[1]丁 森,王 焱,陆蓝翔,等.一株促生抗病的樱花内生细菌的分离、筛选和鉴定[J].南京林业大学学报(自然科学版),2019,43(05):081-88.[doi:10.3969/j.issn.1000-2006.201802023]
 DING Sen,WANG Yan,LU Lanxiang,et al.Isolation, screening and identification of an endophytic bacteria in Ceresas with resistance to Agrobacterium tumefaciens and phosphorus solubilizing ability[J].Journal of Nanjing Forestry University(Natural Science Edition),2019,43(05):081-88.[doi:10.3969/j.issn.1000-2006.201802023]
点击复制

一株促生抗病的樱花内生细菌的分离、筛选和鉴定
分享到:

《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

卷:
43
期数:
2019年05期
页码:
081-88
栏目:
研究论文
出版日期:
2019-09-20

文章信息/Info

Title:
Isolation, screening and identification of an endophytic bacteria in Ceresas with resistance to Agrobacterium tumefaciens and phosphorus solubilizing ability
文章编号:
1000-2006(2019)05-0081-08
作者:
丁 森1王 焱2陆蓝翔1张岳峰2江明明1魏 奇1叶建仁1*
(1.南京林业大学,南方现代林业协同创新中心,南京林业大学林学院,江苏省有害生物入侵预防与控制重点实验室, 江苏 南京 210037; 2.上海市林业总站,上海 200072)
Author(s):
DING Sen1WANG Yan2LU Lanxiang1ZHANG Yuefeng2 JIANG Mingming1WEI Qi1YE Jianren1*
(1.Co-Innovation Center for the Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Jiangsu Key Laboratory for Prevention and Management of Invasive Species, Nanjing 210037, China; 2. Shanghai Forestry Station,Shanghai 200072,China)
关键词:
瓦雷兹芽孢杆菌(YH-20) 根癌农杆菌 拮抗作用 解磷作用 樱花
Keywords:
Bacillus velezensis Agrobacterium tumefaciens antagonism phosphorus removal Ceresas
分类号:
S763.1
DOI:
10.3969/j.issn.1000-2006.201802023
文献标志码:
A
摘要:
【目的】解决樱属植物生产过程中根癌病的发生问题。【方法】采用组织分离法和稀释分离法分别从枝干和叶片中分离内生细菌; 采用滤纸片法选拮抗菌,通过形态学、生理学和分子生物学法对筛选菌株进行种类鉴定; 通过比色法测定筛选菌株的解有机磷的能力; 以特定培养基测定菌株产IAA(anxin)的能力,通过测定OD600值判断耐NaCl的能力。【结果】在樱花体内共分离到32株内生细菌,其中从枝条内分离到28株,从叶片内分离到4株。对32株内生细菌进行抑菌活性的筛选,有2株对根癌农杆菌(Agrobacterium tumefaciens)有明显的抑制作用。另对其中的1株细菌YH-20进行鉴定,鉴定结果为瓦雷兹芽孢杆菌(Bacillus velezensis)。菌株YH-20具有良好的解有机磷和无机磷作用,其在第7天的解无机磷的量为178.43 mg/L,在第7天解有机磷的量为76.50 mg/L; 具有较高的分泌IAA的能力,在第7天IAA的生成量为9.174 μg/mL; 可在含10% NaCl的培养基上正常生长。【结论】筛选获得的瓦雷兹芽孢杆菌YH-20菌株具有抗植物根癌病菌和解磷能力,是一株在林木根癌病的防治上有很好应用前景的菌株。
Abstract:
【Objective】Solve the problem of cherry root-cancer in cherry plant production. 【Method】Endophytic bacteria were isolated from the leaves and branches of cherry plants using dilution separation and tissue separation, respectively. Antagonistic bacteria against Agrobacterium tumefaciens were selected by the filter paper method; strains were identified by morphological, physiological and molecular biology methods. The ability of a strain to screen for organophosphate residues was determined by colorimetry. Additionally, the ability of a strain to produce IAA and to resist NaCl was determined using a specific medium and by measuring bacterial density at OD600, respectively.【Result】A total of 32 endophytic bacteria were isolated from cherry plants, 28 of them were isolated from the shoots and 4 from the leaves. All the 32 endophytic bacteria were screened for antibacterial activity and only two strains showed a significant inhibitory effect over Agrobacterium tumefaciens. One of these bacteria, strain YH-20, was identified as Bacillus velezensis. Strain YH-20 also showed that it was capable of solubilizing organic and inorganic phosphorus. The amount of inorganic and organic phosphorus dissolved after 7 days was 178.43 and 76.50 mg/L, respectively. Strain YH-20 also had the ability to secrete IAA; it produced 9.174 μg/mL IAA after 7 days of incubation. B. velezensis strain YH-20 can grow normally on a medium containing 10% NaCl. 【Conclusion】Therefore, the endophyte, Bacillus velezensis YH-20, obtained through this screening, is an antagonist of A. tumefaciens that has the ability to dissolve phosphorus. Thus, it is a strain with good application prospects in the prevention and treatment of root cancer in the forest.

