Characterization Of Oxalate Decarboxylase Genes In Coniothyrium Minitans: Cloning, Functional Analysis And Roles In Biological Control

Sclerotinia sclerotiorum (Lib.) de Bary is a cosmopolitan fungus infecting more than400plant species including many economically important crops. Previous studies showed that S. sclerotiorum produces oxalic acid (OA), which has been proved to play an important role in pathogenesis of S. sclerotiorum. Coniothyrium minitans Campbell is an important mycoparasitic fungus of S. sclerotiorum. It is reported that C. minitans is an effective biocontrol agent of S. sclerotiorum. Previous studies also indicated that OA has toxic effect on mycelial growth and conidial germination of C. minitans. On the other hand, C. minitans has evolved a mechanism to eliminate the toxic effect of OA by degradation of this toxic chemical. OA degradation by C. minitans also has biocontrol effects in interaction between C. minitans and S. sclerotiorum. However, information about the genes responsible for OA degradation in C. minitans and the mechanisms involved in interaction between C. minitans and S. sclerotiorum through OA degradation remains unknown. Thererfore, a study was conducted to clone oxalate decarboxylase genes in C. minitans (Cmoxdcs), to analyze the function of Cmoxdcl and Cmoxdc2and to understand the roles of the two OXDC genes in OA degradation and in interaction between C. minitans and S. sclerotiorum. Results achieved so far are summarized below:Firstly, two oxalate decarboxylase genes designated as Cmoxdcl (GenBank Ace. No. JF718548) and Cmoxdc2(GenBank Acc. No. JF718549), were cloned in C. minitans by degenerate PCR and RACE techniques. Cmoxdcl is1587bp long containing four exons and three introns. Cmoxdc2is1496bp long containing six exons and five introns. Both Cmoxdcl and Cmoxdc2were predicted to encode two putative polypeptides, designated as CMOXDC1and CMOXDC2, respectively, which contain477and408amino acid residues, respectively. Both CMOXDC1and CMOXDC2have the conserved bicupin primary structure. Phylogenetic analysis showed CMOXDC1and CMOXDC2belong to two highly divergent clades in a phylogenetic tree and were closely related to two hypothetical proteins in Phaeosphaeria nodorum, the causal agent of wheat glume blotch disease. Secondly, transcript patterns of Cmoxdc1and Cmoxdc2were clarified. Results showed that the transcription of Cmoxdc1could be induced by OA and the transcription levels of this gene were higher under OA at16,20and24mM (conditioned pH to2.7,2.5and2.3, respectively) than those under OA at2,4and8mM (conditioned pH to4.8,4.4and3.8, respectively). The transcript levels of Cmoxdc1were gradually decreased with increase of the ambient pH from3to8. The transcript level of Cmoxdc1at pH8was only0.05%of those at pH3. The transcripts of Cmoxdc2were detected in cultures at pH3-8with the highest transcript level occurring at pH5.Thirdly, two Cmoxdc1-disruption mutants designated as ΔCmoxdc1-195and ΔCmoxdc1-198, and one Cmoxdc2-disruption mutant ΔCmoxdc2-23, and their corresponding complementary mutants ΔCmoxdc1-195C, ΔCmoxdc1-198C, and ΔCmoxdc2-23C were obtained. The mutants ΔCmoxdc1-195and ΔCmoxdc1-198showed slower growth rates than WT in PDA amended with OA at4and8mM. The mycelial biomass (dry weight) of ΔCmoxdc1-195and ΔCmoxdc1-198were reduced by20-37%, compared to WT. The pH values of the culture filtrates ranged from3.5to4.4and were significantly lower than those of their complementary mutants and WT. OA was degraded by59-63%in the PDB cultures of ΔCmoxdc1-195and ΔCmoxdc1-198, compared to nearly100%OA degradation by the other mutants and WT. On the other hand, the mutant ΔCmoxdc2-23did not differ or slightly differed from WT in tolerance to OA on PDA. The mycelial dry biomass, pH value of the cultural filtrate and the percentage of OA degradation in PDB did not differ from the complementary mutant ΔCmoxdc2-23C and WT. Therefore, Cmoxdc1is more important than Cmoxdc2for C. minitans in OA degradation.Fourthly, Effects of Cmoxdc1and Cmoxdc2on mycoparasitism and antifungal activity of C. minitans were investigated. The results showed that compared to WT, the mutants ΔCmoxdc1-195and ΔCmoxdc1-198were compromised in infecting colonies of the OA-producing strain A5of S. sclerotiorum on PDA. However, they showed no differences in colonizing or invading colonies of OA-deficient mutant S. sclerotiorum PB. Further study showed that in the presence of OA, the relative transcription levels of the two mycoparasitism-related genes Cmch1(chitinase) and Cmg1(β-1,3-glucanase) in the mutants ACmoxdcl-195and ACmoxdcl-198were significantly reduced. Activity of extra-cellular proteases produced by ΔCmoxdcl-195and ΔCmoxdcl-198was not detected; On the other hand, antifungal activity of ΔCmoxdcl-195and ACmoxdcl-198was enhanced.In conclusion, the present study cloned two new oxalate decarboxylase genes from C. minitans (Cmoxdcl and Cmoxdc2) and demonstrated the importance of Cmoxdcl in OA degradation in C. minitans for the first time. It provides direct genetic evidence showing the importance of OA degradation in regulation of both mycoparasitism and antibiosis of C. minitans against S. sclerotiorum.