中药渣堆肥中解磷细菌的筛选、鉴定及其拮抗作用

doi:10.13560/j.cnki.biotech.bull.1985.2021-0571

摘要/Abstract

摘要：

为开发新型、安全及高效的微生物菌肥,以堆肥2个月后的中药渣为试材,筛选解磷拮抗细菌。用有机磷细菌培养基初步筛选5株解磷细菌;采用溶磷圈法和磷钼蓝比色法测定初筛菌株的解磷作用,解磷作用最优的菌株是YP5;通过形态学特征观察、生理生化试验和16S rDNA序列分析鉴定,确定菌株YP5为铜绿假单胞菌（Pseudomonas aeruginosa）。促生拮抗因子测定结果表明菌株YP5能分泌铁载体和大量胞外蛋白酶;药敏实验结果显示菌株YP5对诺氟沙星、氯霉素、庆大霉素、恩诺沙星、丁胺卡那霉素高度敏感;分别采用平板对峙法和带毒培养基法测定结果表明菌株 YP5发酵液和无菌滤液均对冬瓜根腐病等9种植物病原菌有良好的拮抗作用,抑菌率最高达80%以上。高效液相色谱测定菌株YP5在28℃、KB培养基中抗菌代谢物申嗪霉素的产量最高,为17.73 mg/L。

关键词: 解磷细菌, 铜绿假单胞菌, 解磷, 拮抗

Abstract:

To develop new,safe and efficient microbial fertilizer,Chinese medicine residues after two months composting was used as test material to screen phosphate-solubilizing and antagonistic bacteria. Organophosphorus bacteria medium was used to preliminarily screen 5 strains of phosphate-solubilizing bacteria. Phosphorus solubilizing ring method and phosphorus-molybdenum blue colorimetry were employed to determine phosphorus solubility of the strains. The strain with the best phosphorus solubility was YP5. Based on the colony morphology,physiological and biochemical characteristics,and 16S rDNA sequence analysis,YP5 was identified as Pseudomonas aeruginosa. The results of promoting and antagonist factors showed that YP5 secreted iron carrier and a large amount of extracellular protease. YP5 was highly sensitive to norfloxacin,chloramphenicol,gentamicin,enrofloxacin and amikacin. The results from the plate confrontation method and the virulent medium method revealed that YP5 fermentation broth and cell-free filtrate presented promising antagonism on 9 plant pathogens such as Fusarium solani,and the maximum inhibition rate was > 80%. The highest yield of phenazino-1-carboxylic acid of YP5 in KB medium at 28℃was 17.73 mg/L by high performance liquid chromatography.

Key words: phosphate-solubilizing bacteria, Pseudomonas aeruginosa, phosphate-solubilizing, antagonistic action

胡珊, 梁卫驱, 黄皓, 徐匆, 罗华建, 胡楚维, 黄晓彦, 陈仕丽. 中药渣堆肥中解磷细菌的筛选、鉴定及其拮抗作用[J]. 生物技术通报, 2022, 38(3): 92-102.

HU Shan, LIANG Wei-qu, HUANG Hao, XU Cong, LUO Hua-jian, HU Chu-wei, HUANG Xiao-yan, CHEN Shi-li. Screening,Identification and Antagonism of Phosphate-Solubilizing Bacteria from the Compost Chinese Medicinal Herbal Residues[J]. Biotechnology Bulletin, 2022, 38(3): 92-102.

