盐生植物对黄河三角洲微生物多样性的影响
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摘要
黄河三角洲是我国三大河口三角洲之一,具有巨大土地开发利用潜力,2009年,国务院正式批复了黄河三角洲高效生态经济区发展规划,将其上升为国家战略。但该区濒临渤海,滨海盐渍土广泛分布,土壤含盐量高,盐分主要以氯化物为主,土壤表层盐分大多在0.4%-3%的范围,土壤结构性差,肥力低,极大地限制了该区经济社会的发展。本文针对黄河三角洲地区的实际情况,运用传统的平板培养法和现代分子生物学技术研究了不同盐生植物对黄河三角洲土壤微生物多样性的影响、盐生植物长期种植对黄河三角洲土壤微生物多样性的影响和土壤微生物区系季节动态变化,为研究黄河三角洲盐碱地生态系统的改良和治理阶段土壤微生物学多样性的变化、构建优化的植被恢复技术和提高不同植被的生产力提供理论依据。主要研究内容和结果如下:
1、确立了从盐碱地中高效提取和纯化土壤DNA的方法,并证明了该方法对多种不同样品的适用性
以黄河三角洲盐碱地土壤样品为研究对象,对目前已报道的21种土壤DNA的提取方法和4种纯化方法进行比较研究,探索出了一套高效的从土壤样品中提取、纯化大片段DNA的方法——改良微波法,此方法经济实用,能在4小时以内获得够质够量的土壤DNA用于微生物分子生态学研究,较适合于广大实验室进行土壤DNA提取工作。通过掺菌试验、RFLP、RAPD等试验,表明运用改良微波法可以有效的从不同样品(肥沃土壤、活性污泥、河底淤泥、河沙、盐碱地土壤、哺乳动物粪便和盐芥叶片)中将DNA提取出来,而且提取出的DNA是可以直接进行分子生物学操作的,能够应用于微生物分子生态学研究。
2、明确了不同盐生植物对土壤微生物的影响
种植盐生植物后,通过测定盐生植物根际和空白对照土壤微生物的数量变化,结果表明,种植以上五种盐生植物后,土壤中微生物的总数都明显上升,只是程度不一,微生物总数多少依次为柽柳>二色补血草>马蔺>金银花>中亚滨藜>对照,并且耐盐微生物的数量和比例均有所下降;各样地土壤微生物生理类群的数量表现为:氨化细菌>自生固氮菌>纤维素分解菌>芽孢菌>硫化细菌>反硫化细菌>亚硝化细菌>硝化细菌>反硝化细菌,氨化细菌和自生固氮菌总数处于绝对的优势,纤维素分解菌的数量较多,而且各样地之间生理类群之间差异比较明显,且各样地土壤微生物生理类群微生物的数量多少依次为柽柳>二色补血草>金银花>马蔺>中亚滨藜>对照,土壤中各生理类群不仅有助于保持土壤肥力还可以加强其他根际微生物的生命活动,在本试验中柽柳对土壤微生物的影响最大,可以显著提高其在土壤中的数量,而且土壤中氨化细菌、自生固氮菌、纤维素分解菌和亚硝化细菌的数量是最多的。
RFLP分析和RAPD分析显示,种植盐生植物后,土壤微生物的种类和数量都发生了相应的变化,RFLP分析显示6样地16S rDNA扩增产物经HinfI和Csp6I酶切产生的条带的大小基本一致,只是亮度有所区别;RAPD分子标记分析显示,17条引物共计扩增出331条RAPD条带,98.8%为多态性条带,通过RAPD条带图谱和丰富度分析表明,种植不同盐生植物后,微生物的种类都有所增加,不同样地间的微生物种类也有所区别,其中样地6(柽柳样地)最为明显,6块样地微生物群落的DNA序列Shannon-Wiener指数有所不同,大小依次为样地6>样地5>样地1>样地2>样地3>样地4,但是样地间的Shannon-Wiener指数差异性不大,群落DNA序列的均匀度大小依次为为样地1>样地5>样地3>样地2>样地4>样地6,整体看样地1和样地6均匀度指数差别较大外,各样地间土壤微生物的均匀度无显著差异。
运用传统纯培养方法结合现代分子生物学技术测定五种盐生植物对土壤微生物的影响表明,种植盐生植物后,土壤微生物的种类和数量都发生了相应的变化,其中样地6(柽柳)土壤微生物的种类和数量增加最为显著。
3、探明了盐生植物长期种植对黄河三角洲土壤微生物多样性的影响
采用传统的生物学手段对不同种植年限的盐生植物(两年柽柳、十年柽柳、两年盐地碱蓬和十年盐地碱蓬)根际土壤微生物种类和数量的变化进行研究,结果表明,种植盐生植物后土壤中微生物的数量显著提高,而且柽柳对微生物的影响比较大,可以显著提高微生物的数量,并且耐盐细菌的比例显著下降,随着连续种植时间的增加,微生物总数的减少以及真菌数量的增加,细菌与真菌的比值(即B/F)显著变小,并且并且氨化细菌、自生固氮菌、硝化细菌和亚硝化细菌等生理类群微生物的数量也逐渐下降;盐地碱蓬和柽柳类似,可以提高微生物的数量,但是幅度没有柽柳大,而且随着种植年限的延长,微生物的数量逐步提高,细菌与真菌的比值(即B /F)也是逐步提高,从生理类群的研究结果看,随着种植年限的延长,土壤中的自生固氮菌、硝化细菌、亚硝化细菌、纤维素分解菌和硫化细菌的数量也是在慢慢下降。
4、初步探明了黄河三角洲土壤微生物区系季节动态变化规律
以二色补血草、盐地碱蓬和柽柳根际土壤微生物为研究对象,通过对不同月份(4月、7月和9月)土壤微生物的数量变化研究了微生物区系季节动态变化,结果表明,同一盐生植物根际不同月份之间微生物数量呈现一定的变化规律,细菌在各样地均占绝对优势,比例最大,微生物的数量7月份较多,9月份较少,而且各月份之间差异显著;同一时期,不同盐生植物土壤微生物的数量:柽柳样地>盐地碱蓬样地>二色补血草样地;各微生物生理类群的动态变化均有其自身的规律,但是无论是在4月、7月份还是9月份,各类群数量的多少顺序为:氨化细菌>自生固氮菌>纤维素分解菌>芽孢菌>硫化细菌>反硫化细菌>亚硝化细菌>硝化细菌>反硝化细菌。
Yellow River Delta, one of the three largest river estuary deltas in China, has high potentials of land development and utilization, but the widely distributed saline soils here in the coastal area at Bohai-Sea severely restrict the development of agriculture. The quite high salt content of the saline soils mainly chlorates, ranging from 0.4% to 3% in the surface layer, worsens the soil structure and lowers the soil fertility. Considering the soil properties and other conditions in Yellow River Delta, we used the traditional