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  土壤学报  2018, Vol. 55 Issue (4): 977-986  DOI: 10.11766/trxb201712250571
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引用本文  

王玉书, 刘海, 李佳, 等. 黄连须根浸提液对无机磷细菌的负化感效应. 土壤学报, 2018, 55(4): 977-986.
WANG Yushu, LIU Hai, LI Jia, et al. Negative Allelopathic Effects of Extract of Coptis Chinensis Hair Root on Inorganic Phosphorus-Dissolving Bacteria. Acta Pedologica Sinica, 2018, 55(4): 977-986.

基金项目

国家重点基础研究发展计划(973计划)项目(2013CB127405)资助

通讯作者Corresponding author

黄建国, E-mail: huang99@swu.edu.cn

作者简介

王玉书(1991—),男,山西人,硕士研究生,主要从事土壤微生物研究。E-mail:969014327@qq.com
黄连须根浸提液对无机磷细菌的负化感效应
王玉书1 , 刘海1,2 , 李佳1 , 魏立本1 , 彭丽媛1 , 黄建国1     
1. 西南大学资源环境学院,重庆 400716;
2. 贵州省农业科学院农业科技信息研究所,贵阳 550006
摘要:黄连(Coptis chinensis)是我国人工栽培的传统大宗中药材,种植面积大,连作障碍严重,了解黄连对土壤微生物的化感效应,有益于揭示黄连连作障碍的发生机理,为减轻连作障碍提供科学依据。试验利用固、液培养技术,以3株能溶解无机磷的伯克霍尔德氏菌(Burkholderia Yabunchi B05、B07和B09)为材料,研究了黄连须根浸提液(Extracts of fibrous roots of Coptis chinensis,ERC)对其生长繁殖和溶磷的化感作用。结果表明,在固体培养时,随ERC浓度提高,无机磷细菌(Inorganic phosphorus-dissolving bacteria,iPDB)的菌落直径、溶磷圈直径和溶磷指数降低。在液体培养基中,ERC不同程度地抑制iPDB繁殖生长,数量减少35.98%~52.98%。供试菌株均能分泌氢离子、草酸和柠檬酸,B05和B09还能分别分泌乙酸和苹果酸,B07能分泌乙酸和丁二酸,三株iPDB溶磷量平均值(mg L-1)分别为228.31(B05)、311.64(B07)和174.89(B09)。有机酸和氢离子分泌总量分别与溶磷量呈显著正相关,相关系数依次为0.877和0.604(p≤0.05,n=12)。在ERC抑制iPDB分泌有机酸和氢离子的同时,溶磷量降低21.68%~60.55%。因此,在集约化种植黄连的土壤中,其根系分泌的化感物质可能影响iPDB的种群结构和生理生态功能,降低土壤无机磷的生物有效性,可视为发生连作障碍的潜在原因之一。
关键词黄连    化感效应    无机磷细菌    有机酸    氢离子    

黄连(Coptis chinensis)属毛茛科多年生草本植物,是我国人工栽培的传统大宗中药材植物之一[1-2]。在黄连生长过程中,通过茎叶淋溶、根系分泌和残株腐解等多种途径向土壤生态系统释放小檗碱(berberine)、黄连碱(coptisine)和甲基黄连碱(worenine)等多种化感物质,对土壤生态系统产生持续影响[3-4]。在黄连根系分泌物中,小檗碱含量最高,可达总分泌物的50%以上,且化感作用最强[5]。研究发现,黄连须根浸提液中的小檗碱影响隆线溞的泳动能力、趋光性和存活率;降低斜生栅藻和蛋白核小球藻的叶绿素和蛋白质含量,抑制其生长繁殖,并改变体内超氧化物歧化酶(SOD)和过氧化氢酶(CAT)的活性[6-7]。此外,黄连根系分泌物还破坏种子细胞膜结构,抑制蚕豆、豌豆、绿豆、莴苣和白菜等多种作物的种子发芽,胚乳养分利用和幼苗生长,以及硝酸还原酶和根系活性等[8-9]

