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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2009, Vol. 3 Issue (1) : 16-23     DOI: 10.1007/s11703-009-0005-2
RESEARCH ARTICLE |
Isolation and identification of the antagonistic strain DM-54 of Bacillus amyloliquefacien against Verticillium dahliae, and optimization of antifungal protein producing conditions
Ying ZHU1(), Shuna LI2, Hongshui YUAN2, Xiaojun GUO2, Baocheng ZHU1,2()
1. College of Life Science, Hebei University, Baoding 071002, China; 2. College of Life Science, Agricultural University of Hebei, Baoding 071001, China
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Abstract  

The strains capable of resistance against Verticillium dahliae Kleb were isolated and screened from the soils of cotton fields from several different provinces in China. A strain, coded DM-54, with a rather high antagonistic activity was obtained. Its morphological characteristics, physiological and biochemical properties and a 16 S rDNA sequence of this strain were further studied. The DM-54 strain was finally identified as a kind of Bacillusamyloliquefacien. Through a single factor experiment and an orthogonal experiment, the optimal shaking flask fermentation condition of strain DM-54 was found to be: media composed of 5% dextrin, 3% soy peptone, 0.02% MgSO4, 0.01% CaCl2, initial pH 7.0 and 10% inoculum volume, media volume 30/250 (mL/mL), fermentation temperature at 32oC, rotating speed 200 r?min-1, fermentation time of 48 h. Its antagonistic activity was distinguished to be elevated, at about 39.9%. Our research offers an effective means for the massive production of antagonistic proteins.

Keywords cotton verticilliumwilt      antagonistic bacterium      isolation and identification      Bacillus amyloliquefacien      fermentation     
Corresponding Authors: ZHU Ying,Email:aileenzy@126.com; ZHU Baocheng,Email:zhu2222@126.com   
Issue Date: 05 March 2009
URL:  
http://academic.hep.com.cn/fag/EN/10.1007/s11703-009-0005-2     OR     http://academic.hep.com.cn/fag/EN/Y2009/V3/I1/16
Fig.1  Colonial morphology of stain DM-54
items testedexperimental resultitems testedexperimental result
anaerobic growth-malonate utilization-
growth in pH 5.7 nutrition broth+saline tolerance
production of H2O2 enzyme+2%+
V-P determination+5%+
the pH of V-P determination6.127%+
anti-lysozyme+10%+
amylolysis+12%-
nitrate reduction+sugar and spirits fermentation
nitrite reduction-glucose+
citrate utilization+manicol+
gelatine liquefication+lactose+
methyl red-growth temperature
lacmus milkA5°C-
cellulose decomposition-10°C+
tyrosine hydrolysis-20°C+
casein hydrolysis+30°C+
indole+40°C+
phenylalanine deaminase-50°C-
lecithinase-
Tab.1  Physiological and biochemical characteristics of strain DM-54
Fig.2  Phylogenetic tree of the 16 S rDNA sequence of strain DM-54 and related strains
serial-numberspecific namestrain-numbersimilarity/%
DQ993671Bacillus axarquiensisLMG 2247698.99
DQ993673Bacillus malacitensisLMG 2247798.99
AB021191Bacillus mojavensisIFO1571899.14
AB271744Bacillus subtilisNBRC 1371999.28
AB255669Bacillus amyloliquefaciensNBRC 1553599.43
AB021198Bacillus vallismortisDSM1103199.36
AB021181Bacillus atrophaeusJCM907099.21
CP000002Bacillus licheniformisATCC 1458097.16
AB267380Alkalibacillus silvisoliHN293.28
Tab.2  Comparison of similarities between several norm stains and strain DM-54
carbon sourcemaizenaglucosesucroselactosestarchdextrinmannitolmaltose
inhibition zone diameter/mm16.80±0.2915.70±0.5814.80±0.5816.0016.00±0.5017.67±0.7616.20±1.1615.33±1.04
Tab.3  Effect of carbon source on antagonistic protein production of strain DM-54
nitrogen sourcepeptonebeef extractyeast extracttryptonesoy peptoneureasodium nitratesulfate ammonium
inhibition zone diameter/mm15.60±1.6817.80±1.2618.00±0.5018.00±1.3218.67±0.76000
Tab.4  Effect of nitrogen source on antagonistic protein production of strain DM-54
inorganic saltMn2+Fe2+Na+Mg2+K+Zn2+Cu2+Ca2+
inhibition zone diameter/mm17.80±2.0211.00±0.0013.80±0.7619.30±0.5712.80±1.26017.67±1.5319.70±1.16
Tab.5  Effect of ions on antagonistic protein production of strain DM-54
numberA (dextrin)B (soy peptone)C (CaCl2)D (MgSO4)inhibition zone diameter/mm
10.50.50.010.0113.00±0.00
20.51.00.030.0214.00±1.41
30.52.00.050.0510.00±0.00
40.53.00.070.1011.00±0.00
51.00.50.030.0511.75±1.06
61.01.00.010.1013.50±0.71
71.02.00.070.0111.75±1.06
81.03.00.050.0214.75±1.06
92.00.50.050.1014.75±0.35
102.01.00.070.0513.00±0.00
112.02.00.010.0214.50±2.12
122.03.00.030.0115.75±0.35
135.00.50.070.0216.75±0.35
145.01.00.050.0117.50±3.54
155.02.00.030.1013.50±2.12
165.03.00.010.0516.25±1.06
K112.0014.0614.3114.50
K212.9414.2513.7515.00
K314.5012.6914.2512.75
K416.0014.4413.1313.19
R4.001.751.182.25
Tab.6  Orthogonal experimental design and results of culture media
numbertime/hbottle filling 了size/mLinoculum size/%pHinhibition zone diameter/mm
1243026.017.25±0.35
2245047.016.00±2.83
3247588.015.25±0.35
424100109.016.75±0.35
5483048.019.75±1.06
6485029.019.25±1.06
74875106.017.25±1.06
84810087.016.25±1.77
9723089.017.88±0.18
107250108.018.00±1.41
11727527.016.75±1.06
127210046.015.75±1.77
139630107.021.00±1.41
14965086.018.75±1.06
15967549.015.25±0.35
169610028.014.75±0.35
K116.3118.9717.0017.25
K218.1318.0016.6917.50
K317.0916.1317.0316.94
K417.3315.8818.2517.28
R1.823.091.560.56
Tab.7  Orthogonal experimental design and results of culture conditions
Fig.3  Effect of rotating speed on antagonistic protein activity of strain DM-54
Fig.4  Effect of temperature on antagonistic protein activity of strain DM-54
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