Isolation, Screening and the Optimization of Fermentation Conditions of Biocontrol Bacteria against Eleutherococcus senticosus Black Spot

doi:10.13560/j.cnki.biotech.bull.1985.2024-0400

Abstract

Abstract:

【Objective】The biocontrol bacteria against Eleutherococcus senticosus black spot were isolated, screened and identified, and the fermentation conditions of bacteria were optimized. This study may provide a good source of biocontrol bacteria against E. senticosus black spot.【Method】The serial dilution method was used to isolate bacteria from healthy E. senticosus rhizosphere soil, and the plate confrontation method was used to screen strains with strong antagonistic effects on A. tenuissima, and the inhibitory effects on other 5 pathogenic fungi were measured. The taxonomic status of this strain was clarified based on morphological characteristics, physiological and biochemical characteristics and 16S rDNA gene sequencing. The culture medium components and fermentation conditions were optimized using single-factor experiments and orthogonal experiments. The effect of fermentation broth of this strain on the biocontrol efficiency against E. senticosus black spot was also studied.【Result】Total 419 strains of bacteria were isolated, among which the strain YZ-228 had the highest inhibitory rate of 67.38% against A. tenuissima, the inhibition rate of sterile filtrate against A. tenuissima was 64.32%. And strain YZ-228 had the inhibitory effects against Rhizoctonia solani, Fusarium solani, Alternaria panax, Cylindrocarpon destructans, and Fusarium oxysporum, with an inhibition rate of 62.80%-67.89%. Strain YZ-228 was identified as Bacillus cereus. Soluble starch and beef extract were the best carbon and nitrogen sources for YZ-228. Under the condition that the pH, temperature, inoculum, liquid volume and speed were 7.0, 28℃, 3%, 50 mL/250 mL and 200 r/min, respectively, the bacterial solution fermented for 1 d had a good antagonistic effect on A. tenuissima, with an inhibition rate of 74.19%. It was 11.99% higher than that in beef extract-peptone-yeast extract medium（BPY）. In addition, inoculation of the fermentation broth of strain YZ-228 significantly reduced the disease index of E. senticosus black spot, and its control effect was 59.15%. 【Conclusion】B. cereus YZ-228 has good control effect on E. senticosus black spot and has the potential to be used as a biological control agent for E. senticosus black spot.

Key words: Eleutherococcus senticosus, Alternaria tenuissima, Bacillus cereus, black spot, biological control

DING Yan-zhe, YAO Xin-xin, SUN Zhuo, YANG Li-min, HAN Zhong-ming, WANG Yun-he. Isolation, Screening and the Optimization of Fermentation Conditions of Biocontrol Bacteria against Eleutherococcus senticosus Black Spot[J]. Biotechnology Bulletin, 2024, 40(12): 218-226.