重庆市石柱县是我国黄连的主产区(超过全国的60%产量)。但是当地种植黄连后的土壤必须间隔2~3年才能再次种植,严重影响黄连的持续供应,其原因尚不明确。有研究表明,在种植黄连后的土壤中,磷钾细菌、自生固氮菌、氨化细菌和硝化细菌等多种有益微生物数量和生物量大幅减少,螺杆菌属等病原菌增多,微生物群落多样性指数和均匀度指数降低,说明土壤微生物的固氮、溶磷、解钾、促生等功能受到抑制[10-11],原因可能与黄连根系分泌物对有益菌群的化感效应有关,但迄今为止研究甚少。

无机磷细菌(Inorganic phosphorus-dissolving bacteria, iPDB)聚集的根系周围,能够将难溶性无机磷转化为植物能吸收的有效磷,是重要的土壤有益微生物[12-14]。黄连根系分泌物抑制有益微生物的新陈代谢和生长繁殖,直接影响土壤养分的有效含量,植物促生和抗生物质分泌,增加发生连作病害的风险,造成连作障碍。高通量测序表明,在黄连种植的供试土壤中,伯克霍尔德氏菌属(Burkholderia Yabunchi)的相对丰度较高,故本研究将以该菌属的3株iPDB作为有益菌的代表,通过固、液培养,测定黄连须根浸提液对其生长繁殖、氢离子和有机酸分泌,以及溶磷作用的影响,探讨黄连根系分泌物对有益菌群的化感效应,为揭示黄连连作障碍的发生机理和减轻连作障碍提供科学依据。

1 材料与方法 1.1 供试材料

供试菌株:伯克霍尔德氏菌属(Burkholderia Yabunchi)的3株iPDB(B05、B07和B09),从重庆市缙云山黄壤(106º20′E,29º49'N,pH 4.34)中分离获得,保存于西南大学资源环境学院微生物实验室。

固体培养基(g):琼脂20、葡萄糖10、磷酸三钙2.5、(NH4)2SO4 0.5、NaCl 0.2、MgSO4·7H2O 0.1、KCl 0.2、酵母膏0.5、MnSO4·4H2O和FeSO4·7H2O各0.002,蒸馏水1 000 ml,pH 7.0,液体培养基不含琼脂。

黄连须根:在重庆市石柱县黄水镇(108º30' E,30º15'N),田间采集新鲜黄连须根,小檗碱50.20 mg g-1(干基)。

1.2 研究方法

取保存菌株,固体培养72 h,无菌生理盐水洗涤,配成103个ml-1的菌液。

取新鲜黄连须根,洗净泥土,去离子水浸泡24 h(25±1℃),过滤,配制成每ml含0.1 g原药的黄连须根浸提液(Extracts from fibrous roots of Coptis chinensis,ERC),紫外线灭菌。

蒸汽灭菌(121℃,1.5大气压,30 min,下同)固体培养基。由于1000mgL-1ERC显著抑制蚕豆、莴苣和白菜等幼苗生长[8-9],故当培养基冷却至45~50℃时,加入ERC使其浓度分别达到0(对照)、500(低)、1000(中)和2000(高)mg L-1,相当于含小檗碱0、7.31、14.62和29.24 mg L-1。取10ml培养基于60 mm培养皿中,冷却后在中央分别接种0.1 ml B05、B07和B09菌液,25±1℃暗培养7 d,测量菌落和透明圈直径,计算溶磷指数[15]

$ 溶磷指数 = \frac{{菌落直径+透明圈直径}}{{菌落直径}} $

取100 ml液体培养基于250 ml三角瓶中,蒸汽灭菌,冷却后加ERC形成浓度分别为0、500、1 000和2 000 mg L-1的处理,各接种1 mlB05、B07和B09菌液,于25±1 ℃摇瓶(75 r min-1)暗培养120 h,重复5次。

在培养6、12、18、36、72、120 h,各吸取5 ml液体培养基用于后续实验。首先,取200 μl培养液用XSP-6C显微镜(上海迪诺力泰公司)观测并计数iPDB数量。然后,将剩余培养基10 000 r min-1离心10 min,用PHS—3C精密酸度计测定上清液pH,钼蓝比色法测定无机磷含量[16],计算溶磷量(培养液无机磷含量-对照培养液无机磷含量)×培养液体积[17]。培养结束后用0.1 mol L-1 HCl酸化培养液,日立公司生产的D-7000高效液相色谱仪测定有机酸浓度。色谱条件为:L-7455二级管阵列检测器,RezexRoa-Organic Acid300离子交换柱(美国Phenomenex公司生产),进样量20 μl,流动相2.5 mmol L-1H2SO4,流速0.5 ml min-1,柱温35℃,压力450 psi,Diode Array L-7455紫外检测器,检测波长210 nm。所检测的有机酸包括草酸、柠檬酸、苹果酸、丁二酸和乙酸,出峰时间依次为11.60、13.73、16.05、19.47和23.92 min。

