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"

[1] 李晓妮, 徐娜娜, 于金凤. 中国北方马铃薯黑痣病立枯丝核菌的融合群鉴定. 菌物学报, 2014,33(3):584-593.
LI X N, XU N N, YU J F. Anastomosis groups of Rhizoctonia solani from black scurf of potato in northern China. Mycosystema, 2014,33(3):584-593. (in Chinese)
[2] 曹春梅, 李文刚, 张建平, 张庆平, 郭景山. 马铃薯黑痣病的研究现状. 中国马铃薯, 2009,23(3):171-173.
CAO C M, LI W G, ZHANG J P, ZHANG Q P, GUO J S. Current situation of research on potato black scurf. Chinese Potato Journal, 2009,23(3):171-173. (in Chinese)
[3] 曹春梅, 张智芳, 李文刚, 胡冰, 张利辉. 新型杀菌剂对马铃薯黑痣病病菌的室内毒力测定和田间效果分析. 中国马铃薯, 2011,25(4):246-250.
CAO C M, ZHANG Z F, LI W G, HU B, ZHANG L H. Toxicity bioassay and field trial of novel fungicides against Rhizoctonia solani. Chinese Potato Journal, 2011,25(4):246-250. (in Chinese)
[4] 刘宝玉, 蒙美莲, 胡俊, 张笑宇, 杨明明. 5种杀菌剂对马铃薯黑痣病的病菌毒力及田间防效. 中国马铃薯, 2010,24(5):306-310.
LIU B Y, MENG M L, HU J, ZHANG X Y, YANG M M. Virulence and control efficacy in field of five fungicides on Rhizoctonia solani of potato. Chinese Potato Journal, 2010,24(5):306-310. (in Chinese)
[5] IKEDA S, SHIMIZU A, SHIMIZU M, TAKAHASHI H, TAKENAKA S. Biocontrol of black scurf on potato by seed tuber treatment with Pythium oligandrum. Biological Control, 2012,60(3):297-304.
doi: 10.1016/j.biocontrol.2011.10.016
[6] BEAGLE-RISTAINO J E, PAPAVIZAS G C. Biological control of rhizoctonia stem canker and black scurf of potato. Phytopathology, 1985,75(5):560-564.
doi: 10.1094/Phyto-75-560
[7] HUSSAIN T, KHAN A A. Bacillus subtilis HussainT-AMU and its antifungal activity against potato black scurf caused by Rhizoctonia solani on seed tubers. Biocatalysis and Agricultural Biotechnology, 2020,23:101443.
doi: 10.1016/j.bcab.2019.101443
[8] 马龙, 杨莉莉, 马兴, 贾蕊鸿, 董星辰, 邱慧珍. 马铃薯黑痣病生防菌的筛选鉴定及生长条件研究. 中国马铃薯, 2017,31(4):227-233.
MA L, YANG L L, MA X, JIA R H, DONG X C, QIU H Z. Isolation, identification and growth condition of antagonistic strains against potato black scurf. Chinese Potato Journal, 2017,31(4):227-233. (in Chinese)
[9] KHEDHER S B, KILANI-FEKI O, DAMMAK M, JABNOUN- KHIAREDDINE H, DAAMI-REMADI M, TOUNSI S. Efficacy of Bacillus subtilis V26 as a biological control agent against Rhizoctonia solani on potato. Comptes Rendus Biologies, 2015,338(12):784-792.
doi: 10.1016/j.crvi.2015.09.005
[10] 彭振红. 马铃薯黑痣病生防菌的筛选及抑菌机理的初步研究[D]. 呼和浩特: 内蒙古农业大学, 2012.
PENG Z H. Screening of biocontrol agent against to potato black scurf and its antibacterial mechanism[D]. Hohhot: Inner Mongolia Agricultural University, 2012. (in Chinese)
[11] 王培培. 生防枯草芽胞杆菌NCD-2菌株泛革素合成和生物膜形成相关基因的功能分析[D]. 保定: 河北农业大学, 2015.
WANG P P. Functional analysis of the fengycin biosynthesis and biofilm formation related genes in Bacilius subtilis strain NCD-2[D]. Baoding: Agricultural University of Hebei, 2015. (in Chinese)
[12] 杨继业, 杨帆, 崔冠慧, 孙劲冲, 王雅娜, 刘洪伟, 程辉彩, 张丽萍. 马铃薯早疫病拮抗菌的筛选及其代谢产物的抑菌活性. 中国蔬菜, 2018(5):62-66.
YANG J Y, YANG F, CUI G H, SUN J C, WANG Y N, LIU H W, CHENG H C, ZHANG L P. Screening of antagonistic bacteria against potato early blight and antibacterial activity of its metabolites. China Vegetables, 2018(5):62-66. (in Chinese)
[13] PATEL S, GUPTA R S. A phylogenomic and comparative genomic framework for resolving the polyphyly of the genus Bacillus: Proposal for six new genera of Bacillus species, Peribacillus gen. nov., Cytobacillus gen. nov., Mesobacillus gen. nov., Neobacillus gen. nov., Metabacillus gen. nov. and Alkalihalobacillus gen. nov. International Journal of Systematic and Evolutionary Microbiology, 2020,70:406-438.
doi: 10.1099/ijsem.0.003775
[14] 李宝庆, 鹿秀云, 郭庆港, 钱常娣, 李社增, 马平. 枯草芽孢杆菌BAB-1产脂肽类及挥发性物质的分离和鉴定. 中国农业科学, 2010,43(17):3547-3554.