plate culture method and modern molecular biology techniques to study the effect of different halophytes on the soil microbial diversity, the long-term cultivation of halophytes’effects on soil microbial community, and the seasonal dynamics changes of soil microorganism. Based on these, we can study the changes of microbial diversity of Yellow River Delta saline-alkali soil in the ecosystem improvement and treatment stage, and study the forest quality improvement and ecology recovery technology. The main contents and results of this study are presented as followings:
1. We established a simple, rapid, cost-effective method for the extraction of microbial DNA from different samples, including the saline-alkali soil.
We chose the saline-alkali soil samples from the Yellow River Delta as the research objects, used 21 kinds of extraction methods and four kinds of purification methods to extract and purify the soil DNA, and established a simple, rapid, cost-effective method for the extraction of microbial DNA——microwave method. It could be effectively used in different samples(fertile soil, active sludge, riverbed sullage, sand, alkaline land soil, album canis and leaves of Arabidopsis salsuginea), and the PCR amplification results showed that the extracted DNA can be directly operated in molecular biology, which demonstrated the method has broad applicability.
2. We ascertained the effect of different halophytes on the soil microbial diversity.
After the cultivation of halophytes, the number of soil microbial in the five kinds of halophytes rhizosphere showed that the total number of microorganisms in the soil were significantly increased, but the degree of change was t not the same, the sequence of the influence on the increase of the soil microbial number were: Tamarix chinensis Lour.>Limonium bicolor(Bge.) Kuntz>Iris ensata Thunb>Lonicera japonica>Atriplex centralasiatica Iljin>CK, and the salt-tolerant micro-organisms’number and proportion has declined. The results indicated that the total number of physiological microorganism showed the following rule: Ammonifying>Aerobe azotobacter>Aerobe cellulolytic>Bacillus>Thiobacillus>Desulphurizing bacteria>Nitrosomonas>Nitrifiers>Dennitrifying bacteria. The amount of ammonfying and aerobe azotobacter took absolute dominance, the number of different plots followed as:Tamarix chinensis Lour.>Limonium bicolor(Bge.) Kuntz>Lonicera japonica>Iris ensata Thunb>Atriplex centralasiatica Iljin>CK. Various physiological microorganism groups of soil would not only help maintain soil fertility but also enhance the lives of other rhizosphere microbial activity. In this experiment, Tamarix chinensis Lour. had the greatest influence on soil microorganisms and could significantly increase their number of the soil, and the amounts of ammonification bacteria, aerobe azotobacter, aerobe cellulolytic and Nitrosomonas were the majority.