参考文献/References:

[1] 刘冰, 宋水林, 刘晓丽,等. 柑橘溃疡病生防内生细菌的筛选、鉴定及其活性代谢产物的稳定性[J]. 浙江农业学报, 2015, 27(12):2152-2158.DOI:10.3969/j.issn.1004-1524.2015.12.17. LIU B,SONG S L,LIU X L,et al.Screening,identification of bio-control endophytic bacterium against citrus canker and stability of its bioactive metabolites[J].Acta Agriculturae Zhejiangensis,2015,27(12):2152-2158. [2] 赵凯,肖崇刚,孔德英.内生细菌对番茄青枯病的控病作用及其抗菌谱[J].西南农业大学学报(自然科学版),2006(2):314-318. DOI:10.13718/j.cnki.xdzk.2006.02.036. ZHAO K,XIAO C G,KONG D Y.Controlling effect of endophytic bacteria on ralstonia solannacearum and it santifungal spectrum[J].Journal of Southwest University(Natural Science Edition),2006(2):314-318. [3] 张静娟,周娟,相望年.中国毛白杨根癌土壤杆菌的类型和对土壤杆菌素敏感性的研究[J].微生物学报,1988,28(1):12-18,99.DOI:10.3969/j.issn.1004-1524.2015.12.17. ZHANG J J,ZHOU J,XIANG W N.Study on the types of Agrobacterium tumefaciens and its sensitivity to Agrobacterium in Populus tomentosa in China[J].Actamicrobiologica Sinica,1988,28(1):12-18,99. [4] UTKHEDE R S,SMITH E M.Evaluation of biological and chemical treatments for control of crown gall on young apple trees in the Kootenay valley of British Columbia[J].Journal of Phytopathology,2010,137(4):265-271.DOI:10.1111/j.1439-0434.1993.tb01347.x. [5] 李昱佳,李茜,张志想,等.拮抗根癌土壤杆菌的桃内生细菌的筛选鉴定[J].中国农业科学,2017,50(20):3918-3929.DOI:CNKI:CDMD:2.1017.269470. LI Y J,LI Q,ZHANG Z X,et al.Screening and identification of peach endophytic bacteria with antagonism against Agrobacterium tumefaciens[J]. Scientia Agricultura Sinica,2017,50(20):3918-3929. [6] OLIVEIRA C A,ALVES V M C,MARRIEL I E,et al.Phosphate solubilizing microorganisms isolated from rhizosphere of maize cultivated in an oxisol of the Brazilian Cerrado Biome[J].Soil Biology & Biochemistry,2009,41(9):1782-1787.DOI:10.1016/j.soilbio.2008.01.012. [7] SABATÉ D C, BRANDAN C P, PETROSELLI G, et al. Decrease in the incidence of charcoal root rot in common bean(Phaseolus vulgaris L.)by Bacillus amyloliquefaciens B14, a strain with PGPR properties [J]. Biological Control, 2017,113:1-8.DOI:10.1016/j.biocontrol.2017.06.008. [8] 杨定祥,林巧玲,卢乃会,等.拮抗辣椒疫霉菌海洋细菌菌株SH-27的筛选鉴定及其防病促生作用[J].微生物学通报,2018,45(1):54-63.DOI: 10.13344/j.microbiol.china.170221. YANG D X,LIN Q L,LU N H,et al.Screening and identification of a marine bacterium strain SH-27 against Phytophthora capsici causing pepper phytophthorablight[J]. Microbiology China,2018,45(1):54-63. [9] 石玉莹,宋海慧,苗爽,等.番茄灰霉病和叶霉病拮抗细菌WXCDD51的筛选鉴定及其生防促生作用[J].园艺学报,2017,44(10):1925-1936. DOI:10.16420/j.issn.0513-353x.2017-0182. SHI Y Y,SONG H H,MIAO S,et al.Screening,identification,biocontrol and growth promoting effects of antagonistic bacteria WXCDD51 of tomato gray mold disease and tomato leaf mold disease [J].Acta Horticulturae Sinica,2017,44(10):1925-1936. [10] 东秀珠 蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:370-398. DONG X Z,CAI M Y.Common bacterial system identification manual[M]. Beijing: Science Press,2001:370-398. [11] 孙燕. 微生物学实验指导[M]. 南京:南京大学出版社, 2015. SUN Y,Experimental guidance in microbiology[M]. Nanjing: Nanjing University Press,2015. [12] 江明明. 几株优良内生菌的分离及鉴定[D].