一株拮抗葡萄灰霉病的嗜线虫致病杆菌发酵条件优化

doi:10.6048/j.issn.1001-4330.2022.11.012

新疆农业科学 ›› 2022, Vol. 59 ›› Issue (11): 2688-2695.DOI: 10.6048/j.issn.1001-4330.2022.11.012

• 植物保护・草业・畜牧兽医 • 上一篇下一篇

一株拮抗葡萄灰霉病的嗜线虫致病杆菌发酵条件优化

曹林青¹(), 詹发强²(), 杨蓉¹, 侯新强², 包慧芳, 侯敏², 王宁², 龙宣杞²

1.新疆大学生命科学与技术学院,乌鲁木齐 830046
2.新疆农业科学院微生物应用研究所/新疆特殊环境微生物重点实验室,乌鲁木齐 830091

收稿日期:2022-01-07 出版日期:2022-11-20 发布日期:2022-12-28
通信作者: 詹发强(1980-),男,新疆人,副研究员,硕士生导师,研究方向为农业微生物与生物防治,(E-mail)zfq_xj@126.com
作者简介:曹林青(1996-),女,硕士研究生,研究方向为农业微生物应用,(E-mail)1450743639@qq.com
基金资助:
新疆维吾尔自治区自然科学基金(2019D01A62);新疆农业科学院青年科技骨干创新能力培养(xjnkq-2019016)

Study on the Optimization of Fermentation Conditions of a Xenorhabdus nematophila against Grape Gray Mold

CAO Linqing¹(), ZHAN Faqiang²(), GAO Yujie¹, HOU Xinqiang², BAO Huifang, HOU Min², YANG Rong², WANG Ning², LONG Xuanqi²

1. College of Life Sciences and Technology, Xinjiang University, Urumqi 830046, China
2. Xinjiang Key Laboratory of Special Environmental Microbiology /Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China

Received:2022-01-07 Published:2022-11-20 Online:2022-12-28
Supported by:
Natural Science Foundation of Xinjiang Uygur Autonomous Region(2019D01A62);Innovation Ability Training Project for Young Sci-Tech Backbone Talents of Xinjiang Academy of Agricultural Sciences(xjnkq-2019016)

摘要/Abstract

摘要：

【目的】分析单因素试验和响应面法优化嗜线虫致病杆菌(Xenorhabdus nematophila-ALL)对葡萄灰霉菌抑菌活性的发酵条件,提高拮抗菌(ALL)对灰葡萄孢菌丝的抑制率,为Xenorhabdus nematophila-ALL菌株发酵生产及生物防治应用奠定基础。【方法】以菌丝抑制率为指标,经菌丝生长速率法,单因素试验及Box-Behnken设计对菌株发酵条件进行优化,确定菌株最佳发酵抑制条件。【结果】最佳培养条件为培养基初始pH值改为7.12,装液量为56％,发酵培养时间为77 h。经验证的抑制率为87.70％,比优化之前提高了1.82倍。【结论】发酵条件优化有效提高了嗜线虫致病杆菌对葡萄灰霉菌的抑菌活性。

关键词: 嗜线虫致病杆菌; 灰葡萄孢; 响应面法; 抑制率

Abstract:

【Objective】 This project aims to optimize the fermentation conditions for the antibacterial activity of Xenorhabdus nematophila-ALL (Xenorhabdus nematophila-ALL) strains against Botrytis cinerea through single factor test and response surface methodology in the hope of further increasing the inhibition rate of antagonistic bacteria against Botrytis cinerea hyphae and lay the relevant research foundation for the future large-scale fermentation production and biological control application of Xenorhabdus nematophila-ALL strains. 【Methods】 Using the mycelial growth rate method, taking the mycelial inhibition rate as an indicator, the Box-Behnken design was used to optimize the fermentation conditions of the strains through single factor and orthogonal experiments were conducted to determine the best inhibition conditions for the strains. 【Results】 The results showed that the optimal culture conditions were obtained by response surface analysis based on the single factor experiment: The initial pH of the medium was changed to 7.12, the bottling volume was 56％, and the fermentation culture time was 77h. The verified inhibition rate was 87.70％, which was 1.82 times higher than that before optimization. 【Conclusion】 The response surface optimization test effectively improves the antibacterial activity of Xenorhabdus nematophila.