1.3 数据处理

分别用Excel 2010、SPSS 17.0和SigmaPlot12.5软件对试验数据进行基本计算、统计分析和作图,采用LSD进行多重比较,显著水平为p≤0.05。

2 结果 2.1 ERC对iPDB生长和溶磷的影响

图 1表 1可知,在固体培养时,ERC显著降低B07和B09的菌落直径、溶磷圈直径和溶磷指数,浓度越高,降幅越大;尽管低、中浓度的ERC对B07的生长和溶磷参数无显著影响,但高浓度仍表现出抑制作用。在高浓度的ERC培养基中,透明圈直径、菌落圈直径和溶磷指数的最大降幅依次为51.01%~61.17%、48.51%~66.38%和9.15%~20.77%。此外,在含ERC的培养液中,溶磷量的降幅B09 > B07 > B05。

图 1 不同浓度ERC固体培养基iPDB的生长和溶磷状况 Fig. 1 Growth of iPDB and phosphorus dissolution in the solid culture medium relative to ERC concentration

表 1 固体培养时ERC对iPDB生长和溶磷的影响 Table 1 Effects of ERC on iPDB growth and phosphorus dissolution in the solid culture medium

图 2可见,在培养液中,iPDB生长对ERC的敏感性因菌株不同而异,在培养液中的平均数量变化于15.26×108ml-1(B05)~30.15×108 ml-1(B07)。低、中浓度的ERC对B05生长无显著影响,高浓度则抑制生长;低浓度ERC对B07生长无抑制作用,中、高浓度表现出抑制现象;B09数量随ERC浓度提高而降低,最大降幅为52.98%。

图 2 在不同浓度的ERC培养液中iPDB的数量变化 Fig. 2 Variation of the population of iPDB in the liquid culture medium relative to ERC concentration 注:不同大、小写字母分别表示菌株间和处理间差异显著(p≤0.05) Note: Different capital and lowercase letters indicate significant difference betweenbacterial strains and treatmentsat 0.05 level, respectively
2.2 ERC对iPDB分泌有机酸和氢离子的影响

在液体培养时,ERC对iPDB分泌有机酸和氢离子的影响见表 2

表 2 iPDB培养液中的有机酸和氢离子浓度 Table 2 Concentrations of organic acids(mg L-1)and protons (μg L-1) in the iPDB culture solutions

草酸:B09分泌草酸最多,平均浓度分别是B05和B07的1.72倍和2.21倍。随ERC浓度提高,iPDB分泌草酸速率显著降低,最大降幅依次为31.12%(B05)、73.37%(B07)和40.20%(B09)。

柠檬酸:ERC浓度越高,B07和B09分泌柠檬酸速率越低;但低浓度的ERC对B05分泌柠檬酸无显著影响,中、高浓度则产生抑制作用。此外,柠檬酸分泌速率B07 > B05 > B09。

乙酸:在培养液中,B09不分泌乙酸。低、中浓度的ERC对B07分泌乙酸无显著影响,但高浓度则降低分泌速率;但是,ERC对B05乙酸分泌无显著影响。

苹果酸:在B05和B07的培养液中,未检测出苹果酸。ERC浓度越高,B09培养液中苹果酸含量越低,最大降幅较对照降低24.92%。

丁二酸:在供试菌株中,B05和B09不分泌丁二酸;B07的丁二酸分泌量随ERC浓度增加而降低,较对照降低14.78%~51.61%。

氢离子:ERC浓度提高,B05、B07和B09分泌氢离子的速率持续降低。此外,培养液中的氢离子平均浓度(µg L-1)B07(331.18) > B09(160.56) > B05(11.34)。

