不同改良处理对梅州离子型稀土矿废弃地大豆根瘤菌多样性的影响
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摘要
为评价梅州离子型稀土矿废弃地不同土壤改良措施对大豆根瘤菌多样性的影响,通过nif H-PCR-DGGE技术对不同土壤改良处理条件下大豆根瘤菌群落的多样性进行表征。结果表明:7种不同土壤改良处理的DGGE图谱条带数目不等,位置各异,说明梅州离子型稀土矿废弃地大豆的根瘤菌群落结构受改良措施的影响较大,具有较强的可塑造性。其中T4处理(有机肥+生物炭+钝化剂)的条带数最多,多样性指数最高,说明该处理对增加大豆根瘤菌的多样性效果最好,可作为以后土壤改良的参考依据。聚类分析结果显示:7个处理可分为4个类群,CK单独为一个类群,T1(有机肥)、T2(有机肥+生物炭)为一个类群,T4(有机肥+生物炭+钝化剂)和T5(有机肥+菌剂+蚯蚓)为一个类群,T3(有机肥+钝化剂)和T6(有机肥+菌剂+蚯蚓+生物炭+钝化剂)为一个类群。生物炭与钝化剂组合处理、菌剂与蚯蚓的组合处理对增加大豆根瘤内根瘤菌的多样性效果较显著。通过序列比对和系统发育树可以看出,在梅州黄畲地区稀土采矿迹地,大豆主要与慢生根瘤菌属菌株共生结瘤,也可与中华根瘤菌属菌株共生。大豆根瘤内根瘤菌的多样性与土壤镉含量之间为显著正相关关系,与土壤有机质、氮、磷等营养指标之间为负相关关系,说明根瘤菌可能在提高大豆耐镉方面具有重要作用。
In order to evaluate the effect of different improvement measures on the diversity of rhizobium of soybean(Glycinemax) in the abandoned land of ionic rare earth mine,nif H-PCR-DGGE was used to manifestate the diversity of Rhizobia. Theresults of denaturing gradient gel electrophoresis(DGGE) fingerprint showed that there were differences in the number,thepositions and the brightness of bands depended on different restorative treatments,which showed that different soil amendmentshad a significant effect on the community structure of Rhizobium. The number and diversity index of bands of T4 treatment(fertilizer + biochar + deactivator) were highest,it was best to improve the diversity of rhizobium of soybean. The results ofcluster analysis showed that 7 treatments could be divided into four groups,CK was group 1,T1 and T2 were group 2,T4 andT5 were group 3,T3 and T6 were group 4. The combination of biochar and passivator,the combination of bacterial manureand earthworm had the more significant effect on increasing the diversity of rhizobia in root nodules of soybean. According tosequence comparison and phylogenetic tree,soybean is mainly lived in symbiosis with Bradyrhizobium,and also coexisted withSinorhizobium in the abandoned land of ionic rare earth mine of Meizhou,Guangdong. There was significant positive correla-tion between the diversity of rhizobium and soil cadmium content,and negative correlation among the diversity of rhizobiumand soil organic matter,nitrogen,phosphorus and other nutritional indicators,which suggesting that rhizobium may play animportant role in improving the cadmium tolerance of soybean.
In order to evaluate the effect of different improvement measures on the diversity of rhizobium of soybean(Glycinemax) in the abandoned land of ionic rare earth mine,nif H-PCR-DGGE was used to manifestate the diversity of Rhizobia. Theresults of denaturing gradient gel electrophoresis(DGGE) fingerprint showed that there were differences in the number,thepositions and the brightness of bands depended on different restorative treatments,which showed that different soil amendmentshad a significant effect on the community structure of Rhizobium. The number and diversity index of bands of T4 treatment(fertilizer + biochar + deactivator) were highest,it was best to improve the diversity of rhizobium of soybean. The results ofcluster analysis showed that 7 treatments could be divided into four groups,CK was group 1,T1 and T2 were group 2,T4 andT5 were group 3,T3 and T6 were group 4. The combination of biochar and passivator,the combination of bacterial manureand earthworm had the more significant effect on increasing the diversity of rhizobia in root nodules of soybean. According tosequence comparison and phylogenetic tree,soybean is mainly lived in symbiosis with Bradyrhizobium,and also coexisted withSinorhizobium in the abandoned land of ionic rare earth mine of Meizhou,Guangdong. There was significant positive correla-tion between the diversity of rhizobium and soil cadmium content,and negative correlation among the diversity of rhizobiumand soil organic matter,nitrogen,phosphorus and other nutritional indicators,which suggesting that rhizobium may play animportant role in improving the cadmium tolerance of soybean.