Key words: Xenorhabdus nematophila-ALL; Botrytis cinerea; response surface method; inhibition rate

中图分类号:

S188

曹林青, 詹发强, 杨蓉, 侯新强, 包慧芳, 侯敏, 王宁, 龙宣杞. 一株拮抗葡萄灰霉病的嗜线虫致病杆菌发酵条件优化[J]. 新疆农业科学, 2022, 59(11): 2688-2695.

CAO Linqing, ZHAN Faqiang, GAO Yujie, HOU Xinqiang, BAO Huifang, HOU Min, YANG Rong, WANG Ning, LONG Xuanqi. Study on the Optimization of Fermentation Conditions of a Xenorhabdus nematophila against Grape Gray Mold[J]. Xinjiang Agricultural Sciences, 2022, 59(11): 2688-2695.

图/表 11

图1 不同培养基发酵上清液下灰葡萄孢(B. cinerea)的抑制率变化

Fig.1 Inhibition rate of fermentation supernatants of different media on B. cinerea

图2 不同时间发酵上清液下灰葡萄孢(B.cinerea)的抑制率变化

Fig.2 Inhibition rate of fermentation supernatant at different time on B. cinerea

图3 不同种子上清液下灰葡萄孢(B.cinerea)的抑制率变化

Fig.3 Inhibition rate of seed-age supernatant on B. cinerea

图4 不同接菌量下灰葡萄孢(B.cinerea)的抑制率变化

Fig.4 Inhibition rate of inoculation amount on B. cinerea

图5 装液量对灰葡萄孢(B.cinerea)抑制率变化

Fig.5 Inhibition rate of bottling volume on B. cinerea

图6 不同转速下灰葡萄孢(B.cinerea)的抑制率变化

Fig.6 Inhibition rate of rotation speed on B. cinerea

图7 不同初始pH值下灰葡萄孢(B.cinerea)的抑制率变化

Fig.7 Inhibition rate of initial pH on B. cinerea

表1 因素水平编码

Table 1 Coding of the factors and the corresponding levels

水平 Level	发酵培养时间 Fermentation time(h)	培养基初始pH值 Initial pH of the medium	装液量 Liquid volume (mL)
-1	60	5.5	40
0	72	7	50
1	84	8.5	60

表2 Box-Behnken试验设计

Table 2 Experimental design and results of Box-Behnken

试验号 Test number	因素1 Factor 1 培养基 pH值	因素2 Factor 2 发酵培养时间 (h)	因素3 Factor 3 装液量 (mL)	抑制率 Inhibition rate (％)
1	8.5	72	40	46.22
2	7	60	60	75.45
3	5.5	60	50	46.2
4	8.5	60	50	52.62
5	7	72	50	83.06
6	7	72	50	81.26
7	7	72	50	80.43
8	5.5	72	40	50.24
9	7	72	50	83.12
10	7	72	50	85.38
11	7	60	40	58.33
12	8.5	84	50	64.71
13	5.5	84	50	58.08
14	7	84	40	64.12
15	7	84	60	82.38
16	8.5	72	60	63.7
17	5.5	72	60	57.86

表3 回归模型方差

Table 3 Variance analysis of regression model

方差来源 Source of Variance	平方和 sum of square	自由度 Degree of freedom	均方 Mean square	F值 F value	P值 P value	显著性 Significance
模型	3 100.59	9	344.51	40.53	<0.000 1	＊＊
A-pH	27.64	1	27.64	3.25	0.114 3
B-发酵时间	168.27	1	168.27	19.8	0.003	＊＊
C-装液量	457.23	1	457.23	53.79	0.000 2	＊＊
AB	0.011	1	0.011	1.30E-03	0.972 3
AC	24.3	1	24.3	2.86	0.134 7
BC	0.32	1	0.32	0.038	0.850 5
A²	1 929.38	1	1 929.38	227	<0.000 1	＊＊
B²	143.66	1	143.66	16.9	0.004 5	＊＊
C²	191.2	1	191.2	22.5	0.002 1	＊＊
残差	59.5	7	8.5
失拟项	44.79	3	14.93	4.06	0.104 6
纯误差	14.7	4	3.68
总误差	3 160.09	16