Figures/Tables 8

References 33

[1]	中国科学院中国植物志编辑委员会. 中国植物志-第六十九卷[M]. 北京: 科学出版社, 1990.
	Chinese Academy of Sciences Editorial Committee of Flora of China. Flora of China-Volume 69[M]. Beijing: Science Press, 1990.
[2]	支叶. 刺五加病害的病原学研究及室内药剂筛选[D]. 长春: 吉林农业大学, 2011.
	Zhi Y. Etiology of Acanthopanax diseases and fungicide screening[D]. Changchun: Jilin Agricultural University, 2011.
[3]	Ngo MT, Van Nguyen M, Han JW, et al. Biocontrol potential of Aspergillus species producing antimicrobial metabolites[J]. Front Microbiol, 2021, 12: 804333.
[4]	Ciofini A, Negrini F, Baroncelli R, et al. Management of post-harvest anthracnose: current approaches and future perspectives[J]. Plants, 2022, 11(14): 1856.
[5]	Franke MD, Brenneman TB, Stevenson KL, et al. Sensitivity of isolates of Sclerotium rolfsii from peanut in Georgia to selected fungicides[J]. Plant Dis, 1998, 82(5): 578-583. doi: 10.1094/PDIS.1998.82.5.578 pmid: 30856992
[6]	El-Nahhal I, El-Nahhal Y. Pesticide residues in drinking water, their potential risk to human health and removal options[J]. J Environ Manage, 2021, 299: 113611.
[7]	El Hawari K, Mokh S, Al Iskandarani M, et al. Pesticide residues in Lebanese apples and health risk assessment[J]. Food Addit Contam Part B Surveill, 2019, 12(2): 81-89.
[8]	Droby S. Improving quality and safety of fresh fruits and vegetables after harvest by the use of biocontrol agents and natural materials[J]. Acta Hortic, 2006,(709): 45-52.
[9]	Chow YY, Rahman S, Ting ASY. Evaluating the host defense responses in oil palm to complex biocontrol endophyte-pathogen-host plant interaction via Fluidigm® real-time polymerase chain reaction(RT-PCR)[J]. Biol Contr, 2019, 129: 148-157.
[10]	Wang R, Yu XL, Yin YP, et al. Biocontrol of cucumber Fusarium wilt by Trichoderma asperellum FJ035 dependent on antagonism and spatiotemporal competition with Fusarium oxysporum[J]. Biol Contr, 2023, 186: 105334.
[11]	Hu JL, Zheng MZ, Dang SZ, et al. Biocontrol potential of Bacillus amyloliquefaciens LYZ69 against anthracnose of alfalfa(Medicago sativa)[J]. Phytopathology, 2021, 111(8): 1338-1348.
[12]	Dong WP, Long T, Ma JH, et al. Effects of Bacillus velezensis GUAL210 control on edible rose black spot disease and soil fungal community structure[J]. Front Microbiol, 2023, 14: 1199024.
[13]	Chandran H, Meena M, Swapnil P. Plant growth-promoting rhizobacteria as a green alternative for sustainable agriculture[J]. Sustainability, 2021, 13(19): 10986.
[14]	Chen K, Tian ZH, He H, et al. Bacillus species as potential biocontrol agents against citrus diseases[J]. Biol Contr, 2020, 151: 104419.
[15]	Wang BJ, Pu Q, Zhang YH, et al. Secretion and volatile components contribute to the antagonism of Bacillus velezensis 1-10 against fungal pathogens[J]. Biol Contr, 2023, 187: 105379.
[16]	Pan M, Zhu ML, Jiang HH, et al. The characterization of the inhibitory substances produced by Bacillus pumilus LYMC-3 and the optimization of fermentation conditions[J]. Fermentation, 2023, 9(11): 966.
[17]	Sun Z, Yang LM, Zhang LX, et al. An investigation of Panax ginseng Meyer growth promotion and the biocontrol potential of antagonistic bacteria against ginseng black spot[J]. J Ginseng Res, 2018, 42(3): 304-311.
[18]	冯志珍, 陈太春, 段军娜, 等. 烟草黑胫病拮抗根际芽胞杆菌FB-16的筛选鉴定及其抑菌活性[J]. 植物保护学报, 2012, 39(3): 224-230.
	Feng ZZ, Chen TC, Duan JN, et al. Screening, identification and antifungal activity of antagonistic rhizospheric Bacillus FB-16 against tobacco black shank[J]. J Plant Prot, 2012, 39(3): 224-230.
[19]	单文荣, 李俊霞, 刘花粉. 滤纸片法筛选不同活性物对棉花黄萎病菌抑制效果研究[J]. 中国农学通报, 2010, 26(19): 285-289.
	Shan WR, Li JX, Liu HF. Study on the inhibitory effects of different active materials screened by the filter paper on cotton Verticillium wilt[J]. Chin Agric Sci Bull, 2010, 26(19): 285-289. doi: 10.11924/j.issn.1000-6850.2010-1478
[20]	Garrity G. Bergey's Manual of Systematic Bacteriology:v. 4[M]. New York: Springer Verlag, 2010.
[21]	孙卓, 杨利民. 人参灰霉病拮抗细菌的筛选及鉴定[J]. 植物保护学报, 2016, 43(6): 935-942.
	Sun Z, Yang LM. Isolation and characterization of antagonistic bacteria against ginseng grey mould[J]. J Plant Prot, 2016, 43(6): 935-942.
[22]	Cui XL, Mao PH, Zeng M, et al. Streptimonospora salina gen. nov., sp. nov., a new member of the family Nocardiopsaceae[J]. Int J Syst Evol Microbiol, 2001, 51(Pt 2): 357-363.
[23]	刘畅, 卢宝慧, 刘小畅, 等. 刺五加黑斑病的室内药剂筛选和田间药效试验[J]. 吉林农业大学学报, 2015, 37(3): 281-286.
	Liu C, Lu BH, Liu XC, et al. Indoor screening and field efficacy trails for fungicides to Alternaria leaf spot disease of Acanthopanax senticosus[J]. J Jilin Agric Univ, 2015, 37(3): 281-286.
[24]	Ünlü E, Çalış Ö, Say A, et al. Investigation of the effects of Bacillus subtilis and Bacillus thuringiensis as Bio-agents against powdery mildew(Podosphaera xanthii)disease in zucchini(Cucurbita pepo L.)[J]. Microb Pathog, 2023, 185: 106430.
[25]	Ravindran AP, Lajapathy JM, Lalithakumari SG, et al. Efficacy of Bacillus licheniformis: a biocontrol agent against Colletotrichum gloeosporioides Penz.(Penz. & Sacc.)causing anthracnose in greater yam(Dioscorea alata L.)[J]. Egypt J Biol Pest Contr, 2023, 33(1): 112.
[26]	丁艳哲, 杜立财, 孙卓, 等. 刺五加黑斑病生防细菌分离、筛选及室内防控效果[J]. 华南农业大学学报, 2024, 45(2): 266-272.
	Ding YZ, Du LC, Sun Z, et al. Isolation, screening and indoor control effect of biocontrol bacteria against Acanthopanax senticosus black spot[J]. J South China Agric Univ, 2024, 45(2): 266-272.
[27]	Nunes C, Usall J, Teixidó N, et al. Biological control of postharvest pear diseases using a bacterium, Pantoea agglomerans CPA-2[J]. Int J Food Microbiol, 2001, 70(1/2): 53-61.
[28]	Pan M, Wang YR, Tan JJ, et al. Optimization of fermentation conditions for Bacillus pumilus LYMC-3 to antagonize Sphaeropsis sapinea[J]. Fermentation, 2023, 9(5): 482.
[29]	Dai Y, Wang YH, Li M, et al. Medium optimization to analyze the protein composition of Bacillus pumilus HR10 antagonizing Sphaeropsis sapinea[J]. AMB Express, 2022, 12(1): 61.
[30]	李娟, 夏凯丽, 王远宏, 等. 解淀粉芽孢杆菌LJ1摇瓶发酵条件优化[J]. 生物技术通报, 2015, 31(12): 214-220. doi: 10.13560/j.cnki.biotech.bull.1985.2015.12.031
	Li J, Xia KL, Wang YH, et al. The optimization of fermentation condition in flask for Bacillus amyloliquefaciens LJ1[J]. Biotechnol Bull, 2015, 31(12): 214-220.
[31]	He J, Zhang XY, Wang QH, et al. Optimization of the fermentation conditions of Metarhizium robertsii and its biological control of wolfberry root rot disease[J]. Microorganisms, 2023, 11(10): 2380.
[32]	Pan H, Xiao Y, Xie AL, et al. The antibacterial mechanism of phenylacetic acid isolated from Bacillus megaterium L2 against Agro-bacterium tumefaciens[J]. PeerJ, 2022, 10: e14304.
[33]	Chen K, Tian ZH, Luo Y, et al. Antagonistic activity and the mechanism of Bacillus amyloliquefaciens DH-4 against citrus green mold[J]. Phytopathology, 2018, 108(11): 1253-1262. doi: 10.1094/PHYTO-01-17-0032-R pmid: 29799309