引文
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[7]陈雪丽,王玉峰,李伟群,等.黑土区连作大豆根际微生物群落结构的动态变化[J].大豆科学,2018,37(5):748-755.(Chen X L,Wang Y F,Li W Q,et al. Dynamic evolution of mi-crobial community in the rhizosphere of continuous cropping of soy-bean in black soil[J]. Soybean Science, 2018, 37(5):748-755.)
[8]郭慧娟.大豆快生根瘤菌共生基因的遗传分化及快慢生根瘤菌对土壤微生物的影响比较[D].北京:中国农业大学,2014:1-8.(Guo H J. Replicon-dependent differentiation of sym-biosis-related genes in Sinorhizobium strains and the effect on soilmicrobial community of Sinorhizobium and Bradyrhizobium[D].Beijing:China Agricultural University,2014:1-8.)
[9]关大伟,李力,姜昕,等.长期施肥对黑土大豆根瘤菌群体结构和多样性的影响[J].生物多样性,2015,23(1):68-78.(Guan D W,Li L,Jiang X,et al. Influence of long-term fertiliza-tion on the community structure and diversity of soybean rhizobia inblack soil[J]. Biodiversity Science,2015,23(1):68-78.)
[10] Poly F,Monrozier L J,Bally R. Improvement in the RFLP proce-dure for studying the diversity of nif H genes in communities of ni-trogen fixers in soil[J]. Research in Microbiology,2001,152(1):95-103.
[11]沈建秀.接种根瘤菌后刺槐对镉胁迫的响应[D].杨凌:西北农林科技大学,2017:16-30.(Shen J X. Response of Robiniapseudoacacia to cadmium stress after inoculation with rhizobium[D]. Yangling:Northwest A&F University,2017:16-30.)
[12] Jin Y,Liu H,Luo D,et al. DELLA proteins are common compo-nents of symbiotic rhizobial and mycorrhizal signalling pathways[J]. Nature Communications,2016,7:12433.
[13] Zehr J P,Jenkins B D,Short S M,et al. Nitrogenase gene diver-sity and microbial community structure:A cross-system comparison[J]. Environ Microbiol,2003,5(7):539-554.
[14]唐凤灶.安徽铜陵铜尾矿原生演替过程中的土壤固氮菌研究[D].广州:中山大学,2010:5-30.(Tang F Z. Soil nitrogen-fixing microorganisms in primary succession of copper mine tailingsat Tongling,Anhui,China[D]. Guangzhou:Sun Yat-sen Univer-sity,2010:5-30.)
[15]姚钦.生物炭施用对东北黑土土壤理化性质和微生物多样性的影响[D].长春:中国科学院大学(中国科学院东北地理与农业生态研究所),2017:21-39.(Yao Q. Effect of biochar ad-dition on soil physicochemical properties and microbial diversity ina black soil of northeast China[D]. Changchun:Northeast Institu-te of geography and Agroecology,Chinese Academy of Sciences,2017:21-39.)
[16] Kolton M,Graber E R,Tsehansky L,et al. Biochar-stimulatedplant performance is strongly linked to microbial diversity and met-abolic potential in the rhizosphere[J]. New Phytologist,2017,213(3):1393-1404.
[17]唐行灿,陈金林.生物炭对土壤理化和微生物性质影响研究进展[J].生态科学,2018,37(1):192-199.(Tang X C,Chen JL. Review of the effect of biochar on soil physi-chemical and mi-crobial properties[J]. Ecological Science, 2018, 37(1):192-199.)
[18]何莉莉,杨慧敏,钟哲科,等.生物炭对农田土壤细菌群落多样性影响的PCR-DGGE分析[J].生态学报,2014,34(15):4288-4294.(He L L,Yang H M,Zhong Z K,et al. PCR-DGGEanalysis of soil bacterium community diversity in farmlandinflu-enced by biochar[J]. Acta Ecologica Sinica,2014,34(15):4288-4294.)
[19] Wu J,Wang J,Zhang X,et al. A gyr B-targeted PCR for rapid i-dentification of Paenibacillus mucilaginosus[J]. Applied Microbi-ology and Biotechnology,2010,87(2):739-747.