2.3 ERC对iPDB溶磷量的影响

图 3可见,iPDB菌株不同,在培养液中的溶磷量也不一样,平均值(mg L-1)分别为228.31±18.76(B05)、311.64±30.40(B07)和174.89±38.61(B09)。B05和B09的溶磷量(y)与培养时间(x)的关系可用y=x/(a+bx)描述,B07则可用y=y0+a(1-e-bx)c反应。随ERC浓度提高,B07和B09的溶磷量显著降低。对B05而言,高浓度ERC抑制溶磷;培养时间≥72 h,低、中浓度ERC处理的溶磷量与对照无显著差异。

图 3 不同浓度的ERC培养液中iPDB的溶磷量 Fig. 3 Content of phosphorus dissolved by iPDB in the liquid culture medium relative toERC concentration
2.4 有机酸和氢离子分泌与溶磷量的关系

相关分析表明,在B07和B09培养液中,溶磷量与氢离子浓度和有机酸分泌总量分别呈极显著(p < 0.01)和显著(p < 0.05)正相关;B05的溶磷量与氢离子和有机酸分泌总量的相关性均未达到显著相关水平(表 3)。但是,按3株iPDB进行统计,iPDB的溶磷量均与氢离子和有机酸分泌总量呈显著正相关(r氢离子= 0.604*r有机酸= 0.877**n = 12)。此外,iPDB的氢离子和有机酸分泌总量也呈显著正相关(r= 0.598*n = 12)。

表 3 有机酸和氢离子分泌及溶磷量之间的相关系数 Table 3 Correlation coefficients of P dissolution with release of organic acids and protons
3 讨论

黄连释放的化感物质选择性抑制植物、原生动物和微生物的生长繁殖[6]。前人研究发现,1 g L-1 ERC(相当于小檗碱14.62 mg L-1)可抑制作物幼苗生长,显著降低土壤微生物生物量、细菌数量和种群多样性指数[8-11]。本研究表明,在固、液培养基中,尽管500 mg L-1的低浓度ERC对B05和B07的生长繁殖无显著影响,但1 000、2 000 mg L-1浓度的ERC抑制iPDB生长。可见ERC对iPDB具有生物毒性。医学研究表明,ERC对淋球菌(Neisseria gonorrhoeae)、白色念珠菌(Candida albicans)和肺炎克雷伯氏菌(klebsiella pneumoniae)等病原菌有抑制作用[18-19]。小檗碱是黄连的有效成分之一,能改变铜绿假单胞菌(Pseudomonas aeruginosa)膜脂流动性和膜蛋白构象,抑制其繁殖生长[20];此外,小檗碱还能与副溶血菌(Vibrio parahaemolyticus)的核酸结合,抑制细胞膜蛋白合成,破坏细胞结构[21]。因此,在了解ERC抑制iPDB生长繁殖的基础上,很有必要继续研究有关机理。

iPDB能够有效参与土壤无机磷活化,提高土壤磷的生物有效性[22]。本研究表明,ERC抑制iPDB繁殖生长的同时,不同程度地降低其溶磷指数和溶磷量。由此可以推测,黄连生长过程中根系分泌物的释放将抑制iPDB生长并进一步影响无机磷活化,由此产生连作障碍。

iPDB主要依赖分泌有机酸和氢离子活化土壤难溶性磷酸盐[23]。供试菌株均能不同程度地分泌氢离子、草酸和柠檬酸和特异性分泌乙酸、苹果酸和丁二酸,由此造成溶磷指数和溶磷量差异。由于iPDB主要分泌草酸和柠檬酸,而柠檬酸和草酸对Ca2+、Fe3+和Al3+有极强的络合效应(log K=7.26~25.00[24]),因而柠檬酸和草酸可能是土壤无机磷释放的关键有机酸。此外,草酸和乙酸属于较强的有机酸,能电离出大量的氢离子,促进难溶性磷酸盐溶解[25]。从结果可以看出,iPDB的氢离子和有机酸分泌总量与溶磷量呈显著正相关(r有机酸= 0.877**r氢离子= 0.604*n = 12)。推测iPDB溶磷圈减小,溶磷指数和溶磷量降低的原因可能与ERC抑制氢离子和有机酸分泌有关。

