| To test the efficacy of 52 bacterial isolates conserved in our laboratory in inhibiting growth of F. graminearum in vitro, dual cultures were prepared on PDA, KMB and TSA media respectively. 22 isolates among these tested isolates showed strongly antagonistic activity. 22 promise bacterial isolates were cultured in KMB liquid medium, determinated their antirungal activity. The results showed that the liquid culture of CC41 during 72 hours culturing have strongest inhibitive activity to F. graminearum. Liquid culture was precipitated by (NH4)2SO4 with 70% saturation at 0 ℃ for two hours. The deposits collected through centrifuging were thermostable and show the strong inhibitory action to F. graminearum, the inhibitive value is at 76.2%. The results demonstrated that the antagonistic substance were produced by CC41 are probably one or several kinds of antifungal proteins. It had no effect to antagonistic activity under the different oxygen conditions. In both formulation A and C, CC41 can produce more antagonistic substances than in B.CC41 was a gram-positive, rod, average size of cell of 0.75 × 2. 5 μm. It can produce spores on the spore-bearing medium. According to morphological characteristics, CC41 was considered to be the member of genus Bacillus. According to its biochemical, physiological characteristics, we can confirm that it is belong to one strain of B. subtilis. The result of sequence analysis of 16S rDNA showed thatCC41 shared 99.2% homology with published sequence of B. subtilis (accession number:AB065370). In the phylogenetic tree, both of CC41 and B. subtilis are on the same branch, which showed that strain CC41 is closely related with B. subtilis. So we can make a conclusion that the isolate CC41 is one strain of B. subtilis.Green fluorescent protein gene (GFPuv) and promoter (Lac) were amplified by PCR and sequenced. These will help us for future studying on expression plasmid pRGL construction, GFP marker and colonization of CC41 on barley spikes.
|
PDF Full Text Request