The results of RFLP and RAPD analysis indicated that after the cultivation of halophytes, the type and quantity of soil microbes were changed, RFLP analysis showed that the six plots’16S rDNA amplification products were digested by HinfI and Csp6I giving birth to the similar bands, even though they had different brightness. The RAPD fingerprints showed substantial differences among the different soil samples, and according to apparent changes in the number and size of amplified DNA fragments, the Shannon-weaver index of different plots was Tamarix chinensis Lour.>Lonicera japonica>CK>Limonium bicolor(Bge.) Kuntz>Atriplex centralasiatica Iljin>Iris ensata Thunb, and the Pielou index were CK>Lonicera japonica>Atriplex centralasiatica Iljin>Limonium bicolor(Bge.) Kuntz>Iris ensata Thunb>Tamarix chinensis Lour..
3.Ascertained the effect of long-term cultivation of halophytes on the microbial diversity.
Using the traditional means to determine the rhizosphere microbial changes in the types and quantities of different halophytes growing for long years, the result of the study indicated that the number of micro-organisms significantly increased after halophytes planted, and the Tamarix chinensis Lour. showed greater influence, which could significantly increase the number of micro-organisms, and at the same time the salt-tolerant bacteria’proportion was decreased significantly. After two-year cultivation, the number of microorganisms reached to the maximum, and the ratio of bacteria and fungi (B/F) significantly descended with the continuous cultivation. The total number of micro-organisms and nitrogen-transform bacteria were reduced, meanwhile, the number of fungi increased. The Suaeda heteroptera Kitagawa. had the similar effect, and it could increase the number of micro-organisms.
4. We primarily ascertained the seasonal dynamics of soil microorganism quantities.
There were significant changes of soil microorganism quantities in different seasons, and the microorganisms of different months showed a certain variation in the same halophytes’rhizosphere: the bacteria in every plot had an absolute advantage both in the number and proportion, and the number of micro-organisms were more in July, and less in September. In the same period, the number of soil microorganisms in the halophytes rhizosphere was: Tamarix chinensis Lour. >Suaeda heteroptera Kitagawa.>Limonium bicolor(Bge.) Kuntz. The dynamic changes of physiological microorganism groups had their own laws, but the number of each group still followed the rules: Ammonifying>Aerobe azotobacter > Aerobe cellulolytic > Bacillus > Thiobacillus > Desulphurizing bacteria>Nitrosomonas>Nitrifiers>Dennitrifying bacteria, no matter when it was in April, July or September.
1、确立了从盐碱地中高效提取和纯化土壤DNA的方法,并证明了该方法对多种不同样品的适用性
以黄河三角洲盐碱地土壤样品为研究对象,对目前已报道的21种土壤DNA的提取方法和4种纯化方法进行比较研究,探索出了一套高效的从土壤样品中提取、纯化大片段DNA的方法——改良微波法,此方法经济实用,能在4小时以内获得够质够量的土壤DNA用于微生物分子生态学研究,较适合于广大实验室进行土壤DNA提取工作。通过掺菌试验、RFLP、RAPD等试验,表明运用改良微波法可以有效的从不同样品(肥沃土壤、活性污泥、河底淤泥、河沙、盐碱地土壤、哺乳动物粪便和盐芥叶片)中将DNA提取出来,而且提取出的DNA是可以直接进行分子生物学操作的,能够应用于微生物分子生态学研究。
2、明确了不同盐生植物对土壤微生物的影响
种植盐生植物后,通过测定盐生植物根际和空白对照土壤微生物的数量变化,结果表明,种植以上五种盐生植物后,土壤中微生物的总数都明显上升,只是程度不一,微生物总数多少依次为柽柳>二色补血草>马蔺>金银花>中亚滨藜>对照,并且耐盐微生物的数量和比例均有所下降;各样地土壤微生物生理类群的数量表现为:氨化细菌>自生固氮菌>纤维素分解菌>芽孢菌>硫化细菌>反硫化细菌>亚硝化细菌>硝化细菌>反硝化细菌,氨化细菌和自生固氮菌总数处于绝对的优势,纤维素分解菌的数量较多,而且各样地之间生理类群之间差异比较明显,且各样地土壤微生物生理类群微生物的数量多少依次为柽柳>二色补血草>金银花>马蔺>中亚滨藜>对照,土壤中各生理类群不仅有助于保持土壤肥力还可以加强其他根际微生物的生命活动,在本试验中柽柳对土壤微生物的影响最大,可以显著提高其在土壤中的数量,而且土壤中氨化细菌、自生固氮菌、纤维素分解菌和亚硝化细菌的数量是最多的。