南京:南京林业大学,2016. JIANG M M.Isolation and identification about several strains of endophyticbacteria[D]. Nanjing: Nanjing Forestry University,2016. [13] 鲁如坤. 土壤农业化学分析方法[M]. 北京: 中国农业科技出版社, 2000:56. LU R K. Soil agricultural chemical analysis method[M]. Beijing: China Agricultural Science and Technology Press, 2000:56. [14] PATTEN C L, GLICK B R. Role of Pseudomonas putidaindole acetic acid in development of the host plant root system [J].Appl Environ Microbio, 2002,68:3795-3801.DOI:10.1128/AEM.68.8.3795-3801.2002. [15] 刘露,李丽,闫洪雪,等. 巨大芽孢杆菌的应用研究进展[J].北方农业学报,2016(4):117-120.DOI:10.3969/j.issn.2096-1197.2016.04.25. LIU L,LI L,YAN H X,et al.Research progress of application of Bacillus megaterium[J]. Journal of Northern Agriculture,2016(4):117-120. [16] 卫星,鲁荣,张丹,等.一株耐硝酸盐的巨大芽孢杆菌溶磷特性研究[J].环境科学学报,2015,35(7):2052-2058.DOI:10.13671/j.hjkxxb.2014.0976. WEI X,LU R,ZHANG D,et al. Phosphate solubilizing characteristics of a nitrate-tolerating bacterium,Bacillus megaterium[J].Acta ScientiaeCircumstantiae,2015,35(7):2052-2058. [17] 颜思齐,林桂芸,吴帮承,等. 水稻、小麦、玉米纹枯病菌和棉花立枯病菌对小麦的致病力比较[J]. 植物病理学报,1993,23(3):216-218.DOI:13926/j.cnki.apps.1993.03.008. YAN S Q,LIN G Y,WU B C,et al.Comparison of pathogenicity on wheat among four strains of Rhizoctonia spp. isolated from sheath blight of rice, maize, and wheat, and from soreshin cotton[J].Acta Phytopathologica Sinica,1993,23(3):216-218. [18] 杨晓云,陈志谊,蒋盼盼,等.解淀粉芽孢杆菌B1619对番茄的促生作用[J].中国生物防治学报,2016,32(3):349-356.DOI:10.16409/j.cnki.2095-039x.2016.03.010. YANG X Y, CHEN Z Y,JIANG P P,et al.Growth-promotion effect of Bacillus amyloliquefaciens B1619 on tomato plant[J].Chinese Journal of Biological Control,2016,32(3):349-356. [19] ELLIS J G, KERR A, VAN M M, et al. Agrobacterium: genetic studies on agrocin 84 production and the biological control of crown gall.[J]. Physiological Plant Pathology,1979,15(3):311-319. DOI:10.1016/0048-4059(79)90082-1. [20] 李金云,王慧敏,王建辉.根癌病生防菌E26菌株产生细菌素的初步研究[J].中国农业科学,2004,37(12):1860-1865.DOI:10.3321/j.issn:0578-1752.2004.12.012. LI J Y, WANG H M, WANG J H. A bacteriocin with a broad spectrum activity produced by grapevine crown gall biocontrol strain E26[J].Scientia Agricultura Sinica,2004,37(12):1860-1865. [21] 谢学梅,游积峰,陈培民,等.葡萄根癌病的生物防治技术[J].内蒙古农业科技,1997(5):31. XIE X M,YOU J F,CHEN P M, et al.Biological control technology for grape root cancer[J]. Journal of Northern Agriculture,1997(5):31. [22] HAMMAMI I,RHOUMA A, JAOUADI B, et al. Optimization and biochemical characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for biocontrol of Agrobacterium spp. strains[J]. Letters in Applied Microbiology, 2010, 48(2):253-260. DOI:10.1111/j.1472-765X.2008.02524.x. [23] 万其露. 桃树根癌病病原分离及拮抗菌筛选研究[D].南京:南京林业大学,2016. WAN Q L.The study on peach tree root cancer pathogen and the screening of antagonistic microorganisms[D].Nanjing: Nanjing Forestry University,2016.

备注/Memo

备注/Memo:
收稿日期:2018-02-24 修回日期:2019-04-11 基金项目:农业科技自主创新资金项目(CX(16)1005-6)。 第一作者:丁森(2815971394@qq.com)。*通信作者:叶建仁(jrye@njfu.edu.cn),教授,ORCID(0000-0001-5711-0516)。 引文格式:丁森,王焱,陆蓝翔,等. 一株促生抗病的樱花内生细菌的分离、筛选和鉴定[J]. 南京林业大学学报(自然科学版),2019,43(5):81-88.
更新日期/Last Update: 2019-10-08