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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Frontiers of Agriculture in China  2009, Vol. 3 Issue (1): 16-23   https://doi.org/10.1007/s11703-009-0005-2
  RESEARCH ARTICLE 本期目录
Isolation and identification of the antagonistic strain DM-54 of Bacillus amyloliquefacien against Verticillium dahliae, and optimization of antifungal protein producing conditions
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.

Key wordscotton verticilliumwilt    antagonistic bacterium    isolation and identification    Bacillus amyloliquefacien    fermentation
收稿日期: 2008-07-18      出版日期: 2009-03-05
Corresponding Author(s): ZHU Ying,Email:aileenzy@126.com; ZHU Baocheng,Email:zhu2222@126.com   
 引用本文:   
. Isolation and identification of the antagonistic strain DM-54 of Bacillus amyloliquefacien against Verticillium dahliae, and optimization of antifungal protein producing conditions[J]. Frontiers of Agriculture in China, 2009, 3(1): 16-23.
Ying ZHU, Shuna LI, Hongshui YUAN, Xiaojun GUO, Baocheng ZHU. Isolation and identification of the antagonistic strain DM-54 of Bacillus amyloliquefacien against Verticillium dahliae, and optimization of antifungal protein producing conditions. Front Agric Chin, 2009, 3(1): 16-23.
 链接本文:  
https://academic.hep.com.cn/fag/CN/10.1007/s11703-009-0005-2
https://academic.hep.com.cn/fag/CN/Y2009/V3/I1/16
Fig.1  
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  
Fig.2  
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  
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  
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  
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  
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  
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  
Fig.3  
Fig.4  
1 Buchanan R E, Gibbons N E (1984). Bergey’s Manual of Determinative Bacteriology. Beijing: Science Press, 729–797
2 Cheng L J, Xue Q H, Lai H X (2000). Experimental Technology of Microbiology. Xi'an: World Publishing Corporation (in Chinese)
3 Dong X Z, Cai M Y (2001). Manual of systematic and Determinative Bacteriology. Beijing: Science Press, 349–388 (in Chinese)
4 Hu M (2004). Isolation, purification and characterization of an antifungal protein to Verticillium dahliae of cotton. Dissertation for the Master Degree . Baoding: University of Hebei, 14–18 (in Chinese)
5 Jiang Y, Huang L L, Chen C Q, Qiao H P, Kang Z S (2007). Screen, identification and optimized fermentation condition of an actinomycete strain against pathogenic fungus Fulvia fulva. Acta Microbiologica Sinica , 47(7): 622–627 (in Chinese)
6 Lane D J (1991). 16S/23S rRNA Sequencing. In: Stackebrandt E, Goodfellow, eds. Nucleic Acid Techniques in Bacteria Systematics . Chichester: John Wiley & Sons, 115–147 (in Chinese)
7 Li S Z, Ma P, Liu X Z, Huang H C, Chen X H (2001). Biological control of cotton verticillium wilt by antagonistic bacteria. Journal of Huazhong Agricultural University , 20(5): 42–425 (in Chinese)
8 Liang Q M, Qi D M, Jia J, Hui M, Niu T G (2005). Isolation of antagonistic Bacillus and purification of antifungal protein B110-a. Plant Protection , 8(5): 31–35 (in Chinese)
9 Quan C S, Wang J H, Xu H T, Fan S D (2006). Identification and characterization of a Bacillus amyloliquefaciens with high antifungal activity. Acta Microbiologica Sinica , 46(1): 7–12 (in Chinese)
10 Rainey F A, Ward-Rainey N, Kroppenstedt R M, Stackebrandt E (1996). The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage; proposal of Nocardiopsaceae fam. nov. International Journal of Systematic Bacteriology , 46: 1088–1092
11 Saitou N, Nei M (1987). The Neighbour-joining Method: a New Method for Reconstructing Phylogenetic Trees. Mol Biol Evol , 406–425
12 Sambong K, Jung Y, Hongik K (1995). A phylogenetic analysis of the genus Saccharomonospora conducted with 16 S rRNA gene sequences. International Journal of Systematic Bacteriology , 45(2): 351–356
13 Shen P, Fan X R, Li G W (1999). Microbiology Experiment. Beijing: Higher Education Press (in Chinese)
14 Xia Z J, Gu B K, Wu A M (1996). A study on the antagonistic activity of cotton to Verticillium dahliae induced by the endophytic bacteria in the ectohiaosphere and endorhizosphere of soil. Chinese Journal of Biological Control , 12(1): 7–10 (in Chinese)
15 Yang H, Cai L W, Pan Q B, Chen J P, Shi Q H, Zhang E, Wang H Y, Chen L M, Yang M F, Huo J Q (2006). Research advance of cotton verticillium wilt. Jiangxi Cotton , 28(6): 3–6 (in Chinese)
16 Zhou Y F, Du H F, Yuan H S, Zhang Y L, Zhu B C (2007). Isolation and purification of antifungal protein from Paenibacillus to Verticillium dahliae. Cotton Science , 19(2): 98–101 (in Chinese)
17 Zhou Y F, Zhao X Y, Zhang Y, Feng S L, Wang R Y (2006). Expression of Vitreoscilla hemoglobin gene in Bacillus thuringiensis. Journal of Hebei University (Natural Science Edition) , 26(1): 33–37 (in Chinese)
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