SB24

Fusarium root rot is an important disease of soybean in Ontario, Canada. This study is to select antagonistic bacterial agents as effective alternatives to chemical pesticides for the control of root rots caused by Fusarium oxysporum and F. graminearum. Twenty-two Bacillus subtilis strains from soybean and corn roots were tested in dual cultures for inhibition of mycelial growth of F. oxysporum and F. graminearum. All strains significantly reduced mycelial growth of F. oxysporum by approximately 17 to 48% and of F. graminearum by 10 to 32%. Ten B. subtilis strains selected based on their larger fungal inhibition zones were evaluated against macroconidial germination. These strains inhibited the spore germination of F. oxysporum by 20 to 48% and of F. graminearum by 14 to 32% in cell-free filtrates. Under greenhouse conditions, the efficacy of seed and soil treatments with B. subtilis strains against the two Fusarium root rot pathogens was evaluated based on root rot severity, seedling emergence, plant height, and root dry weight. Six B. subtilis strains (SB01, SB04, SB23, SB24, SB28, and SB33) from soybean roots and two strains (CB01 and CH22) from corn roots significantly reduced the severity of the two Fusarium root rots in seed or soil treatments. Strains SB01, SB04, SB23, and SB24 were the most effective treatments against both pathogens in either seed or soil treatment. When applied as seed treatments, these four strains reduced root rot severity by 43 to 63% and increased emergence by 13 to 17%, plant height by 9 to 18%, and root dry weight by 8.4 to 19%. When used as soil treatments, they reduced root rot severity by 68 to 74% and increased emergence by 14 to 18%, plant height by 11 to 23%, and root dry weight by 16 to 24%. These results suggest that the novel strains of B. subtilis identified in this research can be effective alternatives to fungicides in managing Fusarium root rots of soybean, and a greater level of efficacy may be achieved when they were used as soil treatments than seed treatments.

Objective: To obtain the C7C peptide ligands of Mycobacterium tuberculosis by affinity screening based on the phage-displayed random C7C peptide library, and preliminarily identify the binding capacity of the peptide to Mycobacterium.Methods: Inactive Mycobacterium tuberculosis reference strain H₃₇ Rv was used as the target mol. to screen the Ph. D. -C7C peptide library, and Mycobacterium boris, BCG was used for reverse screening.After 4 rounds of affinity screening, single phages eluted by H₃₇ Rv and BCG were selected for DNA sequencing.The binding affinities of different single phage clones were detectd by ELISA.The cyclic peptides displayed by the phage clones showing the highest appetency were synthesized in vitro with fluorescent markers.Fluorescence microscopy and flow cytometer was used to detect the binding affinities of synthesized cyclic peptides, comparing with linear binding peptides obtained before.Results: After 4 rounds of biopanning, phages that could bind with target mols. were remarkable enriched. 16 common sequences were obtained by sequencing anal. of single phages.With ELISA, phage SB1, SB5, SB8 and SB26 all showed higher affinity with H₃₇ Rv and BCG, the ratio to neg. control of which were > 2.1, but could not bind to the 3 nonmycobacteria, which were identified as the pos. clones.Based on the results of flow cytometer detection, the affinities to H₃₇ Rv of 4 cyclic peptides SB1, SB5, SB24, SB26 were (73.2 ± 6.3)%, (63.2 ± 5.3)%, (32.9 ± 3.1)%, (89.4 ± 7.0)%, and to BCG were (65.6 ± 6.1)%, (48.6±4.5)%, (10.3 ± 1.8)%, (86.6±7.9)%, sep., which were all higher than H8 ((4.0 ± 1.0)%, (5.5 ± 1.2)%).From the results of fluorescence microscopy observation, all of the fluorescent labeled cyclic peptides SB1, SB5, SB24, 5B26 could bind to H₃₇ Rv and showed higher fluorescence intensities, which also had certain affinities to other 18 mycobacteria, but the fluorescence intensities were lower than H₃₇ Rv, and didn't bind to 3 non-mycobacteria.Conclusion: Based on the replacement of linear 7 peptide library with C7C peptide library, new ligands of Mycobacterium tuberculosis were achieved successfully, which showed significantly higher binding affinities to mycobacteria.