Degradation of oxalic acid by Coniothyrium minitans and its effects on production and activity of β-1,3-glucanase of this mycoparasite

Abstract

Degradation of oxalic acid (OA) by the mycoparasitic fungus Coniothyrium minitans (Cm), and the effects of OA degradation on production and activity of β-1,3-glucanase of Cm were investigated. Results showed that OA was degraded by 86–92% by Cm grown at 20 °C for 15 days in modified potato dextrose broth (mPDB) containing OA at 0.8–28 mM. Ambient pH values of these cultures were increased from 3.0–4.8 to 8.3–8.6 due to the degradation of OA and/or accumulation of ammonia by Cm. In dual cultures of Cm and Sclerotinia sclerotiorum (Ss) on potato dextrose agar amended with 0.1% (w/v) bromophenol blue (a pH indicator), spread of Cm onto colonies of Ss was correlated with the elevation of the ambient pH from 2.9 to 6.6, implying that OA degradation by Cm may occur during the interaction between the two fungi. Increase of the ambient pH was also observed on flower petals of oilseed rape inoculated with Cm (5.4 × 10 4 conidia/petal) and Ss (5.4 × 10 3 ascospores/petal) when they were incubated on water agar amended with 0.1% (w/v) bromophenol blue for 6 days, compared to those inoculated with Ss alone. Meanwhile, leaf blight incidence of oilseed rape caused by flower petals inoculated with Cm (1.5 × 10 3–5 conidia/petal) and Ss (4.2 × 10 3 ascospores/petal) was significantly lower ( P < 0.05) than that caused by flower petals inoculated with Ss alone. Results also showed that OA degradation was correlated with the enhanced production of β-1,3-glucanase by Cm and the stimulated activity of this enzyme. In cultures of Cm grown in mPDB amended with OA (16 mM) for 9, 12 and 15 days, OA was degraded by 90%, 91% and 94%, respectively, and the yield of β-1,3-glucanase produced by Cm was increased by 61%, 23% and 52%, respectively, compared to that in Cm cultures grown in mPDB alone. Results in buffered PDB media showed that the yield of β-1,3-glucanase produced by Cm was positively correlated ( R = 0.9439, P < 0.01) with the ambient pH ranging from 3 to 8, implying that the increase in ambient pH caused by OA degradation may be responsible for the enhanced production of β-1,3-glucanase by Cm in OA-containing media. Inhibition by OA of the activity of β-1,3-glucanase produced by Cm was observed and the degree of inhibition was positively correlated to the concentration of OA ranging from 4 to 32 mM, which inversely conditioned the ambient pHs to a range from 1.5 to 3.5. In contrast, the activity of β-1,3-glucanase produced by Cm was not affected by sodium oxalate ranging from 2 to 32 mM, which conditioned the ambient pH to a range from 5.9 to 6.1. The optimum ambient pH for the enzymatic reaction of β-1,3-glucanase of Cm ranged from 4.0 to 6.0. This study suggests that degradation of OA by Cm can nullify the pH effect conditioned by OA and may improve mycoparasitism of Cm on Ss by stimulating production of β-1,3-glucanase by Cm and/or the activity of this enzyme. Degradation of OA by Cm might also be a mechanism by which Cm can protect plants from infection by Ss.