Partial Cloning Conidiation-Associated Gene Of Coniothyrium Minitans And Improving Its Conidia Production In Shaken Liquid Medium

Coniothyrium minitans, an important sclerotial mycoparasite of Sclerotinia sclerotiorum which causes serious lost on many important crops, has great potential for biological control of plant diseases caused by S. sclerotiorum and other Sclerotinia spp. This paper reported that the cloning and functional analysis of a gene obtained from a T-DNA insertion mutant ZS-1T16229 which forms abnormal colony on PDA plate and produces much less conidia than original strain; also in this paper, the improvement of conidia production in shaken liquid medium was studied.Southern blot shows only one copy of T-DNA was insertied in ZS-1T16229, and DNA fragment with a size of 1131 bp of genomic DNA was amplified with TAIL-PCR. Sequencing analysis showed that there might include an incomplete ORF ( nucleotide positions 324-1100) and encoded 259 amino acids containing a conserved domain of Pex2-Pex12. The deduced amino acid sequence shared 59% identical and 74% similarity with Peroxisomal protein Pex2 of Aspergillus fumigatus Af293 which plays important role in metabolism of reactive oxygen species. Therefore the gene cloned here was named CMPEX2. The role of CMPEX2 in ZS-1T16229 could be confirmed indirectly by ZS-1T16229 growing deeply into the substrate when cultured on PDA. The conidia-defency of ZS-1T16229 may be related with the loss of function of CMPEX2.In order to improve conidia production in shaken liquid medium, the culture system of C.minitans strain ZS-1 was studied. The results showed that the biomass and conidia production were significantly improved when little amount of agar added into PDB, and the best achievement was obtained when 0.75‰(w/v) of agar added into PDB. In this condition, the biomass (dry weight) of strain ZS-1 was 616.1±6.2g and the total spores produced was (6.1±0.1)×10⁹ spores/100 ml PDB-la after incubated at 20°C with a rotation of 200 r/min for 10days. Compared with the production of biomass and spores in PDB without any agar, the biomass and spores production were improved by 40.4% and 260.9 % .respectively.The highest production of biomasas and conidia could be obtained at the 8^th day when ZS-1 was incubated in 0.75‰PDB-la; while in PDB the time for the highest production was the 11^th day. Results also showed that the best inoculum dose was 6 ml spores suspension (10⁷spores/ml) into 250ml flask containing 100 ml 0.75‰PDB-la.An orthogonal array design was adopted to investigate the effect of operational parameters including agar dose, inoculum dose and incubation time on the biomass of mycelia and conidia in liquid media. The orthogonal experiment result suggested the best composition to improve the total amount of conidia of C. minitans ZS-1 strain was 6 ml conidial suspension (10⁷spores/ml) into 0.75‰PDB-la medium and the incubation time was 8 days.C. minitans strain CM37, CM38, Chy-1 were used to further confirm the effect of agar in PDB. The results showed that the biomass and conidiation of strains CM37, CM38 were improved significantly in 0.75‰PDB-la; The biomass of strain Chy-1 was singnificantly improved, but it few conidia was produced. These evidence may suggest that agar in PDB could not induce conidiation, but just improve the biomass. Laterly, other fungi, such as Penicillium oxalicum, Magnaporthe grisea, Trichoderma spp and Fusarium graminearum, were tested for growing in 0.75‰PDB-la, and the improvement of biomass were observed.The function of gene (CMPEX2) on conidiation obtained in this work and the effect of agar in PDB on improvement of biomass of C. minitans were discussed.