Activities Of Cell Wall-degrading Enzymes Associated With Gliocladium Spp. Parasitizing On Sclerotia Of Sclerotinia Sclerotiorum And Construction Of CDNA Subtractive Library Of The Mycoparasitism

Gliocladium spp., a group of mycoparasitic fungi, are important biocontrol agents of crop diseases, such as spybean stem rot caused by Sclerotinia sclerotiorum. In order to elucidate the mycoparasitic mechanism of Gliocladium spp. parasitizing on sclerotia of S. sclerotiorum, the correlation of the ability of Gliocladium spp. parasitizing on sclerotia of S. sclerotiorum and the cell wall degrading enzymes was analyzed, and the cDNA subtractive library was constructed.Seventeen Gliocladium strains were selected and inoculated on Sclerotia of S. sclerotiorum, the parasitic ability, inhibitive activity of the metabolites to mycelial growth of S. sclerotiorum and the chitinase and glucanase activities were determined. Results showed that, in contrast to other isolates, HL-1-1, GZH-1-1 and SYP-1-1 were of stronger parasitic ability, metabolites from HL-1-1was of higher inhibitive and glucanase activity, SHW-1-1 was of higher glucanase activity. No significant correlation was found among the parasiting ability, chitinase activity and glucanase activity, and no significant correlation was found, among inhibitory rate of metabolites and the above two cell wall degrading enzymes for the seventeen Gliocladium strains. Relationship was also analyzed among the degradation of the sclerotia powder of S. sclerotiorum by srain HL-1-1 and the chitinase activity and glucanase activity, significant correlation was found among the degradation of the sclerotia powder and the glucanase activity, but the chitinase activity was not. So, chitinase and glucanase played different role during the process of the Gliocladium spp. parasitizing on sclerotia of S. sclerotiorum, and other factors played important role during processes of the parasitism and the fungistasis.In order to understand the genes related with the mycoparasitism, cDNA from Gliocladium HL-1-1 parasitizing on sclerotia of S. sclerotiorum was digested RasΙand used as Tester, mixture of RasΙdigested cDNAs from pure cultures of Gliocladium HL-1-1 and S. sclerotiorum, respectively, was used as Driver to constructe cDNA subtractive library via suppression subtractive hybridization (SSH) technique. 1315 positive clones were confirmed and selected with PCR from the cDNA library,and size of the inserts in all plasmids in the clones was between 300-600bp. 60 sequences were obtained by performing a sequencing and homology searching from 120 randomly selected clones. Among them, some sequences code proteins similar to peroxidase, ribosomal protein L11, cytochrome P450, and heat shock proteins that expressed under certain stresses. 11 gene fragments had no homology with the sequences deposited in NCBI, and therefore were supposed to be novel genes. To ensure the validity of the subtractive hybridization,cDNA from Tester and Driver were used as probes respectively to perform reverse-Northern blotting,and all of the 25 sequences selected were demonstrated as differently expressed gene fragments.