[20] Ma M,Wang Z,Li L,et al. Complete genome sequence of Paeni-bacillus mucilaginosus 3016,a bacterium functional as microbialfertilizer[J]. Journal of Bacteriology, 2012, 194(10):2777-2778.
[21]李敏.铅镉污染土壤的稳定化及蚯蚓联合植物修复[D].上海:华东师范大学,2018:45-54.(Li M. Stabilization andearthworm-assisted phytoremediation of Pb-Cd contaminated soil[D]. Shanghai:East China Normal University,2018:45-54.)
[22]李彦霈,邵明安,王娇.蚯蚓粪施用方式及用量对土壤入渗的影响[J/OL].土壤学报,DOI:10. 11766/trxb201807250261.(Li Y P,Shao M A,Wang J. Effects of pattern and amount ofearthworm cast application on water infiltration in soil[J/OL].Acta Pedologica Sinica,DOI:10. 11766/trxb201807250261.)
[23] Peix A,Ramirez-Bahena M H,Velazquez E,et al. Bacterial as-sociations with legumes[J]. Critiacal Reviews in Plant Sciences,2015,34(1-3SI):17-42.
[24] Clua J,Roda C,Eugenia Z,et al. Compatibility between legumesand rhizobia for the establishment of a successful nitrogen-fixingsymbiosis[J]. Genes,2018,9(3):125.
[2]刘文深,刘畅,王志威,等.离子型稀土矿尾砂地植被恢复障碍因子研究[J].土壤学报,2015,52(4):879-887.(Liu WS,Liu C,Wang Z W,et al. Limiting factors for restoration ofdumping sites of ionic rare earth mine tailings[J]. Acta Pedologi-ca Sinica,2015,52(4):879-887.)
[3]黄华谷,黄铁兰,周兆帅,等.广东三个离子吸附型稀土矿的地球化学特征及开采现状[J].岩矿测试,2014,33(5):737-746.(Huang H G,Huang T L,Zhou Z S,et al. Mining situationand geochemistry characteristics of three ion adsorption rare-earthdeposit in Guangdong province[J]. Rock and Mineral Analysis,2014,33(5):737-746.)
[4]池汝安,田君.风化壳淋积型稀土矿评述[J].中国稀土学报,2007,25(6):641-650.(Chi R A,Tian J. Review ofweathered crust rare earth ore[J]. Journal of the Chinese RareEarth Society,2007,25(6):641-650.)
[5]金姝兰,黄益宗.稀土元素对农田生态系统的影响研究进展[J].生态学报,2013,33(16):4836-4845.(Jin S L,HuangY Z. A review on rare earth elements in farmland ecosystem[J].Acta Ecologica Sinica,2013,33(16):4836-4845.)
[6]连腾祥.大豆光合碳在黑土中的转化过程及细菌群落特征[D].长春:中国科学院研究生院(中国科学院东北地理与农业生态研究所),2016:53-66.(Lian T X. The turnover of pho-tosynthetic carbon of soybean and relevant bacterial communitycharacteristics in the Mollisols[D]. Changchun:Northeast Institu-te of geography and Agroecology,Chinese Academy of Sciences,2016:53-66.)
[7]陈雪丽,王玉峰,李伟群,等.黑土区连作大豆根际微生物群落结构的动态变化[J].大豆科学,2018,37(5):748-755.(Chen X L,Wang Y F,Li W Q,et al. Dynamic evolution of mi-crobial community in the rhizosphere of continuous cropping of soy-bean in black soil[J]. Soybean Science, 2018, 37(5):748-755.)
[8]郭慧娟.大豆快生根瘤菌共生基因的遗传分化及快慢生根瘤菌对土壤微生物的影响比较[D].北京:中国农业大学,2014:1-8.(Guo H J. Replicon-dependent differentiation of sym-biosis-related genes in Sinorhizobium strains and the effect on soilmicrobial community of Sinorhizobium and Bradyrhizobium[D].Beijing:China Agricultural University,2014:1-8.)
[9]关大伟,李力,姜昕,等.长期施肥对黑土大豆根瘤菌群体结构和多样性的影响[J].生物多样性,2015,23(1):68-78.(Guan D W,Li L,Jiang X,et al. Influence of long-term fertiliza-tion on the community structure and diversity of soybean rhizobia inblack soil[J]. Biodiversity Science,2015,23(1):68-78.)