图8 各因素交互作用下B. cinerea 抑制率响应面图及等高线

Fig.8 Response surface plot and contour plot of the influence of the interaction of various factors on the inhibition rate of B. cinerea

参考文献 25

[1]	中国统计年鉴[J]. 北京:中国统计出版社, 2017.
	National Bureau of Statistics. China Statistical Yearbook[J]. Beijing: China Statistics Press, 2017.
[2]	罗琳, 王其慧, 赵海霞, 等. 葡萄灰霉病生防菌株的筛选及其拮抗菌机理初探[J]. 中国酿造, 2017, 36(4): 93-98
	LUO Lin L, Wang Qihui, ZHAO Haixia, et al. Screening of bio-control strain against Botrytis cinerea and preliminary research on its antagonistic mechanisms[J]. China Brewing, 2017, 36(4): 93-98.
[3]	Ding Y L, Yang X Z, Li X H, et al. Effect of SO2 Interval Fumigation on Storage Quality of Red Grape[J]. Applied Mechanics and Materials, 2013, (411):3170-3173.
[4]	周拥军, 陈文煊, 陈杭君, 等. SO2控释保鲜剂对南方巨峰葡萄的贮藏效果研究[J]. 食品科学, 2008, 29(7):445-447.
	ZHOU Yongjun, CHEN Wenxuan, CHEN Hangjun, et al. Study on storage effect of SO₂ controlled release preservative on southern Jufeng grape[J]. Food Science, 2008, 29(7):445-447.
[5]	Gabler F M, Smilanick J L, Mansour M F, et al. Influence of fumigationwith high concentrations of ozone gas on postharvest gray mold and fungicide residues on table grapes[J]. Postharvest Biology and Technology, 2010, 55(2):85-90. DOI URL
[6]	McInerney B V, Greg son R P, Akhurst R J. Biologically active metabolites from Xenorhabdus sp.[J]. Journal of Natural Product, 1991, 54(3): 774-795. DOI URL
[7]	Akhurst R J. Antibiotic activity of Xenorhabdus spp. bacteria symbiotically associated with insect pathogenic nematodes of the families Heterorhabditidae and Steinernematidae[J]. Journal of General Microbiology, 1982, (128): 3061-3065.
[8]	Paul V J, Frautschy S, Fenical W, et al. Antibiotics in microbial ecology : isolation and structure assignment of several new antibacterial compounds from the insect - symbiotic bacteria Xenorhabdus sp.[J]. Journal of Chemical Ecology, 1981, (7): 589-597.
[9]	Maxwell P, Chen G, Webster J M, et al. Culture conditions for Xenorhabdus and Photorhabdus symbionts of entomopathogenic nematodes[J]. Nematologica, 1996, 42(1): 124-130. DOI URL
[10]	Fang X L, Han L R, Cao X Q, et al. Statistical Optimization of Process Variables for Antibiotic Activity of Xenorhabdus bovienii[J]. Plos One, 2012, 7.
[11]	Sa-Uth C, Rattanasena P, Chandrapatya A, et al. Modification of Medium Composition for Enhancing the Production of Antifungal Activity from Xenorhabdus stockiae PB09 by Using Response Surface Methodology[J]. International Journal of Microbiology, 2018:1-10.
[12]	邱德文, 焦宁宁, 刘峥, 等. 致病杆菌d43菌株产素培养基及发酵条件[J]. 中国生物防治, 2006,(1):58-62.
	QIU Dewen, JIAO Ningning, LIU Zheng, et al. Cultural Medium and Fermentation Conditions of Xenorhabdus sp. Strain D43[J]. Chinese Journal of Biological Control, 2006,(1):58-62.
[13]	刘洪敏, 钱海涛, 董辉, 等. 昆虫病原线虫共生菌5-5B培养特性及杀虫活性研究[J]. 中国植保导刊, 2007, 27(4): 5-9.
	LIU Hongmin, QIAN Haitao, DONG Hui, et al. Studies on the cultivation characteristics and insecticidal activity of anentomopathogenic nematode symbiotic bacteria strain 5-5B[J]. China Plant Protection, 2007, 27(4): 5-9.
