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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 (3) : 247-252    https://doi.org/10.1007/s11703-009-0042-x
RESEARCH ARTICLE
Isolation and characterization of Bacillus subtilis EB-28, an endophytic bacterium strain displaying biocontrol activity against Botrytis cinerea Pers.
Shutong WANG1, Tongle HU1, Yanling JIAO1, Jianjian WEI2, Keqiang CAO1()
1. Bio-control Center of Plant Disease and Plant Pests of Hebei Province, College of Plant Protection, Agricultural University of Hebei, Baoding 071001, China; 2. Beijing TEPEC Sunshine Science and Technology Cooperation, Beijing 100086, China
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Abstract

The fungal pathogen Botrytis cinerea Pers. causes severe rotting on tomato fruits during storage and shelf life. As a biological control agent, endophytic bacterium was regarded as an effective alternative to chemical control. Out of 238 endophytic bacterial isolates, three strains (EB-15, EB-28, and EB-122) isolated from Lycopersicum esculentum Mill., Speranskia tuberculata (Bge.) Baill, and Dictamnus dasycarpus Turcz. respectively were found to be strongly antagonistic to the pathogen in vitro and were selected for further in vivo tests. One endophytic bacterium strain, encoded EB-28, was selected from the three in vivo tested isolates. The inhibitive rate of EB-28 reached 71.1% in vitro and 52.4% in vivo. EB-28 was identified as Bacillus subtilis according to its morphological, physiological, and biochemical characteristics and 16S rDNA sequence analysis.

Keywords endophytic bacterium      Bacillus subtilis      Botrytis cinerea Pers      biological control     
Corresponding Author(s): CAO Keqiang,Email:ckq@hebau.edu.cn   
Issue Date: 05 September 2009
 Cite this article:   
Shutong WANG,Tongle HU,Yanling JIAO, et al. Isolation and characterization of Bacillus subtilis EB-28, an endophytic bacterium strain displaying biocontrol activity against Botrytis cinerea Pers.[J]. Front Agric Chin, 2009, 3(3): 247-252.
 URL:  
https://academic.hep.com.cn/fag/EN/10.1007/s11703-009-0042-x
https://academic.hep.com.cn/fag/EN/Y2009/V3/I3/247
Fig.1  Description of the in vitro antagonism assay
Note: (a) One mycelial plug of was placed at the center of the plate, and the bacterial isolates were streaked across each side of the mycelial plug. The distance between the two microorganisms was 2.5 cm. Plates were then incubated at 25°C for 4 days. (b) The percentage growth inhibition of the fungus was calculated by the formula of : (1–2)/1×100, where 1 is the farthest radial distance (measured in millimeters) grown by after 4 days of incubation in the direction of the antagonist (a control value), and 2 is the distance of fungal growth from the point of inoculation to the colony margin in the direction of the antagonist.
endophytic bacteria strainoriginal plantinhibition zone /mminhibitory rate/%
EB-7S. tuberculata1060.00
EB-11S. tuberculata1062.50
EB-15S. tuberculata1170.27
EB-19S. tuberculata958.06
EB-28L. esculentum1271.05
EB-112D. dasycarpus960.61
EB-122D. dasycarpus1069.44
Tab.1  Antagonistic effect of endophytic bacteria strains against
Fig.2  and test of endophytic antagonistic bacteria to
Note: (a) Antagonistic effect of endophytic bacterium isolates EB-122, EB-15, and EB-28 (a1, a2, and a3) on cucumber seedling cotyledons against tomato gray mold (a4 was the untreated control). (b) Antagonistic effect of endophytic bacterial isolates EB-28 (b2) on detached tomato leaves against gray mold (b1 was the untreated control). (c) Dual cultures of pathogen-antagonists showing (c1) a strong inhibition of radial growth of (c2 was the untreated control).
Fig.3  Inhibitive effect of three effective endophytic bacterium isolates against on cucumber cotyledons and tomato leaves
characteristicEB-28characteristicEB-28
colonial morphologycircularcatalase+
cell shaperodH2S production+
spore production+nitrate reduction+
motility+vogues-Proskauer test+
anerobic growth-growth temperature
gram stain+4°C-
growth in presence of:10°C-
2% NaCl+20°C+
5% NaCl-30°C++
7% NaCl-40°C+
10% NaCl-55°C-
Tab.2  Phenotypic characterization of EB-28
Fig.4  
Fig.5  Identification characteristic of EB-28
Note: (a) and (b) Morphology and endospores of EB-28. (c) Electrophoresis of 16S rDNA of EB-28. (d) Neighbor-joining tree based on 16S rDNA (1468 bases) sequences showing the phylogenetic relationship between EB-28 and other related species of the sp. Bootstrap values (expressed as percentages of 100 replications)>70% are given at the nodes.
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