Identification And Antifungal Activity Substances Of Endophytic Bacteria From Maize

Northern Corn Leaf Blight caused by Setosphaeria turcica influences the production and quality of maize. At present, the prevention and control on Northern Corn Leaf Blight is using resistant varieties and chemical fungicides for supporting. But the resistance also depresses when the resistant varieties are used in large areas, it usually has a negative impact when the chemical fungicides are used. Therefore, it is significant to search for new control measures. Biological control is one of the most potential and effective prevention methods. In this study, twenty-two endophytic bacteria were isolated from healthy maize, and throughout further screening, only one which had the inhibitory activity to S. turcica was obtained named as YY1. And the biological characteristics, antifungal properties, antifungal substances and mechanism of strain YY1 were analyzed and researched, so as to improve the theory foundation of in-depth study of antifungal substances, to supply theories and ideas for the application of antagonistic strains. Main results in this paper are as follows:1 Strain YY1 was isolated from healthy maize and could inhibit the growth of S. turcica. It showed wide antifungal spectra. The concentration of spore suspension 2×10⁴ /mL with equal proportion B. subtilis YY1 fermentation liquid could suppress S. turcica after spray-on inoculating. Based on the morphological, physiological and biochemical characteristics and 16S rDNA sequences analysis, strain YY1 was identified as Bacillus subtilis.2 The fermentation conditions were optimized. The results were amylodextrine 3.0%, peptone 2.0%, K₂HPO₄ 0.02%, MnCl₂ 0.01%, inoculation 4%, shaking flask volume at 30 mL/250 mL, 120 rpm under 28℃. The inhibitory activity increased almost two times as before at 30 h.3 The antifungal mechanism of the antifungal crude protein extracts produced by strain YY1 was researched. After crude protein extracts treating, the S. turcica hypha became bead-like hyphal phenotype form filiform. The spore germination could be completely inhibited and the protoplasts were broken gradually at the concentration of 0.78μg/μL. The gene expression and the inhibition ratio in the cAMP signaling pathway were reduced during the process of spore germination and hypha growth. So we inferred that the inhibited molecular mechanism might mainly be mediated by cAMP signaling pathway.4 The antifungal crude protein extracts were thermostable, had a thick skin to proteinase and lost its inhibitory activity under heavy metal Cr³⁺ treating. It had a wide active pH range from 6.0 to 11.0, and the antifungal activity was almost disappeared in acid condition. Antifungal protein YY1-0.3-6 purified and its molecular weight was estimated to be 30 kD by SDS-PAGE. It showed the highest matching degree with enoyl-CoA hydratase by MALDI-TOF MS analysis.5 The lipopeptid substances from B. subtilis YY1 were separated through High Speed Counter Current Chromatography （HSCCC）. The best conditions were: wavelength 214 nm; dissolvent system ethyl acetate: n-butyl alcohol: H2O （4: 1: 5）. The ninth component with the retain time 113¹15 min had inhibitory activity to S. turcica after HSCCC. It contained complex components and required further study and identification.6 Strain YY1 signed with GFP could transmit in the corn after irrigation root method at the seventh day. Therefore, B. subtilis YY1 had strong colonization ability.