[14]	马丽丽, 台莲梅, 许艳丽, 等. 发光杆菌NJ菌株的抑菌活性研究[J]. 植物保护, 2008, 34(3): 25-29.
	MA Lili, TAI Lianmei, XU Yanli, et al. A study on the antifungal activity of Photorhabdus sp. NJ[J]. Plant Protection, 2008, 34(3): 25-29.
[15]	詹发强, 布卡·欧尔娜, 侯敏, 等. 新疆嗜菌异小杆线虫及其共生细菌的初步鉴定研究[J]. 新疆农业科学, 2015, 52(2): 244-251.
	ZHAN Faqiang, Buka Ouerna, HOU Min, et al. Preliminary Identification of Entomopathogenic Nematodes (Heterorhabditis bacteriophora strain Nileke) and Its Symbiotic Bacteria in Xinjiang[J]. Xinjiang Agricultural Sciences, 2015, 52(2): 244-251.
[16]	Akhurst R J, Boemare N E. A numerical taxonomic study of the genus Xenorhabdus (Enterobacteriaceae) and proposed elevation of the subspecies of X. nematophilus to species[J]. Journal of General Microbiology, 1988, 134(7): 1835-1845. PMID
[17]	Liu J, Berry R, Poinar G, et al. Phylogeny of Photorhabdus and Xenorhabdus Species and Strains as Determined by Comparison of Partial 16S rRNA Gene Sequences[J]. International Journal of Systematic Bacteriology, 1997, 47(4): 948. PMID
[18]	Boemare N E, Akllurst R J. Biochemical and physiological characterization of colony form variants in Xenorhabdus spp. (Enterobacteriaceae)[J]. Journal of General Microbiology, 1988, (134):751-761.
[19]	Chen G H, Maxwell P, Dunphy G B, et al. Culture conditions for Xenorhabdus and Photorhabdus symbionts of entomopathogenic nematodes[J]. Nematologica, 1996, (42):124-127.
[20]	郭树奇. pH调控嗜线虫致病杆菌抑菌活性机理的初步研究[D]. 杨凌: 西北农林科技大学, 2015.
	GUO Shuqi. Study on antibacterial mechanism of Xenorhabdus nematophila regulated by pH[D]. Yangling: Northwest A & F University, 2015.
[21]	周启, 王道本. 农用抗生素和微生物杀虫剂[M]. 北京: 中国农业出版社, 1995.
	ZHOU Qi, WANG Daoben. Agricultural antibiotics and microbial insecticides[M]. Beijing: China Agriculture Press, 1995.
[22]	陈坚, 李寅. 发酵过程优化原理与实践[M]. 化学工业出版社, 2002.
	CHEN Jian, LI Yin. The principle and practice of fermentation process optimization[M]. Beijing: Chemical Industry Press, 2002.
[23]	郭蔷薇, 张淑静, 李忝珍, 等. 恒定pH对Xenorhabdus nematophila YL001抑菌活性及抑菌物质合成相关基因表达的影响[J]. 西北农业学报, 2016, 25(9):1413-1419.
	GUO Qiangwei, ZHANG Shujing, LI Tianzhen, et al. Effect of Constant pH on Antimicrobial Activity and Related Gene Expression of Antimicrobial Bioactivities Synthesis of Xenorhabdusnematophila YL001[J]. Acta Agriculturae Boreali-occidentalis Sinica, 2016, 25(9):1413-1419.
[24]	方香玲. 昆虫病原线虫共生菌的鉴定、培养及其抑菌活性研究[D]. 杨凌: 西北农林科技大学, 2008.
	FANG Xianling. Study on identification, culture and antimicrobial activity of entomopathognic bacteria[D]. Yangling: Northwest A & F University, 2008.
[25]	Wang Y H, Feng J T, Zhang Q, et al. Optimization of fermentation condition for antibiotic production by Xenorhabdus nematophila with response surface methodology[J]. Journal of Applied Microbiology, 2010, 104(3):735-744. DOI URL

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一株拮抗葡萄灰霉病的嗜线虫致病杆菌发酵条件优化

Study on the Optimization of Fermentation Conditions of a Xenorhabdus nematophila against Grape Gray Mold

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