花生化感物质降解菌的筛选、鉴定与降解特性研究
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摘要
为筛选高效化感物质降解菌,以花生根系分泌物中的化感物质苯甲酸作为唯一碳源,采用富集培养和稀释平板法从连作花生长期定位试验地的健康花生植株根际土壤中筛选高效花生化感物质降解菌,经形态观察、生理生化分析和16S rDNA序列同源性比较以及系统发育分析对降解菌进行鉴定,并通过单因素试验对降解菌进行降解特性研究。结果表明:筛选到的2株高效降解菌分别为木糖氧化无色杆菌(Achromobacter xylosoxidans HJ-2)和硝基还原假单胞菌(Pseudomonas nitroreducens HJ-3)。降解菌HJ-2降解的适宜pH为6.0～9.0,适宜温度为30～45℃,适宜NaCl浓度为0～6%;降解菌HJ-3降解的适宜pH为6.0～8.0,适宜温度为30～35℃,适宜NaCl浓度为0～4%。两株降解菌都能够降解苯乙酮、硬脂酸、棕榈酸、乳酸、3,5-二甲基苯甲醛和丙三醇等化感物质。研究表明,降解菌HJ-2和HJ-3对苯甲酸有显著降解效果,两个菌株均具有广谱降解化感物质的特性,且降解菌HJ-2对不良环境适应能力较强。
Allelopathy is one of the most important factors inducing peanut replant disease. Thus, screening out highly efficient degrading bacteria is an essential means to inhibit peanut allelopathy. Benzoic acid has been considered one of the main allelochemicals in peanut root-exudates. Using benzoic acid as the sole carbon source, degrading bacteria were screened from the rhizosphere soil of healthy peanut plants, in a long-term experiment of peanut monoculture, through enrichment culture and dilution-plate methods. The degrading bacteria were identified by morphological characteristics and 16S rDNA homology comparison. The degrading properties of bacteria were analyzed by single factor tests. Two allelochemical-degrading bacteria were isolated and named strain HJ-2 and strain HJ-3, which belonged to Achromobacter xylosoxidans and Pseudomonas nitroreducens respectively. The appropriate degrading conditions for HJ-2 were pH 6.0～9.0,30～45 ℃, and 0～6% NaCl. For strain HJ-3, the appropriate degrading conditions were pH 6.0～8.0, 30～35 ℃, and 0～4% NaCl. Besides the high efficiency of benzoic acid degradation, both strains degraded some other allelochemicals, including acetophenone, stearic acid, palmitic acid, lactic acid, 3,5-dimethylbenzaldehyde, and glycerol. The HJ-2 and HJ-3 strains both had broad-spectrum degradation characteristics. However, due to its stronger environment adaption, the HJ-2 strain provided theoretical significance for the governance of peanut replant disease in the future.
Allelopathy is one of the most important factors inducing peanut replant disease. Thus, screening out highly efficient degrading bacteria is an essential means to inhibit peanut allelopathy. Benzoic acid has been considered one of the main allelochemicals in peanut root-exudates. Using benzoic acid as the sole carbon source, degrading bacteria were screened from the rhizosphere soil of healthy peanut plants, in a long-term experiment of peanut monoculture, through enrichment culture and dilution-plate methods. The degrading bacteria were identified by morphological characteristics and 16S rDNA homology comparison. The degrading properties of bacteria were analyzed by single factor tests. Two allelochemical-degrading bacteria were isolated and named strain HJ-2 and strain HJ-3, which belonged to Achromobacter xylosoxidans and Pseudomonas nitroreducens respectively. The appropriate degrading conditions for HJ-2 were pH 6.0～9.0,30～45 ℃, and 0～6% NaCl. For strain HJ-3, the appropriate degrading conditions were pH 6.0～8.0, 30～35 ℃, and 0～4% NaCl. Besides the high efficiency of benzoic acid degradation, both strains degraded some other allelochemicals, including acetophenone, stearic acid, palmitic acid, lactic acid, 3,5-dimethylbenzaldehyde, and glycerol. The HJ-2 and HJ-3 strains both had broad-spectrum degradation characteristics. However, due to its stronger environment adaption, the HJ-2 strain provided theoretical significance for the governance of peanut replant disease in the future.
引文
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[4]Huang L F,Song L X,Xia X J,et al.Plant-soil feedbacks and soil sickness:From mechanisms to application in agriculture[J].Journal of Chemical Ecology,2013,39(2):232-242.
[5]刘苹,赵海军,李庆凯,等.三种酚酸类化感物质对花生根际土壤微生物及产量的影响[J].中国油料作物学报,2018,40(1):101-109.LIU Ping,ZHAO Hai-jun,LI Qing-kai,et al.Effects of three phenolic acid allelochemicals on rhizosphere soil microbes and pod yield of peanut(Arachis hypogaea L.)[J].Chinese Journal of Oil Crop Sciences,2018,40(1):101-109.
[6]李培栋,王兴祥,李奕林,等.连作花生土壤中酚酸类物质的检测及其对花生的化感作用[J].生态学报,2010,30(8):2128-2134.LI Pei-dong,WANG Xing-xiang,LI Yi-lin,et al.The contents of phenolic acids in continuous cropping peanut and their allelopathy[J].Acta Ecologica Sinica,2010,30(8):2128-2134.