LI B Q, LU X Y, GUO Q G, QIAN C D, LI S Z, MA P. Isolation and identification of lipopeptides and volatile compounds produced by Bacillus subtilis strain BAB-1. Scientia Agricultura Sinica, 2010,43(17):3547-3554. (in Chinese)
[15] 申永铭, 郭成瑾, 王喜刚, 沈瑞清, 陈爱昌, 胡小平. 土壤中立枯丝核菌AG3菌核的荧光定量PCR快速检测. 菌物学报, 2017,36(10):1383-1391.
SHEN Y M, GUO C J, WANG X G, SHEN R Q, CHEN A C, HU X P. Rapid detection of Rhizoctonia solani AG3 sclerotia in soil by quantitative real-time PCR. Mycosystema, 2017,36(10):1383-1391. (in Chinese)
[16] 庄国宏. 产抗菌脂肽GD菌株筛选及脂肽分离、鉴定与应用研究[D]. 扬州: 扬州大学, 2014.
ZHUANG G H. Screening of GD strain producing antimicrobial lipopeptide studying on its separation, identification and application[D]. Yangzhou: Yangzhou University, 2014. (in Chinese)
[17] 邓建良, 刘红彦, 刘玉霞, 刘新涛, 倪云霞, 高素霞, 李国庆. 解淀粉芽孢杆菌YN-1抑制植物病原真菌活性物质鉴定. 植物病理学报, 2010,40(2):202-209.
DENG J L, LIU H Y, LIU Y X, LIU X T, NI Y X, GAO S X, LI G Q. Identification of the antifungal substances from Bacillus amyloliquefaciens strain YN-1. Acta Phytopathologica Sinica, 2010,40(2):202-209. (in Chinese)
[18] 季冠宁, 汪志鹏, 焦加国, 胡峰, 李辉信. 枯草芽胞杆菌M29产抑菌物质培养条件的优化及抑菌物质组成初探. 中国生物防治学报, 2019,35(6):930-939.
JI G N, WANG Z P, JIAO J G, HU F, LI H X. Optimization of culture conditions for antifungal substances produced by Bacilius subtilis M29 and preliminary study on the composition of antifungal substances. Chinese Journal of Biological Control, 2019,35(6):930-939. (in Chinese)
[19] NADEEM H, AYAZ F, ZHOU H, ULLAH K I, ANAM M, MUHAMMAD Z, YANG N, SUN Y, TANG C M. Antagonistic potential of novel endophytic Bacillus strains and mediation of plant defense against Verticillium wilt in upland cotton. Plants, 2020,9(11):1438.
doi: 10.3390/plants9111438
[20] 王军强, 王连国, 郭荣君, 马桂珍, 李世东. 枯草芽胞杆菌B006产表面活性素的培养条件优化. 生物技术通报, 2017,33(4):214-221.
WANG J Q, WANG L G, GUO R J, MA G Z, LI S D. Optimization of culture conditions for the enhancement of surfactin production from Bacillus substilis B006. Biotechnology Bulletin, 2017,33(4):214-221. (in Chinese)
[21] 王珍. 油菜菌核病生防菌的筛选及其作用机理的研究[D]. 武汉: 华中农业大学, 2010.
WANG Z. Studies on screening of antagonistic biocontrol agents and their mechanism of action of Sclerotinia sclerotiorum[D]. Wuhan: Huazhong Agricultural University, 2010. (in Chinese)
[22] 赵新贝, 王娟, 上官妮妮, 刘红彦, 马青. 番茄灰霉病生防细菌TD-7的鉴定、发酵条件优化及其防治效果. 中国生物防治学报, 2019,35(2):226-239.
ZHAO X B, WANG J, SHANGGUAN N N, LIU H Y, MA Q. Identification, fermentation condition optimization and control efficiency of biocontrol bacterium TD-7 against the tomato grey mould. Chinese Journal of Biological Control, 2019,35(2):226-239. (in Chinese)
[23] 王帅, 高圣风, 高学文, 王娜, 王光强. 枯草芽孢杆菌脂肽类抗生素发酵和提取条件. 中国生物防治学报, 2007,23(4):342-347.
WANG S, GAO S F, GAO X W, WANG N, WANG G Q. Fermentation optimization in lipopeptide productivity of Bacillus subtilis G1. Chinese Journal of Biological Control, 2007,23(4):342-347. (in Chinese)
[24] TANG Q Y, BIE X M, LU Z X, LV F X, TAO Y, QU X X. Effects of fengycin from Bacillus subtilis fmbJ on apoptosis and necrosis in Rhizopus stolonifer. Journal of Microbiology, 2014,52(8):675-680.
doi: 10.1007/s12275-014-3605-3
[25] VANITTANAKOM N, LOEFFLER W, KOCH U, JUNG G. Fengycin—A novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. Journal of Antibiotics, 1986,39(7):888-901.
doi: 10.7164/antibiotics.39.888
[26] MAGET-DANA R, PEYPOUX F. Iturins, a special class of pore- forming lipopeptides: Biological and physicochemical properties. Toxicology, 1994,87(1/3):151-174.
doi: 10.1016/0300-483X(94)90159-7
[27] ONGENA M, JOURDAN E, ADAM A, PAQUOT M, BRANS A, JORIS B, ARPIGNY J L, THONART P. Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants. Environmental Microbiology, 2007,9(4):1084-1090.
doi: 10.1111/emi.2007.9.issue-4
[28] MAGET-DANA R, THIMON L, PEYPOUX F, PTAK M. Surfactin/ iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturin A. Biochimie, 1992,74(12):1047-1051.
doi: 10.1016/0300-9084(92)90002-V
[29] SANSINENEA E, ORTIZ A. Secondary metabolites of soil Bacillus spp. Biotechnology Letters, 2011,33(8):1523-1538.
doi: 10.1007/s10529-011-0617-5
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