Screening, identification and optimization of fermentation conditions of antagonistic Xenorhabdus bovienii 445 against Aspergillus niger

doi:10.6048/j.issn.1001-4330.2022.05.020

Abstract

Abstract:

【Objective】 Entomopathogenic nematode symbiosis have broad antimicrobial spectrum and effective antibacterial effects, so screening strains with highly antagonistic effects against Aspergillus niger from the existing commensal bacteria resources will provide more choices for seeking safe and efficient biopreservation materials. 【Methods】 The existing entomopathogenic nematode symbiosis were preliminarily screened and re screened by plate confrontation method and agar diffusion method respectively, so as to screen the highly antagonistic symbiotic bacteria, identify the highly antagonistic symbiotic bacteria by physiological and biochemical analysis and 16S rRNA sequence evolution analysis, and optimize the strain culture conditions by single factor test and response surface analysis. The control efficiency for Aspergillus niger in Red Globe grape was verified by injury inoculation method. 【Results】 A total of 20 antagonistic symbiotic bacteria were obtained through preliminary isolation and screening, and a symbiotic bacterium (445) with significant antibacterial effect was obtained through re screening. Through physiological, biochemical and molecular biological analysis, it was identified as pathogenic bacterium Xenorhabdus bovienii, Genebank ID: OK560680 through physiological, biochemical and molecular biological analysis. It was clustered in the same branch as strain Xenorhabdus bovienii strain Xb139 (MG995576.1), with a similarity of 99.79%. Through single factor test and response surface analysis, the optimal culture conditions for the antibacterial activity of strain 445 were as follows: inoculation amount of 3.06 %, pH 7.0, liquid volume of 100.15 mL / 250 mL. Under these conditions, the diameter of antibacterial circle was 29.67±0.28 mm, and the antibacterial titer was 10.59 cm/mL, which was 39.16% higher than that before optimization. In the normal temperature antiefficiency experiment of grapes, the fermentation broth of Xenorhabdus bovienii 445 had a better control effect on Aspergillus niger, and the control effect was 63.50 % after 3 days. 【Conclusion】 In this study, entomopathogenic nematode symbiotic bacteria were used to control Aspergillus niger infection during grape storage for the first time, and a highly effective entomopathogenic nematode symbiotic bacteria against Aspergillus niger was screened. Its fermentation broth has good inhibitory effect on Aspergillus niger and good biocontrol potential, which provides new biocontrol materials and research ideas for biocontrol of postharvest pathogen Aspergillus niger.

Key words: Entomopathogenic nematode; Symbiotic bacteria; Aspergillus niger; Optimization of fermentation conditions; Biocontrol

摘要：

【目的】从现有共生细菌资源中筛选对黑曲霉具有高拮抗作用的菌株,为寻求安全高效的生物保鲜材料提供更多选择。【方法】采用平板对峙法和琼脂扩散法,分别对现有昆虫病原线虫共生菌进行初筛和复筛,对筛选出的高效拮抗共生细菌进行生理生化以及16S rRNA 序列进化分析鉴定,通过单因素试验和响应面分析法优化菌株培养条件,利用损伤接种法在红地球葡萄上验证对黑曲霉防治效果。【结果】分离筛选共获得20株拮抗共生菌,复筛得到1株抑菌效果显著的共生细菌(445),经生理生化及分子生物学分析为伯氏致病杆菌Xenorhabdus bovienii,GeneBank ID:OK560680,与菌株Xenorhabdus bovienii strain Xb139 (MG995576.1)聚于同一分支,相似性达99.79%。获得445菌株抑菌活性的最优培养条件为接种量3.06 %、pH 7.0、装液量100.15 mL/250 mL,抑菌圈直径为(29.67±0.28) mm,抑菌效价为10.59 cm/mL,比优化前提高39.16%。Xenorhabdus bovienii 445发酵液对黑曲霉具有较好防治效果,3 d后防效为63.50%。【结论】筛选得到1株高效拮抗黑曲霉的昆虫病原线虫共生细菌,其发酵液对黑曲霉有良好的抑制效果,具有较好的生防潜能。

关键词: 昆虫病原线虫, 共生细菌, 黑曲霉, 发酵优化, 生物防治

CLC Number:

S188

GAO Yujie, ZHAN Faqiang, CHEN Cheng, BAO Huifang, YANG Rong, WANG Ning, HOU Xinqiang, HOU Min, SHI Yingwu, LONG Xuanqi. Screening, identification and optimization of fermentation conditions of antagonistic Xenorhabdus bovienii 445 against Aspergillus niger[J]. Xinjiang Agricultural Sciences, 2022, 59(5): 1203-1215.