iPDB合成与分泌有机酸和氢离子的速率关系到培养液中的有机酸和氢离子含量。在iPDB培养液中加入ERC,不同程度地降低溶液有机酸和氢离子浓度,说明其合成与分泌可能受到抑制。在微生物糖代谢过程中,ERC抑制丙酮酸氧化,乙酰CoA生成减少,影响三羧酸循环(Tricarboxylic acid cycle, TCA)正常进行,直接涉及柠檬酸和乙酸的生物合成,它们同时也是草酸合成的直接(乙酸)和间接前体(柠檬酸)[19, 25]。黄连富含小檗碱、黄连碱和巴马汀等多种化感物质,其中小檗碱能与它们的主要靶点——膜蛋白发生非特异性结合,导致细胞膜的通透性改变,进而弥散进入细胞内,与细胞核膜上的磷脂结合,对细胞的毒性极强;小檗碱还抑制某些微生物的DNA复制、RNA转录、蛋白质和脂质的生物合成,影响有机酸合成酶的基因表达或活性[26-27]。此外,iPDB利用ATP提供的能量,通过质子泵将H+释放到细胞膜外,ERC影响膜电子传递和ATP合成与分解等,可能降低H+分泌速率[28-29]。因此,ERC中的化感成分可能妨碍iPDB合成分泌有机酸和氢离子。

4 结论

ERC对iPDB呈负化感效应,不同程度地抑制iPDB生长繁殖,有机酸和氢离子分泌,以及无机磷溶解。在种植黄连的土壤中,其根系分泌的化感物质可能影响iPDB的种群结构和功能,降低土壤无机磷的生物有效性,可视为发生连作障碍的潜在原因。