图/表 11

参考文献 37

[1]	钟传青. 解磷微生物溶解磷矿粉和土壤难溶磷的特性及其溶磷方式研究[D]. 南京:南京农业大学, 2004.
	Zhong CQ. Studies on solubilizing effects on phosphate rock powder and insoluble phosphorus in soil of P-solubilizing microorganisms and their mechanism[D]. Nanjing:Nanjing Agricultural University, 2004.
[2]	Westheimer F. Why nature chose phosphates[J]. Science, 1987, 235(4793):1173-1178. pmid: 2434996
[3]	曹宁, 张玉斌, 陈新平. 中国农田土壤磷平衡现状及驱动因子分析[J]. 中国农学通报, 2009, 25(13):220-225.
	Cao N, Zhang YB, Chen XP. Spatial-temporal change of phosphorus balance and the driving factors for agroecosystems in China[J]. Chin Agric Sci Bull, 2009, 25(13):220-225.
[4]	Gustafsson JP, Mwamila LB, Kergoat K. The pH dependence of phosphate sorption and desorption in Swedish agricultural soils[J]. Geoderma, 2012, 189/190:304-311. doi: 10.1016/j.geoderma.2012.05.014 URL
[5]	Roberts TL, Johnston AE. Phosphorus use efficiency and management in agriculture[J]. Resour Conserv Recycl, 2015, 105:275-281. doi: 10.1016/j.resconrec.2015.09.013 URL
[6]	阳洁, 江院, 王晓甜, 等. 几株高效溶磷解钾药用稻内生固氮菌的筛选与鉴定[J]. 农业生物技术学报, 2016(2):186-195.
	Yang J, Jiang Y, Wang XT, et al. Screening and identification of several endophytic diazotrophs with high capability of phosphate solubilizing and potassium decomposing from Oryza officinalis[J]. Chin J Agric Biotechnol, 2016(2):186-195.
[7]	Bhattacharyya PN, Jha DK. Plant growth-promoting rhizobacteria(PGPR):emergence in agriculture[J]. World J Microbiol Biotechnol, 2012, 28(4):1327-1350. doi: 10.1007/s11274-011-0979-9 URL
[8]	Gupta M, Kiran S, Gulati A, et al. Isolation and identification of phosphate solubilizing bacteria able to enhance the growth and aloin-A biosynjournal of Aloe barbadensis Miller[J]. Microbiol Res, 2012, 167(6):358-363. doi: 10.1016/j.micres.2012.02.004 URL
[9]	Holguin G, Vazquez P, Bashan Y. The role of sediment microorganisms in the productivity, conservation, and rehabilitation of mangrove ecosystems:an overview[J]. Biol Fertil Soils, 2001, 33(4):265-278. doi: 10.1007/s003740000319 URL
[10]	Oliveira CA, Alves VMC, Marriel IE, et al. Phosphate solubilizing microorganisms isolated from rhizosphere of maize cultivated in an oxisol of the Brazilian Cerrado Biome[J]. Soil Biol Biochem, 2009, 41(9):1782-1787. doi: 10.1016/j.soilbio.2008.01.012 URL
[11]	秦利均, 杨永柱, 杨星勇. 土壤溶磷微生物溶磷、解磷机制研究进展[J]. 生命科学研究, 2019, 23(1):59-64, 86.
	Qin LJ, Yang YZ, Yang XY. Advances in mechanisms of soil phosphorus solubilization and dissolution by phosphate solubilizing microorganisms[J]. Life Sci Res, 2019, 23(1):59-64, 86.
[12]	陈慧, 郝慧荣, 熊君, 等. 地黄连作对根际微生物区系及土壤酶活性的影响[J]. 应用生态学报, 2007, 18(12):2755-2759. pmid: 18333450
	Chen H, Hao HR, Xiong J, et al. Effects of successive cropping Rehmannia glutinosa on rhizosphere soil microbial flora and enzyme activities[J]. Chin J Appl Ecol, 2007, 18(12):2755-2759. pmid: 18333450
[13]	江曙, 段金廒, 严辉, 等. 当归根际微生物种群结构与生态分布的研究[J]. 中国中药杂志, 2009, 34(12):1483-1488.
	Jiang S, Duan JA, Yan H, et al. Population structure and ecological distribution of rhizospheric microorganisms of Angelica sinensis[J]. China J Chin Mater Med, 2009, 34(12):1483-1488.