高宇洁, 詹发强, 陈澄, 包慧芳, 杨蓉, 王宁, 侯新强, 侯敏, 史应武, 龙宣杞. 黑曲霉拮抗菌Xenorhabdus bovienii445筛选、鉴定及发酵优化[J]. 新疆农业科学, 2022, 59(5): 1203-1215.

Figures/Tables 18

Table 1 Test strains of Entomopathogenic nematode symbiotic bacteria

菌株编号 Strain No.	种 Species
B5	Photorhabdus luminescens B5
445	Xenorhabdus bovienii 445
NLKSD 3-1	Photorhabdus temperata NLKSD 3-1
YC	Xenorhabdus bovienii YC
ALL	Xenorhabdus nematophila ALL
sf-scp	Xenorhabdus bovienii SCP
sf-ZMCH	Xenorhabdus bovienii sf-ZMCH
Turkey 40-4	Xenorhabdus bovienii Turkey40-4
B4-3	Xenorhabdus bovienii B4-3
482	Photorhabdus laumondii 482
B4-2	Xenorhabdus bovienii B4-2
sf-ZMBHW	Xenorhabdus bovienii ZMBHW
B30	Xenorhabdus bovienii B30
B13	Xenorhabdus bovienii B13
422	Xenorhabdus bovienii 422
B24	Xenorhabdusbovienii B24
421	Xenorhabdus bovienii 421
B4-1	Xenorhabdus bovienii B4-1
757	Xenorhabdus bovienii 757
sf-ZMHYL	Xenorhabdu bovienii ZMHYL

Table 2 Factors and levels of Box-Behnken design

因素 Design	水平Levels
因素 Design	-1	0	1
接种量A Lnoculation Amocont (%)	2.5	3	3.5
pH B	6.5	7.0	7.5
装液量C Liquid loading (mL)	90	100	110

Fig.1 Antagonistic effects of different symbiotic bacteria strains on Aspergillus niger

Table 3 Inhibition rate of Aspergillus niger by different symbiotic strains

菌株编号 Strain No.	抑制率 Inhibition rate(%)
B5	65.00±1.47^a
445	64.92±2.05^a
NLKSD 3-1	64.30±1.95^ab
YC	63.58±0.50^abc
ALL	62.86±2.98^abcd
sf-scp	62.25±1.67^abcd
sf-ZMCH	61.86±1.58^bcd
Turkey 40-4	61.75±1.36^bcd
B4-3	61.50±3.53^bcde
482	61.44±0.55^bcde
B4-2	61.35±3.20^bcde
sf-ZMBHW	61.12±1.20^cde
B30	61.02±0.94^cde
B13	60.96±1.74^cde
422	60.95±1.80^cde
B24	60.68±1.67^cde
421	60.20±1.02^de
B4-1	60.00±1.38^de
757	59.84±1.26^de
sf-ZMHYL	58.49±1.55^e

Fig.2 The antagonistic effect of different symbiotic bacterial strains on Aspergillus niger Note: 1: 445; 2: YC; 3: B5; 4: NLKSD3-1; 5: scp; 6: ALL.

Table 4 The inhibitory effect of different symbiotic strains on Aspergillus niger

菌株 Strain No.	抑菌圈直径 Inhibition zone diameter (mm)	抑菌效价 Anti-bacterial potency(cm/mL)
445	23.72±0.61^a	7.61
YC	23.33±0.50^b	7.41
B5	20.42±0.46^c	5.96
NLKSD3-1	13.96±0.59^d	2.73
sf-scp	13.20±0.51^e	2.35
ALL	0	-

Fig.3 A: Colonial morphology of Xenorhabdus bovienii 445; B: Gram staining of Xenorhabdus bovienii 445

Table 5 Main physiological and biochemical test of Xenorhabdus bovienii 445

生理生化试验 Physiological and biochemical test	结果 Results
革兰氏染色	-
葡萄糖氧化发酵试验	发酵型
V-P 试验	-
甲基红试验	-
耐盐性试验	2%
油脂水解试验	+
淀粉水解试验	-
硫化氢产生试验	-
接触酶试验	-
明胶液化试验	-
葡萄糖酸盐氧化试验	+
脲酶试验	-
蔗糖发酵试验	-
脂酶试验	-
乳糖发酵试验	+
甘露醇发酵试验	-

Fig.4 Phylogenetic tree of strain 445 based on 16S rRNA genesequence Note: Numbers at branch nodes present bootstrap value; The GeneBank accession number of aligned sequences are shown in the brackets; Bar: Nucleotide divergence

Fig.5 The growth curve of Xenorhabdus bovienii 445

Fig.6 Effects of different initial seed ages (A),inoculum concentration (B), pH (C), liquid loading (D), time (E), and temperature (F) on the inhibitory activity of Xenorhabdus bovienii 445

