Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (12): 2559-2569.doi: 10.3864/j.issn.0578-1752.2021.12.007

• PLANT PROTECTION • Previous Articles     Next Articles

Identification of the Antifungal Active Compounds from Bacillus amyloliquefaciens Strain HMB33604 and Its Control Efficacy Against Potato Black Scurf

LI YangFan1(),SHAO MeiQi2,LIU CHANG3,GUO QingGang1,WANG PeiPei1,CHEN XiuYe1,SU ZhenHe1,MA Ping1()   

  1. 1Plant Protection Institute of Hebei Academy of Agricultural and Forestry Sciences/IPM Centre of Hebei Province/Key Laboratory of IPM on Crops in Northern Region of North China, Ministry of Agriculture and Rural Affairs, Baoding 071000, Hebei
    2College of Plant Protection, Agricultural University of Hebei, Baoding 071000, Hebei
    3College of Plant Protection, Nanjing Agricultural University, Nanjing 210095
  • Received:2020-08-27 Accepted:2020-10-15 Online:2021-06-16 Published:2021-06-24
  • Contact: Ping MA E-mail:760044035@qq.com;pingma88@126.com

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Abstract:

【Objective】Potato black scurf caused by Rhizoctonia solani AG-3 is one of the most important potato diseases in China, resulting in considerable economic losses. Microbial fungicides are confirmed as practical and environmental friendly way to suppress plant soil-borne diseases. To develop microbial fungicide for potato black scurf, bacterium with potential biocontrol ability for potato black scurf was obtained and the antifungal mechanism was identified for the biocontrol agent. Results of this study will be useful for optimizing the fermentation of biocontrol agent, as well as application of microbial fungicide developed for the bacterium.【Method】Potential biocontrol agents were screened by dual culture in vitro as well as suppressing potato black scurf in vivo. Bacteria were identified by Biolog Microbiological Identification System, 16S rDNA sequence, as well as phylogenetic tree inferred from the alignment sequence of gyrA, gyrB, rpoB and rpoC. The fermentation broth, cell free supernatant and bacterial cell suspension of biocontrol agent were evaluated for suppressing potato black scurf under greenhouse condition. The lipopeptide produced by the biocontrol agent was isolated by high performance liquid chromatography (HPLC) and the antifungal active compounds were identified by UPLC-Triple TOF-MS/MS. The DNA copies of R. solani in the potato rhizosphere was calculated by real-time PCR.【Result】Three bacterial strains with potential biocontrol abilities for potato black scurf were selected from 2 106 antagonistic strains, and Bacillus amyloliquefaciens strain HMB33604 showed the best suppressing ability with 52.9% biocontrol efficacy for potato black scurf. Both the fermentation broth and cell free supernatant showed significant biocontrol abilities against potato black scurf with 52.2% and 66.4% biocontrol efficacy, respectively. However, only 16.9% biocontrol efficacy was achieved by the cell suspension, so the antifungal compounds produced by HMB33604 played an important role in suppressing potato black scurf. The lipopeptides fengycin, iturin A, and surfactin were separated and purified by HPLC, and only the fengycin and iturin A showed strong inhibitory abilities against the growth of R. solani, as well as causing abnormal hyphal growth in vitro. The number of R. solani was significantly reduced by 60.3% and 64.0% of the fermentation broth and cell free supernatant treatments, respectively, when compared to the control. Meanwhile, R. solani number was only reduced by 10.3% of the cell suspension treatment.【Conclusion】B. amyloliquefaciens strain HMB33604 was successfully selected as a promising biocontrol agent for potato black scurf, and the antifungal active compounds produced by strain HMB33604 played an important role in suppressing potato black scurf. The lipopeptides fengycin and iturinA were the main antifungal active compounds in strain HMB33604 and caused abnormal hyphal growth and decreased the number of R. solani in the potato rhizosphere.

Key words: potato black scurf, Bacillus amyloliquefaciens, antifungal compound, lipopeptide antibiotics, biological control

Table 1

Biocontrol efficacy of antagonistic bacteria on potato black scurf"

序号
Serial number		 菌株
Strain		 采集信息
Sampling information		 病情指数
Disease index		 防治效果
Control efficacy (%)
1 对照 CK 80.6±9.1a -
2 嘧菌酯Azoxystrobin (250 g·L-1)
100倍稀释液100× diluent		 11.5±2.6c 85.7
3 HMB33604 河北围场马铃薯根际土壤
Potato rhizosphere soil in Hebei paddock		 37.9±4.3b 52.9
4 HMB28363 西藏墨竹工卡山区土壤
Mountain soil of Mozhu Gongka, Tibet		 43.5±2.2b 45.9
5 HMB32830 河北邯郸小麦根际土壤
Rhizosphere soil of wheat in Handan, Hebei		 44.9±8.1b 44.1

Fig. 1

Phylogenetic tree for strain HMB33604 based on 16S rDNA gene sequence"

Fig. 2

Phylogenetic tree for strain HMB33604 based on gyrA, gyrB, rpoB, rpoC gene sequences"

Table 2

Control efficacy of fermentation broth, supernatant and bacterial suspension of strain HMB33604 on potato black scurf"

处理
Treatment		 病情指数
Disease index		 防治效果
Control efficacy (%)
对照Control 54.0±16.6a -
发酵液Fermentation broth 25.8±5.7b 52.2
上清液Supernatant 18.2±6.4b 66.4
菌体悬浮液Bacterial suspension 44.8±1.7a 16.9

Fig. 3

HPLC analysis of the lipopeptide extracted from strain HMB33604"

Fig. 4

UPLC-Triple TOF-MS analysis of the three purified compounds in the lipopeptide of strain HMB33604"

Fig. 5

Inhibitory effect of surfactin, fengycin and iturinA on the growth of R. solani"

Fig. 7

Standard curve for real-time PCR"

Fig. 6

Effects of fengycin and iturinA from strain HMB33604 on the hypha of R. solani"

Fig. 8

Number of R. solani in the potato rhizosphere soil"

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