参考文献
[1]
董宇, 张英丰, 杨庆, 等. 黄连提取物在大鼠肠外翻实验中的吸收研究. 中国中药杂志, 2008, 33(9): 1056-1060.
Dong Y, Zhang Y F, Yang Q, et al. Absorption of extractive Rhizoma Coptidis in rat everted gut scas (In Chinese). China Journal of Chinese Materia Medica, 2008, 33(9): 1056-1060. (1)
[2]
余园媛, 王伯初, 彭亮, 等. 黄连的药理研究进展. 重庆大学学报(自然科学版), 2006, 29(2): 107-111.
Yu Y Y, Wang B C, Peng L, et al. Advances in pharmacological studies of Coptis chinesis (In Chinese). Journal of Chongqing University(Natural Science Edition), 2006, 29(2): 107-111. (1)
[3]
李雪改, 杨立国, 陈丽霞, 等. 黄连水提液化学成分的分离与鉴定. 沈阳药科大学学报, 2012(3): 193-198.
Li X G, Yang L G, Chen L X, et al. Chemical consitituents from the decoction of Coptis chinensis Franch (In Chinese). Journal of Shenyang Pharmaceutical University, 2012(3): 193-198. (1)
[4]
孙健, 马吉胜, 金瑾, 等. 黄连解毒汤各成分的HPLC-UV/MS定性与定量测定方法研究. 药学学报, 2006, 41(4): 380-384.
Sun J, Ma J S, Jin J, et al. Qualitative and quantitative determination of the main components of Huanglianjiedu decoction by HPLC-UV/MS (In Chinese). Acta Pharmaceutica Sinica, 2006, 41(4): 380-384. (1)
[5]
匡艳辉, 朱晶晶, 王智民, 等. 一测多评法测定黄连中小檗碱、巴马汀、黄连碱、表小檗碱、药根碱含量. 中国药学杂志, 2009, 44(5): 390-394.
Kuang Y H, Zhu J J, Wang Z M, et al. Simultaneous quantitative analysis of five alkaloids in rhizoma of Coptis chinensis by multi-components assay by single marker (In Chinese). Chinese Pharmaceutical Journal, 2009, 44(5): 390-394. (1)
[6]
陈亚楠, 袁玲. 黄连根茎浸提物对隆线溞的急性毒性作用. 环境科学, 2015, 36(10): 3892-3895.
Chen Y N, Yuan L. Aucte toxicity of Coptis chinensis rhizome extracts to Daphnia carinata (In Chinese). Environmental Science, 2015, 36(10): 3892-3895. (2)
[7]
陈亚楠, 袁玲. 黄连根茎浸提物对绿藻的毒理作用. 环境科学, 2015, 36(5): 1655-1661.
Chen Y N, Yuan L. Toxicity of Coptis chinensis rhizome extracts to green algae (In Chinese). Environmental Science, 2015, 36(5): 1655-1661. (1)
[8]
李倩, 吴叶宽, 袁玲, 等. 黄连须根浸提液对2种豆科植物的化感效应. 中国中药杂志, 2013, 38(6): 806-811.
Li Q, Wu Y K, Yuan L, et al. Allelopathic effects of extracts from fibrous roots of Coptis chinensis on two leguminous species (In Chinese). China Journal of Chinese Materia Medica, 2013, 38(6): 806-811. (3)
[9]
王亚麒, 陈丹梅, 袁玲. 黄连须根浸提液对莴苣、绿豆和白菜的化感效应. 草业学报, 2015, 24(6): 142-149.
Wang Y Q, Chen D M, Yuan L. Allelopathic effect of extracts from the fibrous roots of Coptis chinensis on Lactuca compositae, Vigna radinte and Brassica rapapekinensis (In Chinese). Acta Prataculturae Sinica, 2015, 24(6): 142-149. DOI:10.11686/cyxb2014287 (2)
[10]
祁建军, 姚槐应, 李先恩, 等. 磷脂脂肪酸法分析地黄根际土壤微生物多样性. 土壤, 2008, 40(3): 448-454.
Qi J J, Yao H Y, Li X E, et al. Analysis of soil microbial diversity in Rehmannia glutinosa rhizosphere with phospholipid fatty acid method (In Chinese). Soils, 2008, 40(3): 448-454. (1)
[11]
李阳波, 何林卫, 张薇, 等. 黄连须根浸提液对土壤微生物及酶活性的影响. 中国中药杂志, 2014, 39(21): 4205-4210.
Li Y B, He L W, Zhang W, et al. Effect of fibrous root extract of Coptis chinensis on soil microbes and enzyme activities (In Chinese). China Journal of Chinese Materia Medica, 2014, 39(21): 4205-4210. (2)
[12]
邵玉芳, 樊明寿, 乌恩, 等. 植物根际解磷细菌与植物生长发育. 中国农学通报, 2007, 23(4): 241-244.
Zhao Y F, Fan M S, Wu E, et al. Phosphate-solubilizing bacteria in rhizosphere and its effect on plant growth (In Chinese). Chinese Agricultural Science Bulletin, 2007, 23(4): 241-244. (1)
[13]
Illmer P, Schinner F. Solubilization of inorganic phosphates by micro-organisms isolated from forestsoils . Soil Biology & Biochemistry, 1992, 24(2): 389-395. (0)
[14]
Gaind S, Gaur A C. Thermotolerant phosphate solubilization micro-organism and their interaction with mung bean . Plant and Soil, 1991, 133(1): 141-149. DOI:10.1007/BF00011908 (1)
[15]
罗世琼. 黄花蒿土壤微生物与抗疟相关成分的关联性研究. 重庆: 西南大学, 2010
Luo S Q. Study on correlation between soil microorganism and anti-malaria –related compounds of Artemisia annua L. (In Chinese). Chongqing: Southwest University, 2010 http://cdmd.cnki.com.cn/Article/CDMD-10635-1013265847.htm (1)
[16]
鲁如坤. 土壤农业化学分析方法. 北京: 中国农业科学技术出版, 2000: 76-79.
