Degradation Of Phytotoxin Oxalic Acid By The Mycoparasite Coniothyrium Minitans And Its Biocontrol Effects

Sclerotinia rot of oilseed caused by Sclerotinia sclerotiorum is a widespread disease. Coniothyrium minitans is an antagonistic fungus of S.sclerotiorum.Many studies indicated that C.minitans has a great potential in controlling sclerotinia disease caused by S.sclerotiorum in greenhouses and fields.Oxalic acid(OA) is a phytotoxin produced by S.sclerotiorum.Previous studies showed that OA played a determinant role in pathogenesis of S.sclerotiorum.As a mycoparasite of S.sclerotiorum,C.minitans should be tolerant to OA through an unknown mechanism.In this study,degradation of OA by C.minitans and the effects of this process on suppression of S.sclerotiorum were investigated.Results achieved so far were summarized below:Firstly,the capacity of OA degradation by C.minitans was investigated by incubation of C.minitans in modified potato dextrose broth(mPDB) amended with OA at 0.8-80 mmol/L.Results showed that after incubation at 20℃for 15 days,C.minitans could grow in mPDB containing 0.8-32 mmol/L of OA and thus degrade OA by 85%-92%.The pH values of the cultural filtrates increased from 2.9-6.3 to 7.7-8.6. Mycelial growth by C.minitans was completely inhibited in mPDB containing 40 or 80 mmol/L of OA.Production of ammonia by C.minitans was observed in mPDB alone or mPDB amended with OA at 0.8-32 mmol/L and was partially responsible for the increase of pH in cultures of C.minitans.Further analysis showed that the element responsible for OA degradation was located within mycelia of C.minitans,as the cultural filtrate of C.minitans could not convert OA.Activity of oxalate decarboxylase was detected in extracts of mycelia incubated in mPDB alone or mPDB amended with OA at 16 mmol/L.Secondly,factors affecting OA degradation by C.minitans were characterized.The factors investigated include strains of C.minitans,ambient pH and carbon/nitrogen sources.Results showed that two different strains of C.minitans:ZS-1 and Chy-1 were not different in degradation of OA in mPDB amended OA at 32 or 40 mmol/L. The initial ambient pH greatly affected degradation of oxalate by C.minitans.In mPDB amended with ammonia oxalate(AO)(16 mmol/L) or sodium oxalate(SO)(16 mmol/L),the ambient pH of the media was neutral to slightly alkaline.The percentage of degradation of oxalate was less than 5%in the 15-day-old cultures of C.minitans in these media.In mPDB amended with ammonia oxalate(AO)(16 mmol/L) plus OA at 16 mmol/L or sodium oxalate(SO)(16 mmol/L) plus OA at 16 mmol/L,the ambient pH of the media decreased to 3.4-3.5.The percentage of degradation of oxalate increased to 14.1%-36.1%in cultures of C.minitans(15 d).In mPDB amended with ammonia oxalate (AO)(16 mmol/L) plus HCl at 0.2%(v/v) or sodium oxalate(SO)(16 mmol/L) plus HCl at 0.2%,the ambient pH of the media decreased to 2.8-2.9.The percentage of degradation of oxalate increased to 63.1%-89.5%after incubation in these media for 15 days.On the contrary,in mPDB amended with oxalic acid(8,16,24 or 32 mmol/L) and adjusted to be neutral with NaOH,the percentage of degradation of oxalate decreased from 82.8%-97.1%to 13.0%-21.5%.Degradation of OA by C.minitans was observed in Czapek mineral salts amended with each of the five carbon sources(glucose,fructose,maltose,lactose and soluble starch) with alanine as the constant nitrogen source or each of the four nitrogen sources (potassium nitrate,ammonia nitrate,alanine and glutamic acid) with glucose as the constant carbon source,indicating that carbon or nitrogen source may be not the important factors affecting OA degradation by C.minitans.In sclerotial extract or mycelial extract of S.sclerotiorum amended with OA(8 or 16 mmol/L),a complete degradation of OA by C.minitans was also recorded after incubation for 15 days.Thirdly,effects of degradation of OA by C.minitans on S.sclerotiorum were studied by analysis of production and activity of mycoparasitism-related extra-cellular enzymes (β-1,3-glucanase,chitinase and protease),and antifungal substances by this mycoparasitic fungus.Regarding the production ofβ-1,3-glucanase by C.minitans,results showed that after incubation for 9,12 and 15 days,the yield ofβ-1,3-glucanase in mPDB amended with 16 mmol/L of OA was significantly higher than that in mPDB alone.At that time, OA was degraded by 90%-94%in the treatment of mPDB+OA(16 mmol/L),thereby enhancing the ambient pH to 8.3-8.5.In the pH-buffered experiment,production ofβ-1,3-glucanase by C.minitans increased with the increase of the ambient pH from 3.0-8.0,implying that the beneficial effect of OA degradation by C.minitans lies in enhancement of ambient pH.Regarding the activity ofβ-1,3-glucanase produced by C.minitans,results showed that the optimum ambient pH for this enzyme was 4-6.In the enzymatic system ofβ-1,3-glucanase without amendment of OA(control),the ambient pH was 7.0 and the activity of this enzyme was 621.8μg glucose/mL/min.However,with the increase of OA from 4 to 32 mmol/L,the ambient pH decreased to 3.5-1.5 and the activity ofβ-1,3-glucanase decreased to 503.5-55.9μg glucose/mL/min.When sodium oxalate was amended in the enzymatic system ofβ-1,3-glucanase of C.minitans at the concentrations ranging from 2 to 32 mmol/L,the ambient pH and the activity ofβ-1,3-glucanase changed slightly,compared to the control treatment without addition of this chemical. Therefore,it is the acidic ambient pH created by OA that can inhibit the activity ofβ-1,3-glucanase produced by C.minitans.Regarding the production of chitinase by C.minitans,results showed that the yield of this enzyme in cultures of C.minitans amended with OA at 0.8-24 mmol/L was significantly higher than that in the cultures of C.minitans without amendment of OA. The ambient pH for these 15-day-old cultures of C.minitans amended with OA was 7-8.Regarding the activity of chitinase produced by C.minitans,results showed that the optimal pH for activity of this enzyme was 8.0.In the enzymatic system of chitinase of C. minitans amended with OA at 4-48 mmol/L,the activity was inhibited and the inhibitory degree was negatively correlated with the concentration of OA.Activity of protease was determined using gelatin-amended plate.Results showed that the optimal pH value for production of protease by C.minitans was 6-7.The highest activity of protease was detected at pH 8.0.Production and activity of protease were inhibited by high concentration of oxalic acid. Degradation of OA results in increase of ambient pH,which is not favorable for production of anti-fungal substance(AFS) by C.minitans.In another word,when OA is degraded partly or not degraded,ambient pH is low.Acidic environment is favorable for production of AFS.Results of this study suggest that OA plays an important role in regulation of the antagonistic mode of action against S.sclerotiorum.The presence of OA is favorable for production of AFS responsible for antibiosis,whereas the absence of OA is favorable for production and activity of mycoparasitism-related enzymes responsible for mycoparasitism.