Table 6 Effect of different test sites on the antibacterial activity of strain 445

试验编码 Experi mental coding	编码水平 Encoding level			抑菌圈直径 Bacteriostatic sphere diameter (mm)
试验编码 Experi mental coding	A	B	C	抑菌圈直径 Bacteriostatic sphere diameter (mm)
1	-1	1	0	24.46
2	1	0	1	25.44
3	-1	0	1	25.06
4	0	1	1	25.78
5	0	0	0	27.79
6	1	-1	0	24.30
7	1	1	0	26.23
8	0	0	0	28.23
9	0	0	0	27.46
10	0	-1	1	23.12
11	0	0	0	27.31
12	0	0	0	27.44
13	0	-1	-1	25.31
14	-1	0	-1	23.65
15	-1	-1	0	24.44
16	1	0	-1	24.57
17	0	1	-1	25.38

Table 7 Significance of the variance in the quadratic regression model

方差来源 Source of variance	平方和 Quadratic sum	自由度 Degree of freedom	均方 Mean square	F值 F value	P值 P value	显著性 Significance
模型	34.96	9	3.88	10.03	0.003	**
A-接种量	1.07	1	1.07	2.77	0.14
B- PH	2.74	1	2.74	7.07	0.032 5	*
C-装液量	0.03	1	0.03	0.077	0.788 8
AB	0.91	1	0.91	2.35	0.168 8
AC	0.07	1	0.073	0.19	0.677 5
BC	1.68	1	1.68	4.33	0.076
A²	9.51	1	9.51	24.55	0.001 6	**
B²	6.96	1	6.96	17.96	0.003 9	**
C²	9.01	1	9.01	23.26	0.001 9	**
剩余	2.71	7	0.39
失拟项	2.16	3	0.72	5.22	0.072 1
纯误差	0.55	4	0.14
总离差	37.68	16
R²=0.928 0 $R_{A d j}^{2}$ =0.835 5

Table 7 Significance of the variance in the quadratic regression model

方差来源 Source of variance	平方和 Quadratic sum	自由度 Degree of freedom	均方 Mean square	F值 F value	P值 P value	显著性 Significance
模型	34.96	9	3.88	10.03	0.003	**
A-接种量	1.07	1	1.07	2.77	0.14
B- PH	2.74	1	2.74	7.07	0.032 5	*
C-装液量	0.03	1	0.03	0.077	0.788 8
AB	0.91	1	0.91	2.35	0.168 8
AC	0.07	1	0.073	0.19	0.677 5
BC	1.68	1	1.68	4.33	0.076
A²	9.51	1	9.51	24.55	0.001 6	**
B²	6.96	1	6.96	17.96	0.003 9	**
C²	9.01	1	9.01	23.26	0.001 9	**
剩余	2.71	7	0.39
失拟项	2.16	3	0.72	5.22	0.072 1
纯误差	0.55	4	0.14
总离差	37.68	16
R²=0.928 0 $R_{A d j}^{2}$ =0.835 5

Fig.7 Influence of interaction of two factors on the diameter of bacteriostatic zone

Fig.8 Comparison of the antibacterial activity before and after the optimization Note: A and B are the optimized antibacterial effect; C and D are the antibacterial effect before optimization

Table 8 Incidence rate of treatment group and control group within 3-7 days

时间 Time (d)	Incidence(%)
时间 Time (d)	处理组	CK
3	0.00	6.00
4	32.00	54.00
5	42.00	62.00
6	75.00	76.00
7	100.00	100.00

Table 9 The inhibitory effect of Xenorhabdus bovienii 445 on Aspergillus niger within 3-7 days

Time (d)	CK	处理组
Time (d)	Lesion diameter (mm)	Lesion diameter (mm)	Control effect (%)
3	7.19±0.10^a	0.00	-
4	7.61±0.32^a	6.01±0.40^a	61.30
5	8.80±0.03^a	6.93±0.45^b	49.21
6	10.89±0.99^a	8.65±0.48^b	38.03
7	12.57±0.29^a	10.47±0.0 ${6^{b}}^{c}$	27.74

Table 9 The inhibitory effect of Xenorhabdus bovienii 445 on Aspergillus niger within 3-7 days

Time (d)	CK	处理组
Time (d)	Lesion diameter (mm)	Lesion diameter (mm)	Control effect (%)
3	7.19±0.10^a	0.00	-
4	7.61±0.32^a	6.01±0.40^a	61.30
5	8.80±0.03^a	6.93±0.45^b	49.21
6	10.89±0.99^a	8.65±0.48^b	38.03
7	12.57±0.29^a	10.47±0.0 ${6^{b}}^{c}$	27.74

Fig.9 Control effect of Xenorhabdus bovienii 445 on Aspergillus niger in grape fruit

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