting a role for the gene in response to FA. Since UV-mutants are not regarded as genetically modified organisms (GMO) and their circulation and use is not subjected to restrictions that apply to strains derived by genetic transformation, the improved strain Th908-5 could be readily available for application in the field.