病原真菌Pathogenic fungus	抑菌率Inhibitory/%
茄立枯丝核菌R. solani	62.80±1.72b
茄腐皮镰孢菌F. solani	67.89±0.70a
链格孢菌A. panax	64.94±1.29ab
毁灭柱孢菌C. destructans	65.24±2.20ab
尖镰孢菌F. oxysporum	65.24±0.81ab

病原真菌Pathogenic fungus	抑菌率Inhibitory/%
茄立枯丝核菌R. solani	62.80±1.72b
茄腐皮镰孢菌F. solani	67.89±0.70a
链格孢菌A. panax	64.94±1.29ab
毁灭柱孢菌C. destructans	65.24±2.20ab
尖镰孢菌F. oxysporum	65.24±0.81ab

处理Treatment	因素 Factor				抑菌率 Inhibitory/%
处理Treatment	温度Temperature/℃	时间Time/d	装液量Liquid content/mL	转速Speed/（r·min^-1）	抑菌率 Inhibitory/%
1	25	1	30/250	150	71.14±1.27
2	25	2	50/250	180	63.21±1.27
3	25	2.5	80/250	200	61.89±0.43
4	28	1	50/250	200	74.19±0.93
5	28	2	80/250	150	62.50±0.43
6	28	2.5	30/250	180	64.63±2.66
7	30	1	80/250	180	69.51±1.06
8	30	2	30/250	200	61.38±1.96
9	30	2.5	50/250	150	62.20±0.61
BPY基础发酵液 BPY basic fermentation					62.20±0.61
k1	65.41	71.61	65.72	65.28
k2	67.11	62.36	66.53	65.78
k3	64.36	62.91	64.63	65.82
R	2.74	9.25	1.90	0.54
最优组合 Optimal combination	k2	k1	k2	k3

处理Treatment	因素 Factor				抑菌率 Inhibitory/%
处理Treatment	温度Temperature/℃	时间Time/d	装液量Liquid content/mL	转速Speed/（r·min^-1）	抑菌率 Inhibitory/%
1	25	1	30/250	150	71.14±1.27
2	25	2	50/250	180	63.21±1.27
3	25	2.5	80/250	200	61.89±0.43
4	28	1	50/250	200	74.19±0.93
5	28	2	80/250	150	62.50±0.43
6	28	2.5	30/250	180	64.63±2.66
7	30	1	80/250	180	69.51±1.06
8	30	2	30/250	200	61.38±1.96
9	30	2.5	50/250	150	62.20±0.61
BPY基础发酵液 BPY basic fermentation					62.20±0.61
k1	65.41	71.61	65.72	65.28
k2	67.11	62.36	66.53	65.78
k3	64.36	62.91	64.63	65.82
R	2.74	9.25	1.90	0.54
最优组合 Optimal combination	k2	k1	k2	k3

处理 Treatment	病情指数 Disease index	防治效果 Control effect/%
YZ-228	21.48±2.57 b	59.15±4.88
CK	52.59±5.13 a	-