Characterization Of Antifungal Substances Produced By The CmCRZ Mutant Of The Biocontrol Fungus Coniothyrium Minitans

Sclerotinia sclerotiorum(Lib.)de Bary,the causal agent of sclerotinia stem rot of rapeseed,has caused huge economic losses in China.The mycoparasite Coniothyrium minitans is a promising biocontrol fungus of S.sclerotiorum.Previous studies in our laboratory found an obvious inhibition zone between the CmCRZ(calcineurin-responsive zinc finger)knockout mutant and S.sclerotiorum in dual culture.Meantime,the extracellular oxidase activity of ΔCmCRZ was significantly enhanced,and the expression of the laccase gene Cmlcc3 was significantly up-regulated in ΔCmCRZ.However,the characterization of antifungal substances(AFS)produced by ΔCmCRZ remains unknown.Meanwhile,whether the increased antifungal activity of ΔCmCRZ is related to laccase Cmlcc3 is unclear.Therefore,our study was conducted to investigate the characterization and biocontrol potential of AFS which produced by ΔCmCRZ.The results are as follows:1.Results of the dual culture test and the inhibition of S.sclerotiorum infection on rapeseed leaves showed that the antifungal activity of ΔCmCRZ AFS against S.sclerotiorum was significantly higher than that of the wild type ZS-1.2.The time dynamics of AFS produced by ΔCmCRZ in modified Modified CzapekDox(MCD)broth were determined.The antifungal activity of AFS in wild type andΔCmCRZ cultures was positively correlated with the incubation time(3 d to 15 d).After incubation for 6 and 9 days,the inhibitory rates of ΔCmCRZ AFS(7.54% and 8.89%)were significantly lower than those of wild type(22.99% and 41.66%).However,after incubation for 15 days,the inhibitory rate of ΔCmCRZ AFS(81.05%)was significantly higher than that of wild type(71.14%).3.The antifungal activity of ΔCmCRZ and wild type AFS against seven species of phytopathogenic fungi were compared.Among the seven fungi,five fungi were more sensitive to ΔCmCRZ AFS,including S.sclerotiorum,Botrytis cinerea,Colletotrichum higginsianum,Fusarium graminearum and Rhizoctonia solani.However,the antifungal activity of AFS against the other two fungi(Alternaria alternata and Fusarium oxysporum)was no significant difference between wild type and ΔCmCRZ.4.AFS of ΔCmCRZ was sensitive to high temperature.After treatment at 80°C or121°C for 30 minutes,the inhibitory rate of ΔCmCRZ AFS decreased significantly to 26.37%and 2.22%,respectively,which was significantly lower than that of wild type(62.52% and25.43%).In a wide range of p H values(2-13),only under the acidic conditions(p H2～p H4),the antifungal activity of ΔCmCRZ AFS was stable.5.The laccase gene Cmlcc3 was knocked out in wild type and ΔCmCRZ,respectively.Deletion of Cmlcc3 resulted in a significant decrease in the laccase activity and the antifungal activity of AFS in wild type and ΔCmCRZ.These results indicated that laccase Cmlcc3 is involved in the antifungal effect of C.minitans against S.sclerotiorum.However,disruption of Cmlcc3 had no significant effect on the colony morphology,growth rate and mycoparasitic ability of wild type and ΔCmCRZ.In addition,knockout of Cmlcc3 also resulted in increased sensitivity of wild type and ΔCmCRZ to oxalic acid(8 m M).In summary,the differences of AFS on antifungal activity,production time dynamics,thermal stability and p H stability between ΔCmCRZ and wild type were compared.Results showed that the antifungal activity of ΔCmCRZ AFS was enhanced,but the thermal stability and p H stability of ΔCmCRZ AFS was decreased.We found that the laccase gene Cmlcc3 is involved in laccase activity and the antifungal activity of AFS in wild type and ΔCmCRZ,indicating that laccase Cmlcc3 plays a role in the antifungal activity of C.minitans against S.sclerotiorum.