刺五加黑斑病生防细菌分离、鉴定、优选及发酵条件优化

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

摘要/Abstract

摘要：

【目的】从刺五加根际土壤中筛选、分离并鉴定对刺五加黑斑病具有生防潜力的细菌，在此基础上优化其发酵条件，为刺五加黑斑病生物防治提供良好的生防菌源。【方法】采用稀释涂布法从健康刺五加根际土壤中分离细菌，平板对峙法筛选对细极链格孢菌（Alternaria tenuissima）具有较强拮抗效果的菌株，测定其对另外5种病原真菌的抑制作用；通过形态特征、生理生化特征及16S rDNA基因测序明确该菌株的分类学地位；以单因素试验和正交试验方法进行培养基组分及发酵条件优化；此外，探讨该菌株发酵液对刺五加黑斑病的防治效果。【结果】分离获得419株细菌中菌株YZ-228对细极链格孢菌的抑制率最高，达67.38%，无菌滤液的抑制率为64.32%。菌株YZ-228对茄立枯丝核菌（Rhizoctonia solani）、茄腐皮镰孢菌（Fusarium solani）、链格孢菌（A. panax）、毁灭柱孢菌（Cylindrocarpon destructans）、尖镰孢菌（Fusarium oxysporum）均具有抑制效果，抑制率为62.80%-67.89%。菌株YZ-228初步鉴定为蜡样芽孢杆菌（Bacillus cereus）。可溶性淀粉5 g/L和牛肉膏5 g/L是YZ-228最佳碳源和氮源，在pH 7.0、温度28℃、接种量3%、装液量50 mL/250 mL、转速200 r/min条件下，发酵1 d的菌液对细极链格孢菌拮抗效果较好，抑制率达74.19%，较牛肉膏蛋白胨酵母膏培养基发酵液（BPY）提高11.99%。此外，接种菌株YZ-228发酵液显著降低了刺五加黑斑病的病情指数，防效达59.15%。【结论】蜡样芽孢杆菌YZ-228对刺五加黑斑病有较好的防治效果，具有作为刺五加黑斑病生物防治剂的潜力。

关键词: 刺五加, 细极链格孢菌, 蜡样芽孢杆菌, 黑斑病, 生物防治

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

丁艳哲, 姚鑫鑫, 孙卓, 杨利民, 韩忠明, 王云贺. 刺五加黑斑病生防细菌分离、鉴定、优选及发酵条件优化[J]. 生物技术通报, 2024, 40(12): 218-226.

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.

图/表 8

图1 菌株YZ-228对细极链格孢菌的拮抗效果

Fig. 1 Inhibitory effect of antagonistic strain YZ-228 on A. tenuissima

表1 菌株YZ-228的抑菌谱测定

Table 1 Antifungal spectrum of strain YZ-228

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

图2 菌株YZ-228的生物学特性 A：菌落形态；B：革兰氏染色（100×）；C：芽孢染色（100×）

Fig. 2 Biological characteristics of strain YZ-228 A: Colony morphology. B: Gram stain（100×）. C: Spore stain（100×）

图3 基于16S rDNA构建了菌株YZ-228的系统发育树

Fig. 3 Phylogenetic tree of strain YZ-228 constructed based on 16S rDNA

图4 发酵培养基组分对YZ-228抑菌活性的影响 A：碳源；B：氮源；不同小写字母表示P<0.05水平差异显著，下同

Fig. 4 Effects of fermentation medium components on the antibacterial activity of YZ-228 A: Carbon source. B: Nitrogen source. Different lower letters indicate significant different at P<0.05. The same below

图5 发酵条件对YZ-228抑菌活性的影响

Fig. 5 Effects of fermentation condition on the antibacterial activity of YZ-228

表2 菌株YZ-228 L9（34）正交试验结果

Table 2 Result of orthogonal experiment of L9（34）of strain YZ-228

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

表3 YZ-228发酵液对刺五加黑斑病的防治效果

Table 3 Control effect of YZ-228 fermentation liquid against A. tenuissima

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	-

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