一株增强中华绒螯蟹抗病力的固氮红细菌SY5的分离鉴定与表征

doi:10.13560/j.cnki.biotech.bull.1985.2022-0043

摘要/Abstract

摘要：

为丰富在中华绒螯蟹病害防控中具有潜在应用价值的微生物资源，以类胡萝卜素产生能力作为评价指标，从养殖池底泥中分离获得一株优良的好氧光合细菌SY5，在形态与生理生化鉴定以及分子鉴定的基础上对其进行药敏特性分析，并进一步评价了其对中华绒螯蟹抗弗氏柠檬酸杆菌感染的保护效果。结果表明，菌株SY5为固氮红细菌（Rhodobacter azotoformans），革兰氏染色阴性，在营养琼脂平板上的菌落特征为深红色、圆形、边缘整齐、光滑、湿润，与固氮红细菌YLK20的亲缘关系最近，对阿莫西林、新霉素、庆大霉素、四环素、多黏菌素B、恩诺沙星、环丙沙星、卡那霉素、氨苄西林、罗红霉素、磷霉素、链霉素高度敏感，对多西环素中度敏感；在饲料中添加终浓度为（6.0×10⁵）-（6.0×10⁹）CFU/g时对中华绒螯蟹抗弗氏柠檬酸杆菌感染的有效保护率为60.0%-100%。这些研究结果为固氮红细菌SY5作为中华绒螯蟹养殖用饲料添加剂的后期开发奠定了基础。

关键词: 固氮红细菌, 中华绒螯蟹, 表征, 弗氏柠檬酸杆菌

Abstract:

In order to enrich microbial resources with the potential application in the prevention and control of diseases in Eriocheir sinensis, strain SY5 of aerobic photosynthetic bacterium was isolated from the sediment of culture pond with carotenoid production ability as the evaluation index, was identified based on morphological, biochemical and molecular identification, and its antibiotic susceptibility and protective effect on E. sinensis against Citrobacter freundii infection were further evaluated. The results indicated that the SY5 isolate was identified phenotypically and molecularly as Rhodobacter azotoformans. It was Gram negative, formed colonies with the characteristics of dark red, round, smooth, moist and regular-edged on nutrient agar plate, showed the closest genetic relationship with R. azotoformans YLK20, and demonstrated highly sensitive to amoxicillin, neomycin, gentamicin, tetracycline, polymyxin B, enrofloxacin, ciprofloxacin, kanamycin, ampicillin, roxithromycin, fosfomycin, streptomycin and moderately sensitive to doxycycline. Besides, it demonstrated effective protection rates of 60.0% to 100% at final doses of 6.0×10⁵ to 6.0×10⁹ CFU/g diets against C. freundi infection in E. sinensis. These data lays a foundation for the later development of R. azotoformans SY5 as a feed additive for E. sinensis aquaculture.

Key words: Rhodobacter azotoformans, Eriocheir sinensis, characterization, Citrobacter freundii

曹海鹏, 张书萌, 刁菁, 许拉, 盖春蕾. 一株增强中华绒螯蟹抗病力的固氮红细菌SY5的分离鉴定与表征[J]. 生物技术通报, 2022, 38(11): 277-285.

CAO Hai-peng, ZHANG Shu-meng, DIAO Jing, XU La, GAI Chun-lei. Isolation，Identification and Characterization of Rhodobacter azotoformans SY5 with Enhancing Effect on the Disease Resistance of Eriocheir sinensis[J]. Biotechnology Bulletin, 2022, 38(11): 277-285.

图/表 6

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项目Test	结果Result		项目Test	结果Result
项目Test	SY5	固氮红细菌R. azotoformans^[13]	项目Test	SY5	固氮红细菌R. azotoformans^[13]
甲酸钠Sodium formate	+	+	半乳糖醇 Dulcitol	+	+
乙酸钠Sodium acetate	+	+	甘露醇Mannitol	+	+
丙酸钠Sodium propionate	-	-	柠檬酸钠Trisodium citrate dihydrate	+	+
甘油Glycerol	+	+	酒石酸钠Sodium tartaric	-	-
葡萄糖Glucose	+	+	葡萄糖酸钠Sodium citrate	+	+
果糖Fructose	+	+	乙醇Ethanol	+	+
过氧化氢酶Catalase	+	+	硫代硫酸钠Sodium thiosulfate	-	-
甘露糖Mannose	+	+	硫化钠Sodium sulfide	-	-
苯甲酸钠Sodium benzoic	-	-	3%NaCl生长 3%NaCl growth	+	+

项目Test	结果Result		项目Test	结果Result
项目Test	SY5	固氮红细菌R. azotoformans^[13]	项目Test	SY5	固氮红细菌R. azotoformans^[13]
甲酸钠Sodium formate	+	+	半乳糖醇 Dulcitol	+	+
乙酸钠Sodium acetate	+	+	甘露醇Mannitol	+	+
丙酸钠Sodium propionate	-	-	柠檬酸钠Trisodium citrate dihydrate	+	+
甘油Glycerol	+	+	酒石酸钠Sodium tartaric	-	-
葡萄糖Glucose	+	+	葡萄糖酸钠Sodium citrate	+	+
果糖Fructose	+	+	乙醇Ethanol	+	+
过氧化氢酶Catalase	+	+	硫代硫酸钠Sodium thiosulfate	-	-
甘露糖Mannose	+	+	硫化钠Sodium sulfide	-	-
苯甲酸钠Sodium benzoic	-	-	3%NaCl生长 3%NaCl growth	+	+