[14]	吴伟. 潞党参根际产ACC脱氨酶细菌的多样性调查及高效菌株的接种效应[D]. 临汾:山西师范大学, 2018.
	Wu W. Diversity of ACC deaminase activity bacterium in rhizosphere soil and inoculation effects of Codonopsis pilosula with high efficient strain[D]. Linfen:Shanxi Normal University, 2018.
[15]	吴伟, 张鹏飞, 张桂萍, 等. 连翘根际高效解有机磷细菌的筛选鉴定及促生长特性研究[J]. 西南林业大学学报:自然科学, 2018, 38(3):93-100.
	Wu W, Zhang PF, Zhang GP, et al. Screening, identification and growth promoting characteristics of high efficient organic phosphate-mineralizing bacterium from rhizosphere soils of Forsythia suspensa[J]. J Southwest For Univ:Nat Sci, 2018, 38(3):93-100.
[16]	李亚丹, 郭义东, 刘逆夫, 等. 微生物复合菌群联合降解中药药渣的研究进展[J]. 化学与生物工程, 2015, 32(4):12-14, 25.
	Li YD, Guo YD, Liu NF, et al. Review on the degradation of traditional Chinese medicine slag by composite microbial flora[J]. Chem Bioeng, 2015, 32(4):12-14, 25.
[17]	贺超, 王文全, 侯俊玲. 中药药渣生物有机肥的研究进展[J]. 中草药, 2017, 48(24):5286-5292.
	He C, Wang WQ, Hou JL. Research progress on bio-organic fertilizer from Chinese materia Medica residues[J]. Chin Tradit Herb Drugs, 2017, 48(24):5286-5292.
[18]	鲁耀雄, 高鹏, 崔新卫, 等. 中药渣堆肥过程中氮素转化及相关微生物菌群变化的研究[J]. 农业现代化研究, 2018, 39(3):527-534.
	Lu YX, Gao P, Cui XW, et al. Study on nitrogen transformation and related microbial community changes during the composting process of Chinese medicinal herbal residues[J]. Res Agric Mod, 2018, 39(3):527-534.
[19]	王明欢, 张小娜, 林冰, 等. 中药药渣中固氮菌、解磷菌、解钾菌的筛选[J]. 中成药, 2020, 42(2):531-533.
	Wang MH, Zhang XN, Lin B, et al. Screening of nitrogen-fixing bacteria, phosphorus-releasing bacteria and potassium-releasing bacteria in the residue of traditional Chinese medicine[J]. Chin Tradit Pat Med, 2020, 42(2):531-533.
[20]	Holt JG, Krieg NR, Sneath PHA, et al. Bergey’s manual of determinative bacteriology[M]. 9th ed. Baltimore:Williams and Wilkins, 1994.
[21]	要雅倩, 成娜娜, 李培根, 等. 解淀粉芽胞杆菌Bacillus amyloliquefaciens T-6的分离鉴定及抗病促生潜力[J]. 生物技术通报, 2020(9):202-210. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0847
	Yao YQ, Cheng NN, Li PG, et al. Isolation and identification of Bacillus amyloliquefaciens T-6 and its potential of resisting disease and promoting growth[J]. Biotechnol Bull, 2020(9):202-210. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0847
[22]	王欢, 韩丽珍. 4株茶树根际促生菌菌株的鉴定及促生作用[J]. 微生物学通报, 2019, 46(3):548-562.
	Wang H, Han LZ. Identification of four plant growth-promoting rhizobacteria isolated from tea rhizosphere[J]. Microbiol China, 2019, 46(3):548-562.
[23]	荣良燕, 姚拓, 赵桂琴, 等. 产铁载体PGPR菌筛选及其对病原菌的拮抗作用[J]. 植物保护, 2011, 37(1):59-64.
	Rong LY, Yao T, Zhao GQ, et al. Screening of siderophore-producing PGPR bacteria and their antagonism against the pathogens[J]. Plant Prot, 2011, 37(1):59-64.
[24]	穆香轶, 张璇, 胡有贞, 等. 库尔勒香梨黑头病拮抗菌的筛选和鉴定[J]. 微生物学通报, 2020, 47(6):1795-1806.
	Mu XY, Zhang X, Hu YZ, et al. Screening and identification of an antagonistic bacterium against blackhead disease of Korla fragrant pear[J]. Microbiol China, 2020, 47(6):1795-1806.
[25]	周莲, 蒋海霞, 金凯明, 等. 高产申嗪霉素和吩嗪-1-酰胺的水稻根际铜绿假单胞菌PA1201分离、鉴定与应用潜力[J]. 微生物学报, 2015, 55(4):401-411.