Lu R K. Analytical methods for soil and agro-chemistry (In Chinese) (In Chinese). Beijing: China Agricultural Science and Technology Press, 2000: 76-79. (1)
[17]
李娟, 王文丽, 卢秉林, 等. 甘肃省河西高钙土溶磷菌筛选及其溶磷能力初步研究. 干旱地区农业研究, 2008, 26(2): 8-9.
Li J, Wang W L, Lu B L, et al. Selection and measurement of phosphate-solubilizing micro-organisms and their phosphate-dissolving ability in high calcium soil of Hexi corridor in Gansu Province (In Chinese). Agricultural Research in the Arid Areas, 2008, 26(2): 8-9. (1)
[18]
Cernakova M, Kostalova D. Antimicrobial activity of berberine-A constituent of Mahonia aquifolium . Folia Microbiologica, 2002, 47(4): 375-378. DOI:10.1007/BF02818693 (1)
[19]
Mitani N, Murakami K, Yamaura T, et al. Inhibitory effect of berberine on the mediastinal lymp node metastasis produced by orthotopic implantation of Lewis lung carcinoma . Cancer Letters, 2001, 165: 35-42. DOI:10.1016/S0304-3835(00)00710-2 (2)
[20]
王平, 夏飞, 叶丽华, 等. 黄连对铜绿假单胞菌生物学特性的影响及机制. 中药药理与临床, 2013, 39(2): 83-86.
Wang P, Xia F, Ye L H, et al. Effects of on biological performance and its mechanisms in Pseudomonas aeruginosa (In Chinese). Pharmacology and Clinics of Chinese Materia Medica, 2013, 39(2): 83-86. (1)
[21]
谢丽玲, 赵水灵, 余飞, 等. 黄连素对3种水产动物致病菌的抑制作用研究. 南方水产科学, 2013, 9(4): 45-49.
Xie L L, Zhao S L, Yu F, et al. Antibacterial effects of berberine on three aquatic pathogens in vitro (In Chinese). South China Fisherise Science, 2013, 9(4): 45-49. (1)
[22]
赵晨曦, 刘前刚, 张志元. 磷钾细菌解磷解钾能力的研究. 湖南农业大学学报(自然科学版), 2004, 30(6): 519-521.
Zhao C X, Liu Q G, Zhang Z Y. Study on the ability of phosphoric bacteria and potassic bacteria in decomposing of phosphate and silicate (In Chinese). Journal of Hunan Agricultural University (Natural Sciences), 2004, 30(6): 519-521. (1)
[23]
Kimy H, Bae B, Choung Y K. Optimization of biological phosphorus removal from contaminated sediments with phosphate-solubilizing microorganisms . Journal of Bioscience and Bioengineering, 2005, 99(1): 23-29. DOI:10.1263/jbb.99.23 (1)
[24]
宋金凤, 宋利臣, 崔晓阳, 等. 低分子有机酸/盐对森林暗棕壤铁的释放效应. 土壤通报, 2008, 39(2): 315-320.
Song J F, Song L C, Cui X Y, et al. Effects of low molecular weight organic acids/salts on iron release of dark brown forest soil (In Chinese). Chinese Journal of Soil Science, 2008, 39(2): 315-320. (1)
[25]
金朝霞. 草酸盐高效降解菌的筛选及其酶学性质研究. 辽宁大连: 大连理工大学, 2008: 13-15
Jin Z X. Sereening and enzyme properties of a highly effective oxalate-degrading strain (In Chinese). Dalian, Liaoning: Dalian University of Technology, 2008: 13-15 http://www.wanfangdata.com.cn/details/detail.do?_type=degree&id=Y1419438 (2)
[26]
Nandi R, Debnath D, Maiti M. Interactions of berberine with poly(A)and tRNA . Biochimica et Biophysica Acta, 1990, 1049(3): 339-342. DOI:10.1016/0167-4781(90)90107-D (1)
[27]
Kuo C L, Chou C C, Yung B, YUN Y- M.. Berberine complexes with DNA in the berberine-induced apoptosis in human leukemic HL-60 cells . Cancer Letters, 1995, 93: 193-200. DOI:10.1016/0304-3835(95)03809-B (1)
[28]
张志辉, 邓安珺, 于金倩, 等. 黄连碱药理活性研究进展. 中国中药杂志, 2013, 38(17): 2750-2754.
Zhang Z H, Deng A J, Yu J Q, et al. Advance in studies on pharmacological activity of coptisine hydrochloride (In Chinese). China Journal of Chinese Materia Medica, 2013, 38(17): 2750-2754. (1)
[29]
陈哲, 吴敏娜, 秦红灵, 等. 土壤微生物溶磷分子机理研究进展. 土壤学报, 2009, 46(5): 925-931.
Chen Z, Wu M N, Qing H L, et al. Advances in research on molecular mechanisms of phosphate-solubilizing microorganisms in soil (In Chinese). Acta Pedologica Sinica, 2009, 46(5): 925-931. DOI:10.11766/trxb200803140522 (1)
Negative Allelopathic Effects of Extract of Coptis Chinensis Hair Root on Inorganic Phosphorus-Dissolving Bacteria
WANG Yushu1 , LIU Hai1,2 , LI Jia1 , WEI Liben1 , PENG Liyuan1 , HUANG Jianguo1     
1. College of Resources and Environment, Southwest University, Chongqing 400716, China;
2. Institute of Agricultural Science and Technology Information, Guizhou Academy of Agricultural Sciences, Guiyang 550006, China