[10] Poly F,Monrozier L J,Bally R. Improvement in the RFLP proce-dure for studying the diversity of nif H genes in communities of ni-trogen fixers in soil[J]. Research in Microbiology,2001,152(1):95-103.
[11]沈建秀.接种根瘤菌后刺槐对镉胁迫的响应[D].杨凌:西北农林科技大学,2017:16-30.(Shen J X. Response of Robiniapseudoacacia to cadmium stress after inoculation with rhizobium[D]. Yangling:Northwest A&F University,2017:16-30.)
[12] Jin Y,Liu H,Luo D,et al. DELLA proteins are common compo-nents of symbiotic rhizobial and mycorrhizal signalling pathways[J]. Nature Communications,2016,7:12433.
[13] Zehr J P,Jenkins B D,Short S M,et al. Nitrogenase gene diver-sity and microbial community structure:A cross-system comparison[J]. Environ Microbiol,2003,5(7):539-554.
[14]唐凤灶.安徽铜陵铜尾矿原生演替过程中的土壤固氮菌研究[D].广州:中山大学,2010:5-30.(Tang F Z. Soil nitrogen-fixing microorganisms in primary succession of copper mine tailingsat Tongling,Anhui,China[D]. Guangzhou:Sun Yat-sen Univer-sity,2010:5-30.)
[15]姚钦.生物炭施用对东北黑土土壤理化性质和微生物多样性的影响[D].长春:中国科学院大学(中国科学院东北地理与农业生态研究所),2017:21-39.(Yao Q. Effect of biochar ad-dition on soil physicochemical properties and microbial diversity ina black soil of northeast China[D]. Changchun:Northeast Institu-te of geography and Agroecology,Chinese Academy of Sciences,2017:21-39.)
[16] Kolton M,Graber E R,Tsehansky L,et al. Biochar-stimulatedplant performance is strongly linked to microbial diversity and met-abolic potential in the rhizosphere[J]. New Phytologist,2017,213(3):1393-1404.
[17]唐行灿,陈金林.生物炭对土壤理化和微生物性质影响研究进展[J].生态科学,2018,37(1):192-199.(Tang X C,Chen JL. Review of the effect of biochar on soil physi-chemical and mi-crobial properties[J]. Ecological Science, 2018, 37(1):192-199.)
[18]何莉莉,杨慧敏,钟哲科,等.生物炭对农田土壤细菌群落多样性影响的PCR-DGGE分析[J].生态学报,2014,34(15):4288-4294.(He L L,Yang H M,Zhong Z K,et al. PCR-DGGEanalysis of soil bacterium community diversity in farmlandinflu-enced by biochar[J]. Acta Ecologica Sinica,2014,34(15):4288-4294.)
[19] Wu J,Wang J,Zhang X,et al. A gyr B-targeted PCR for rapid i-dentification of Paenibacillus mucilaginosus[J]. Applied Microbi-ology and Biotechnology,2010,87(2):739-747.
[20] Ma M,Wang Z,Li L,et al. Complete genome sequence of Paeni-bacillus mucilaginosus 3016,a bacterium functional as microbialfertilizer[J]. Journal of Bacteriology, 2012, 194(10):2777-2778.
[21]李敏.铅镉污染土壤的稳定化及蚯蚓联合植物修复[D].上海:华东师范大学,2018:45-54.(Li M. Stabilization andearthworm-assisted phytoremediation of Pb-Cd contaminated soil[D]. Shanghai:East China Normal University,2018:45-54.)
[22]李彦霈,邵明安,王娇.蚯蚓粪施用方式及用量对土壤入渗的影响[J/OL].土壤学报,DOI:10. 11766/trxb201807250261.(Li Y P,Shao M A,Wang J. Effects of pattern and amount ofearthworm cast application on water infiltration in soil[J/OL].Acta Pedologica Sinica,DOI:10. 11766/trxb201807250261.)
[23] Peix A,Ramirez-Bahena M H,Velazquez E,et al. Bacterial as-sociations with legumes[J]. Critiacal Reviews in Plant Sciences,2015,34(1-3SI):17-42.
[24] Clua J,Roda C,Eugenia Z,et al. Compatibility between legumesand rhizobia for the establishment of a successful nitrogen-fixingsymbiosis[J]. Genes,2018,9(3):125.