	Zhou L, Jiang HX, Jin KM, et al. Isolation, identification and characterization of rice rhizobacterium Pseudomonas aeruginosa PA1201 producing high level of biopesticide “Shenqinmycin”and phenazine-1-carboxamide[J]. Acta Microbiol Sin, 2015, 55(4):401-411.
[26]	Shahid I, Han J, Hardie D, et al. Profiling of antimicrobial metabolites of plant growth promoting Pseudomonas spp. isolated from different plant hosts[J]. 3 Biotech, 2021, 11(2):1-14. doi: 10.1007/s13205-020-02554-1 URL
[27]	赵炽娜. 申嗪霉素-缬氨酸耦合物在小麦植株中的传导性研究[D]. 荆州:长江大学, 2020.
	Zhao CN. Study on the systemicity of phenazine-1-carboxylic acid-valine conjugates in wheat plants[D]. Jingzhou:Yangtze University, 2020.
[28]	Hameeda B, Harini G, Rupela OP, et al. Growth promotion of maize by phosphate-solubilizing bacteria isolated from composts and macrofauna[J]. Microbiol Res, 2008, 163(2):234-242. pmid: 16831538
[29]	Nuraini Y, Abadi AL Soemarno, et al. Potential of legume and maize composts to stimulate population of nitrogen- fixing bacteria, phosphate-solubilizing bacteria and in dole acetic acid production[J]. Journal of Agriculture and Food Technology, 2011, 1(12):218-226.
[30]	Atekan, Nuraini Y, Handayanto E, et al. The potential of phosphate solubilizing bacteria isolated from sugarcane wastes for solubilizing phosphate[J]. Journal of Degraded and Mining Lands Management, 2014, 1(4):175-182.
[31]	伍善东, 刘清术, 付祖姣, 等. 解有机磷拮抗细菌的筛选及其解磷特性和拮抗作用[J]. 江苏农业学报, 2017, 33(4):843-847.
	Wu SD, Liu QS, Fu ZJ, et al. Screening of the bacteria-degrading organic phosphate and their degradative characteristics and antagonism[J]. Jiangsu J Agric Sci, 2017, 33(4):843-847.
[32]	白变霞, 陈艳彬, 任嘉红. 蜡状芽孢杆菌CLY07菌株的解有机磷特性研究[J]. 西南林业大学学报, 2016(4):75-81.
	Bai BX, Chen YB, Ren JH. Study on Organophosphate-solubilizing characteristics of Bacillus cereus CLY07[J]. J Southwest For Univ, 2016(4):75-81.
[33]	Roychowdhury R, Qaiser TF, Mukherjee P, et al. Isolation and characterization of a Pseudomonas aeruginosa strain PGP for plant growth promotion[J]. Proc Natl Acad Sci India Sect B:Biol Sci, 2019, 89(1):353-360. doi: 10.1007/s40011-017-0946-9 URL
[34]	Linu MS, Asok AK, Thampi M, et al. Plant growth promoting traits of indigenous phosphate solubilizing Pseudomonas aeruginosa isolates from chilli(Capsicumannuum L.)rhizosphere[J]. Commun Soil Sci Plant Anal, 2019, 50(4):444-457. doi: 10.1080/00103624.2019.1566469 URL
[35]	谯天敏, 朱天辉, 李姝江. 铜绿假单胞菌ZB27的定殖能力及对杂交竹梢枯病的防控作用[J]. 植物保护学报, 2011, 38(2):133-138.
	Qiao TM, Zhu TH, Li SJ. Colonization of Pseudomonas aeruginosa ZB27 and its control effect on hybrid bamboo blight[J]. Acta Phytophylacica Sin, 2011, 38(2):133-138.
[36]	谯天敏, 张静, 赵芳, 等. 铜绿假单胞菌发酵条件优化及抗菌物质研究[J]. 南京林业大学学报:自然科学版, 2014, 38(5):45-50.
	Qiao TM, Zhang J, Zhao F, et al. Study on optimization of fermentation conditions of Pseudomonas aeruginosa and its antibacterial substances[J]. J Nanjing For Univ:Nat Sci Ed, 2014, 38(5):45-50.
[37]	张亚, 蒋程, 易龙, 等. 铜绿假单胞菌次生代谢产物的研究进展[J]. 化学与生物工程, 2015, 32(1):8-11.
	Zhang Y, Jiang C, Yi L, et al. Research progress of secondary metabolites produced by Pseudomonas aeruginosa[J]. Chem Bioeng, 2015, 32(1):8-11.