抗生素 Antibiotics	抑菌圈直径判断标准 Assessment standard of inhibition zone diameter /mm			药物含量 Content /（μg·disc^-1）	抑菌圈直径 Diameter of inhibition zone/mm	敏感性 Susceptibility
抗生素 Antibiotics	不敏感R Resistance	中度敏感I Intermediate sensitivity	高度敏感S Sensitivity	药物含量 Content /（μg·disc^-1）	抑菌圈直径 Diameter of inhibition zone/mm	敏感性 Susceptibility
阿莫西林Amoxicillin	≤13	14-17	≥18	10	24.30±0.78	S
新霉素Neomycin	≤12	13-16	≥17	30	24.05±0.83	S
庆大霉素Gentamicin	≤12	13-14	≥15	10	18.20±0.44	S
四环素Tetracycline	≤14	15-18	≥19	30	19.22±0.56	S
多黏菌素B Polymyxin B	≤8	9-11	≥12	30	15.21±0.82	S
恩诺沙星Enrofloxacin	≤15	16-18	≥19	5	26.55±0.70	S
环丙沙星Ciprofloxacin	≤15	16-20	≥21	5	24.20±0.65	S
卡那霉素Kanamycin	≤13	14-17	≥18	30	24.40±0.45	S
氨苄西林Ampicillin	≤13	14-16	≥17	10	22.50±0.25	S
罗红霉素Roxithromycin	≤10	11-15	≥16	15	16.50±1.06	S
磷霉素Fosfomycin	≤12	13-15	≥16	200	23.12±0.60	S
链霉素Streptomycin	≤11	12-14	≥15	10	25.25±0.85	S
多西环素Doxycycline	≤17	18-22	≥23	30	20.85±0.65	I

抗生素 Antibiotics	抑菌圈直径判断标准 Assessment standard of inhibition zone diameter /mm			药物含量 Content /（μg·disc^-1）	抑菌圈直径 Diameter of inhibition zone/mm	敏感性 Susceptibility
抗生素 Antibiotics	不敏感R Resistance	中度敏感I Intermediate sensitivity	高度敏感S Sensitivity	药物含量 Content /（μg·disc^-1）	抑菌圈直径 Diameter of inhibition zone/mm	敏感性 Susceptibility
阿莫西林Amoxicillin	≤13	14-17	≥18	10	24.30±0.78	S
新霉素Neomycin	≤12	13-16	≥17	30	24.05±0.83	S
庆大霉素Gentamicin	≤12	13-14	≥15	10	18.20±0.44	S
四环素Tetracycline	≤14	15-18	≥19	30	19.22±0.56	S
多黏菌素B Polymyxin B	≤8	9-11	≥12	30	15.21±0.82	S
恩诺沙星Enrofloxacin	≤15	16-18	≥19	5	26.55±0.70	S
环丙沙星Ciprofloxacin	≤15	16-20	≥21	5	24.20±0.65	S
卡那霉素Kanamycin	≤13	14-17	≥18	30	24.40±0.45	S
氨苄西林Ampicillin	≤13	14-16	≥17	10	22.50±0.25	S
罗红霉素Roxithromycin	≤10	11-15	≥16	15	16.50±1.06	S
磷霉素Fosfomycin	≤12	13-15	≥16	200	23.12±0.60	S
链霉素Streptomycin	≤11	12-14	≥15	10	25.25±0.85	S
多西环素Doxycycline	≤17	18-22	≥23	30	20.85±0.65	I

浓度 Concentration/（CFU · g^-1）	蟹数 Crab No. /ind.	累计死亡数Total number of deaths /ind.							平均死亡率 Average mortality/%
浓度 Concentration/（CFU · g^-1）	蟹数 Crab No. /ind.	1 d	2 d	3 d	4 d	5 d	6 d	7 d	平均死亡率 Average mortality/%
0	10	0	3	4	6	6	8	9	83.33±11.55
	10	0	2	3	3	5	7	9
	10	0	1	2	3	5	5	7
6.0×10⁵	10	0	1	1	1	2	2	2	33.33±11.55
	10	0	1	1	1	2	3	4
	10	0	0	1	1	1	2	4
6.0×10⁶	10	0	0	0	1	1	1	2	13.33±5.77
	10	0	0	0	0	0	1	1
	10	0	0	0	0	0	0	1
6.0×10⁷	10	0	0	0	0	0	0	1	6.66±5.77
	10	0	0	0	0	0	0	1
	10	0	0	0	0	0	0	0
6.0×10⁸	10	0	0	0	0	0	0	0	0±0
	10	0	0	0	0	0	0	0
	10	0	0	0	0	0	0	0
6.0×10⁹	10	0	0	0	0	0	0	0	0±0
	10	0	0	0	0	0	0	0
	10	0	0	0	0	0	0	0