Abstract: 【Objective】 Coptis chinensis is a medicinal herb traditionally cultivated and used for many centuries in China. As it is now cultivated extensively in large areas and often continuously for years in a row, sustainable development of the industry has come across serious obstacles caused by mono-cropping. it is, therefore, essential to explore allelopathic effects of this medicinal plant on soil microorganisms, in order to understand mechanisms of the occurrence of obstacles in the mono-cropping and provide certain scientific basis for lifting the obstacles.【Method】 To that end, an indoor experiment was carried out culturing three strains of inorganic phosphorus dissolving bacteria, B05, B07 and B09 of Burkholderia Yabunchi bacteria, using the solid and liquid culturing techniqu, During the experiment, extract of fibrous roots of C. chinensis (ERC) was spiked to explore allelopathic effects of the extract on growth, reproduction, and tricalcium phosphate dissolving capacity of the three strains. Fresh C. chinensis roots were soaked in deionized water at 25±1℃ for 24 h to extract active components, and then the solution was filtrated through a 50 μmol L-1filter, and diluted to ERC 0.1 g L-1. For spiking of the extract, the experiment was designed to have four treatments, i.e. 0 (CK), 500 (low concentration), 1 000 (middle concentration), and 2 000 mg L-1(high concentration).【Result】 Results show that the higher the concentration of the ERC, the smaller the size of bacterial colonies, the diameter of P-dissolving halos and the lower the P-dissolving index on the solid culture medium. ERC also inhibited the growth and reproduction of iPDB in variable degrees, reducing the population of iPDB by 35.98%~52.98% in the liquid culture medium as compared with the blank control (without ERC). All the three strains of bacteria tested in the liquid culture released protons, oxalate, and citrate into the solution. Besides, acetate was found in the culture solution with B05, malate with B09, and acetate and succinate with B07. In addition, iPDB varied greatly in tricalcium phosphate dissolving capacity in the culture solution. The mean amount of P dissolved was 228.31 mg L-1 for B05, 311.64 mg L-1 for B07, and 174.89 mg L-1 for B09. Inorganic P dissolution by these bacteria was positively related to efflux of proton (r = 0.877, p ≤ 0.05, n = 12) and organic acids (r = 0.604, p ≤ 0.05, n = 12). When ERC inhibited efflux of protons and organic acids in culture solutions, it reduced P dissolution by 21.68%~60.55%.【Conclusion】 During the growing period, C. chinensis roots released allelopathic chemicals, which affected adversely community structure and physiological and ecological functions of the iPDB, thus reducing bio-availability of inorganic P in the soil, which could be one of the possible reasons why obstacles occur in mono-cropping of C. chinensis.
Key words: Coptis chinensis    Allelopathy    Inorganicphosphate-dissolving bacteria    Organic acids    Protons    
图 1 不同浓度ERC固体培养基iPDB的生长和溶磷状况 Fig. 1 Growth of iPDB and phosphorus dissolution in the solid culture medium relative to ERC concentration
表 1 固体培养时ERC对iPDB生长和溶磷的影响 Table 1 Effects of ERC on iPDB growth and phosphorus dissolution in the solid culture medium
图 2 在不同浓度的ERC培养液中iPDB的数量变化 Fig. 2 Variation of the population of iPDB in the liquid culture medium relative to ERC concentration 注:不同大、小写字母分别表示菌株间和处理间差异显著(p≤0.05) Note: Different capital and lowercase letters indicate significant difference betweenbacterial strains and treatmentsat 0.05 level, respectively
表 2 iPDB培养液中的有机酸和氢离子浓度 Table 2 Concentrations of organic acids(mg L-1)and protons (μg L-1) in the iPDB culture solutions
图 3 不同浓度的ERC培养液中iPDB的溶磷量 Fig. 3 Content of phosphorus dissolved by iPDB in the liquid culture medium relative toERC concentration
表 3 有机酸和氢离子分泌及溶磷量之间的相关系数 Table 3 Correlation coefficients of P dissolution with release of organic acids and protons
黄连须根浸提液对无机磷细菌的负化感效应
王玉书 , 刘海 , 李佳 , 魏立本 , 彭丽媛 , 黄建国