[7]Deng J J,Zhang Y L,Hu J W,et al.Autotoxicity of phthalate esters in tobacco root exudates:Effects on seed germination and seedling growth[J].Pedosphere,2017,27(6):1073-1082.
[8]Yu J Q,Matsui Y.Phytotoxic substances in root exudates of cucumber(Cucumis sativus L.)[J].Journal of Chemical Ecology,1994,20(1):21-31.
[9]Li X G,Ding C F,Hua K,et al.Soil sickness of peanuts is attributable to modifications in soil microbes induced by peanut root exudates rather than to direct allelopathy[J].Soil Biology and Biochemistry,2014,78:149-159.
[10]王小兵,骆永明,刘五星,等.花生根分泌物的鉴定及其化感作用[J].生态学杂志,2011,30(12):2803-2808.WANG Xiao-bing,LUO Yong-ming,LIU Wu-xing,et al.Identification of peanut root exudates and their allelopathic effects[J].Chinese Journal of Ecology,2011,30(12):2803-2808.
[11]袁云云,咸洪泉,洪永聪,等.花生根系分泌物的鉴定及其化感效应分析[J].花生学报,2011,40(3):24-29.YUAN Yun-yun,XIAN Hong-quan,HONG Yong-cong,et al.Identification of peanut root exudates and the analysis of its allelopathy effect[J].Journal of Peanut Science,2011,40(3):24-29.
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[13]Dong L L,Xu J,Li Y,et al.Manipulation of microbial community in the rhizosphere alleviates the replanting issues in Panax ginseng[J].Soil Biology and Biochemistry,2018,125:64-74.
[14]Liu S,Qin F,Yu S.Eucalyptus urophylla,root-associated fungi can counteract the negative influence of phenolic acid allelochemicals[J].Applied Soil Ecology,2018,127:1-7.
[15]Chen S Y,Guo L Y,Bai J G,et al.Biodegradation of p-hydroxybenzoic acid in soil by Pseudomonas putida CSY-P1 isolated from cucumber rhizosphere soil[J].Plant and Soil,2014,389(1):197-210.
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[17]何志刚,汪仁,王秀娟,等.花生自毒物质降解菌的筛选及其降解效果初步研究[J].中国农学通报,2014,30(21):224-227.HE Zhi-gang,WANG Ren,WANG Xiu-juan,et al.Preliminary study of screening of peanut autotoxicity degrading microorganism and the degradation effect[J].Chinese Agricultural Science Bulletin,2014,30(21):224-227.
[18]Pramanik M H R,Nagai M,Asao T,et al.Effects of temperature and photoperiod on the phytotoxic root exudates of cucumber(Cucumis sativus)in hydroponic culture[J].Journal of Chemical Ecology,2000,26(8):1953-1967.
[19]贺强礼,刘文斌,杨海君,等.一株苯酚降解菌的筛选鉴定及响应面法优化其降解[J].环境科学学报,2016,36(1):112-123.HE Qiang-li,LIU Wen-bin,YANG Hai-jun,et al.Isolation,identification of a phenol-degradation using response surface methodology[J].Acta Scientiae Cirecumstantiae,2016,36(1):112-123.
[20]Etal E H.Bergey′s manual of determinative bacteriology[M].9th ed.Lippincott,Williams&Wilkins,1994.
[21]Janso J E,Carter G T.Phylogenetically unique endophytic actinomycetes from tropical plants possess great biosynthetic potential[J].Applied&Environmental Microbiology,2010,76(13):4377-4386.
[22]Lloyd-Jones G,Laurie A D,Hunter D W F,et al.Analysis of catabolic genes for naphthalene and phenanthrene degradation in contaminated New Zealand soils[J].FEMS Microbiology Ecology,2010,29(1):69-79.
[23]李晓芝,王占武,张翠绵,等.设施蔬菜连作障碍成因分析与防治对策[J].河北农业科学,2006,10(1):113-116.LI Xiao-zhi,WANG Zhan-wu,ZHANG Cui-mian,et al.The problems caused by monoculture and the solutions in protected vegetables cultivation[J].Journal of Hebei Agricultural Sciences,2006,10(1):113-116.
[24]刘虹,张朋朋,刘娜,等.三种细菌降解直链烷烃的效果及降解动力学研究[J].安全与环境工程,2017,24(3):66-70.LIU Hong,ZHANG Peng-peng,LIU Na,et al.Degradation efficiency and dynamics of straight-chain alkane degraded by three kinds of bacteria[J].Safety&Environmental Engineering,2017,24(3):66-70.
[25]李怡,李勤奋,邓晓,等.苯甲酰脲类杀虫剂降解菌的分离鉴定及其降解特性研究[J].应用与环境生物学报,2018,24(4):928-933.LI Yi,LI Qin-fen,DENG Xiao,et al.Isolation,identification,and the biodegradation characteristics of a benzoylurea insecticides-degrading strain[J].Chinese Journal of Applied&Environmental Biology,2018,24(4):928-933.
[26]成洁,杜慧玲,张天宝,等.四环素类抗生素降解菌的分离与鉴定[J].核农学报,2017,31(5):884-888.CHENG Jie,DU Hui-ling,ZHANG Tian-bao,et al.Isolation and identification of tetracyclines degrading bacteria[J].Journal of Nuclear Agricultural Sciences,2017,31(5):884-888.
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