Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (1): 55-64.doi: 10.3864/j.issn.0578-1752.2020.01.005

• PLANT PROTECTION • Previous Articles     Next Articles

FliZ Regulated the Biofilm Formation of Bacillus subtilis Bs916 and Its Biocontrol Efficacy on Rice Sheath Blight

HuaFei ZHOU1,2,HongFu YANG1,KeBing YAO1,YiQing ZHUANG1,ZhaoLin SHU1,ZhiYi CHEN3()   

  1. 1 Zhenjiang Institute of Agricultural Sciences in Hilly Region of Jiangsu, Jurong 212400, Jiangsu
    2 College of Plant Protection, Nanjing Agricultural University, Nanjing 210095
    3 Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
  • Received:2019-07-01 Accepted:2019-08-04 Online:2020-01-01 Published:2020-01-19
  • Contact: ZhiYi CHEN E-mail:njaujaas@163.com

Abstract:

【Objective】The objective of this study is to discover and identify new regulatory genes on biofilm formation of Bacillus subtilis Bs916, detect its effect on biofilm formation of Bs916 and biocontrol efficacy on rice sheath blight.【Method】The single knockout mutant of Bs916 at fliZ was construct by homologous recombination, and its defects in biofilm formation were verified by dry weight analysis. The anti-bacterial effect of fliZ mutant and Bs916 on rice sheath blight pathogen (Rhizoctonia solani) was detected by flat panel. The relative production of 3 lipopeptide antibiotics (LPs) surfactin, bacillomycin L, and fengycin in fliZ mutant and Bs916 was detected by HPLC. The GFP-labeled strains of Bs916 and fliZ mutant were constructed by green fluorescent labeling, the colonization ability of them in rice stalks was observed, and the biocontrol efficacy of fliZ mutant and Bs916 on rice sheath blight was detected.【Result】The single knockout mutant of Bs916 at fliZ was successfully constructed. Compared with the three-dimensional structure biofilm of the control group Bs916, fliZ mutant only formed a planar two-dimensional structure biofilm, appeared broken form, which proved that it had significant defects in biofilm formation. Quantitative analysis of the dry weight of biofilms showed that the biofilm dry weight of fliZ mutant was only 23% of the control group Bs916, which further verified that the biofilm formation ability of fliZ mutant was significantly decreased. The motility test found that the expanded diameter of fliZ mutant was only 32% of Bs916, which proved that the swimming ability of fliZ mutant was significantly reduced. The bacteriostatic test showed that the antibacterial bandwidth of the two strains was basically the same, and it is proved that the antibacterial activity of fliZ mutant against R. solani was not significantly different from that of Bs916. The relative production of three LPs bacillomycin L, surfactin, and fengycin in fliZ mutant and Bs916 was successfully detected. Compared with Bs916, the relative production of bacillomycin L was significantly increased by 1 time in fliZ mutant, but the relative production of surfactin and fengycin was not significantly different from that of Bs916. The colonization test of rice stalks showed that the number of fliZ mutant was significantly lower than that of Bs916, and there was no significant aggregation effect near the rice sheath blight lesions, and presented an unordered state, which proved that the colonization ability of fliZ mutant on rice stalks was significantly lower than that of Bs916. The field biocontrol trials against rice sheath blight showed that biocontrol efficacy of fliZ mutant ranged from 6.0% to 20.7% on days 6-15, which was significantly lower than that of Bs916 (36.0%-57.6%). It was proved that the biocontrol efficacy of fliZ mutant on rice sheath blight was significantly reduced.【Conclusion】The new regulatory gene fliZ of Bs916 biofilm identified in this study is located in the signal pathway controlling flagellar movement, directly acts on swimming and expansion of the bacteria, and can significantly control the biofilm formation and its biocontrol efficacy on rice sheath blight.

Key words: Bacillus subtilis Bs916, regulated genes, biofilm, lipopeptide antibiotics (LPs), colonization, rice sheath blight, biocontrol efficacy

Table 1

Primers used in this study"

引物Primer 序列Sequence (5′-3′) 来源Source
SpecF TTTGGATCCCTGCAGCCCTGGCGAATG 本试验This study
SpecR TTTGAATTCAGATCCCCCTATGCAAGG 本试验This study
FliZF TTTAAGCTTTACATCCGTTCCCTGCTTTT 本试验This study
FliZR TTTGGATCCGGCCTTTCTTCTTTCCTTCA 本试验This study

Fig. 1

Verification of biofilm formation defects of ΔfliZ mutant"

Fig. 2

Swimming detection of ΔfliZ mutant and Bs916"

Fig. 3

Antibacterial ability detection of ΔfliZ mutant and Bs916"

Fig. 4

Relative production of 3 lipopeptide antibiotics of ΔfliZ mutant and Bs916"

Fig. 5

Observation on the colonization ability of rice stalk of ΔfliZ mutant and Bs916"

Table 2

Biocontrol efficacy on rice sheath blight of ΔfliZ mutant and Bs916 (%)"

菌株Strain 6 d 9 d 12 d 15 d
Bs916 57.6a 51.2a 43.0a 36.0a
ΔfliZ 20.7b 11.9b 7.3b 6.0b
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