菌株 Strain	溶磷圈直径 Diameter of phosphate Solubilizing halo D/mm	菌落直径 Colony diameter d /mm	D/d
YP1	13.43±0.24d	4.77±0.22b	2.83±0.15c
YP2	18.53±0.33b	5.13±0.13b	3.62±0.15b
YP3	14.60±0.23c	6.47±0.24a	2.26±0.05d
YP4	18.17±0.17b	6.13±0.07a	2.96±0.03c
YP5	21.67±0.24a	5.10±0.25b	4.27±0.20a

菌株 Strain	溶磷圈直径 Diameter of phosphate Solubilizing halo D/mm	菌落直径 Colony diameter d /mm	D/d
YP1	13.43±0.24d	4.77±0.22b	2.83±0.15c
YP2	18.53±0.33b	5.13±0.13b	3.62±0.15b
YP3	14.60±0.23c	6.47±0.24a	2.26±0.05d
YP4	18.17±0.17b	6.13±0.07a	2.96±0.03c
YP5	21.67±0.24a	5.10±0.25b	4.27±0.20a

生理生化试验Physiological and biochemical test	检测结果Result	生理生化试验Physiological and biochemical tests	检测结果Result
硝酸盐还原 Nitrate reduction	-	色氨酸酶Tryptophanase	-
精氨酸双水解酶 Arginine dihydrolase	+	脲酶Urease	-
明胶液化 Gelatine liquefication	+	β-半乳糖苷酶β-galactosidase	-
阿拉伯糖Arabinose fermentation	-	甘露糖Mannose	-
麦芽糖Maltose fermentation	-	葡萄糖酸盐Gluconate	+
己二酸Adipic Acid	+	苹果酸Malic acid	+
苯乙酸Phenylacetic acid	-	氧化酶Oxidase	+
葡萄糖酸Gluconic acid	-	七叶灵水解 Hydrolysis of aescin	-
葡萄糖 Glucose fermentation	+	N-乙酰-葡萄糖胺n-acetyl-glucosamine	+
柠檬酸Citric acid	+	甘露醇Mannitol	+
癸酸 Capric acid	+

生理生化试验Physiological and biochemical test	检测结果Result	生理生化试验Physiological and biochemical tests	检测结果Result
硝酸盐还原 Nitrate reduction	-	色氨酸酶Tryptophanase	-
精氨酸双水解酶 Arginine dihydrolase	+	脲酶Urease	-
明胶液化 Gelatine liquefication	+	β-半乳糖苷酶β-galactosidase	-
阿拉伯糖Arabinose fermentation	-	甘露糖Mannose	-
麦芽糖Maltose fermentation	-	葡萄糖酸盐Gluconate	+
己二酸Adipic Acid	+	苹果酸Malic acid	+
苯乙酸Phenylacetic acid	-	氧化酶Oxidase	+
葡萄糖酸Gluconic acid	-	七叶灵水解 Hydrolysis of aescin	-
葡萄糖 Glucose fermentation	+	N-乙酰-葡萄糖胺n-acetyl-glucosamine	+
柠檬酸Citric acid	+	甘露醇Mannitol	+
癸酸 Capric acid	+

抗菌素Antibiotics	含药量Drug concentration/（μg·μL^-1）	抑菌圈直径Inhibitory zone diameter d/mm	敏感性Sensitivity
氨苄西林Ampicillin	0.5	0	R
诺氟沙星Norfloxacin	0.5	32.00±0.00	S
氯霉素Chloramphenicol	1.5	17.67±0.33	S
利福平 Rifampin	0.25	0	R
亚胺培南Imipenem	0.5	0	R
卡那霉素 Kanamycin	1.5	8.67±0.67	R
庆大霉素 Gentamicin	0.5	19.00±0.58	S
四环素Tetracycline	1.5	0	R
萘啶酸Nalidixic acid	1.5	13.33±0.33	I
链霉素Streptomycin	0.5	12.00±0.00	I
恩诺沙星Enrofloxacin	0.5	21.33±0.67	S
丁胺卡那霉素Amikacin	1.5	20.67±0.33	S
多西环素Doxycycline	1.5	8.67±0.33	R
万古霉素Vancomycin	1.5	0	R
阿莫西林Amoxicillin	1	0	R