Functional Analysis Of Nitrogen Metabolism Regulatory Gene CmareA In Coniothyrium Minitans

Sclerotinia sclerotiorum(Lib.)de Bary,a pathogen for sclerotinia stem rot of oilseed rape,causes severe reduction in oilseed rape yield,resulting in huge economic loss.Coniothyrium minntans,an obligate parasite fungus of S.sclerotiorum,has great exploitive value for biologically controlling sclerotinia disease of oilseed rape.Our laboratory previously found that C.minntans are capable of degrading toxic oxalic acid,which secreted from S.sclerotiorum.And during the process,ammonia was simultaneously produced,to alleviate acidifying environment,which may indicate an accompanied nitrogen metabolism regulation.However,the gene involved this process and its function has not been reported in literature,and the role of the nitrogen metabolism regulatory gene(cmareA)plays in growth and development,mycoparasite ability,oxalic acid degradation of C.minitans and antifungal activity when against S.sclerotiorum are not clear.In this study,we can analyze the function of nitrogen metabolism regulatory gene cmareA in C.minitans.Results are summarized below:Partial sequence of gene areA was cloned from wild type Chy-1 and we subsequently obtained the whole gene sequence by sequence alignment,which was named as cmareA.The cmareA equence was 2811 bp,and 2754 bp when excluding one intron,with open reading frame encoding 917 amino acids.Phylogenetic tree analysis showed that the amino acids encoded by this gene had a most close relationship with its encoded by Parastagonospora nodorum,with the homology being as high as 85%.Gene expression analysis demonstrated that,only in the case of preference nitrogen deficiency or lacking nitrogen resources,the transcription of gene cmareA would be activated and then be expressed.The disrupted mutants(?cmareA-129,?cmareA-133)and their complementary transformants(?cmareA-129 C,?cmareA-133C)were successfully obtained using Split-Marker technology.Analyses of the biological characteristics found,the deletion of gene cmareA reduced growth rate of C.minitans on PDA,decreasing conidium production.Furthermore,such deletion would lead to reduce mycelial growth of C.minitans when nitrate or ammonium salt served as nitrogen resource.The ability of mutant in parasitizing S.sclerotiorum was determined.Results showed that the ability of parasitizing mycelium and sclerotium of S.sclerotiorum was decreased in the mutants.With the examination of the activity of enzymes related with mycoparasitism(including extracellular protease,glucanase and chitinase),it is found thatthe reduced mycoparasite ability may associate with decreased chitinase activity,which was decreased in cmareA mutant.The ability of degrading oxalic acid was quantified using high performance liquid chromatography.Results showed cmareA deletion decreased oxalic acid degradation ability as well.Cultivated on medium based on various nitrogen resources,the disrupted mutants were found to be numerically decreased in producing antifungal substrates due to the decreased growth rate.In addition,in the experiment of studying interaction effect between nitrogen resources and pH on antifungal activity of C.minitans,we found that acidified environment and gene cmareA are prerequisites for C.minitans to produce AFS on medium with nitrates as nitrogen resource.Taking together,the nitrogen metabolism regulation factor cmareA plays an important role in interaction between C.minitans and S.sclerotiorum,including the growth and development and sporulation of C.minitans,and the mycoparasite ability,oxalic acid degradation and antifungal activity of C.minitans when against S.sclerotiorum.