RFLP分析和RAPD分析显示,种植盐生植物后,土壤微生物的种类和数量都发生了相应的变化,RFLP分析显示6样地16S rDNA扩增产物经HinfI和Csp6I酶切产生的条带的大小基本一致,只是亮度有所区别;RAPD分子标记分析显示,17条引物共计扩增出331条RAPD条带,98.8%为多态性条带,通过RAPD条带图谱和丰富度分析表明,种植不同盐生植物后,微生物的种类都有所增加,不同样地间的微生物种类也有所区别,其中样地6(柽柳样地)最为明显,6块样地微生物群落的DNA序列Shannon-Wiener指数有所不同,大小依次为样地6>样地5>样地1>样地2>样地3>样地4,但是样地间的Shannon-Wiener指数差异性不大,群落DNA序列的均匀度大小依次为为样地1>样地5>样地3>样地2>样地4>样地6,整体看样地1和样地6均匀度指数差别较大外,各样地间土壤微生物的均匀度无显著差异。
运用传统纯培养方法结合现代分子生物学技术测定五种盐生植物对土壤微生物的影响表明,种植盐生植物后,土壤微生物的种类和数量都发生了相应的变化,其中样地6(柽柳)土壤微生物的种类和数量增加最为显著。
3、探明了盐生植物长期种植对黄河三角洲土壤微生物多样性的影响
采用传统的生物学手段对不同种植年限的盐生植物(两年柽柳、十年柽柳、两年盐地碱蓬和十年盐地碱蓬)根际土壤微生物种类和数量的变化进行研究,结果表明,种植盐生植物后土壤中微生物的数量显著提高,而且柽柳对微生物的影响比较大,可以显著提高微生物的数量,并且耐盐细菌的比例显著下降,随着连续种植时间的增加,微生物总数的减少以及真菌数量的增加,细菌与真菌的比值(即B/F)显著变小,并且并且氨化细菌、自生固氮菌、硝化细菌和亚硝化细菌等生理类群微生物的数量也逐渐下降;盐地碱蓬和柽柳类似,可以提高微生物的数量,但是幅度没有柽柳大,而且随着种植年限的延长,微生物的数量逐步提高,细菌与真菌的比值(即B /F)也是逐步提高,从生理类群的研究结果看,随着种植年限的延长,土壤中的自生固氮菌、硝化细菌、亚硝化细菌、纤维素分解菌和硫化细菌的数量也是在慢慢下降。
4、初步探明了黄河三角洲土壤微生物区系季节动态变化规律
以二色补血草、盐地碱蓬和柽柳根际土壤微生物为研究对象,通过对不同月份(4月、7月和9月)土壤微生物的数量变化研究了微生物区系季节动态变化,结果表明,同一盐生植物根际不同月份之间微生物数量呈现一定的变化规律,细菌在各样地均占绝对优势,比例最大,微生物的数量7月份较多,9月份较少,而且各月份之间差异显著;同一时期,不同盐生植物土壤微生物的数量:柽柳样地>盐地碱蓬样地>二色补血草样地;各微生物生理类群的动态变化均有其自身的规律,但是无论是在4月、7月份还是9月份,各类群数量的多少顺序为:氨化细菌>自生固氮菌>纤维素分解菌>芽孢菌>硫化细菌>反硫化细菌>亚硝化细菌>硝化细菌>反硝化细菌。
Yellow River Delta, one of the three largest river estuary deltas in China, has high potentials of land development and utilization, but the widely distributed saline soils here in the coastal area at Bohai-Sea severely restrict the development of agriculture. The quite high salt content of the saline soils mainly chlorates, ranging from 0.4% to 3% in the surface layer, worsens the soil structure and lowers the soil fertility. Considering the soil properties and other conditions in Yellow River Delta, we used the traditional plate culture method and modern molecular biology techniques to study the effect of different halophytes on the soil microbial diversity, the long-term cultivation of halophytes’effects on soil microbial community, and the seasonal dynamics changes of soil microorganism. Based on these, we can study the changes of microbial diversity of Yellow River Delta saline-alkali soil in the ecosystem improvement and treatment stage, and study the forest quality improvement and ecology recovery technology. The main contents and results of this study are presented as followings:
1. We established a simple, rapid, cost-effective method for the extraction of microbial DNA from different samples, including the saline-alkali soil.
We chose the saline-alkali soil samples from the Yellow River Delta as the research objects, used 21 kinds of extraction methods and four kinds of purification methods to extract and purify the soil DNA, and established a simple, rapid, cost-effective method for the extraction of microbial DNA——microwave method. It could be effectively used in different samples(fertile soil, active sludge, riverbed sullage, sand, alkaline land soil, album canis and leaves of Arabidopsis salsuginea), and the PCR amplification results showed that the extracted DNA can be directly operated in molecular biology, which demonstrated the method has broad applicability.
2. We ascertained the effect of different halophytes on the soil microbial diversity.
After the cultivation of halophytes, the number of soil microbial in the five kinds of halophytes rhizosphere showed that the total number of microorganisms in the soil were significantly increased, but the degree of change was t not the same, the sequence of the influence on the increase of the soil microbial number were: Tamarix chinensis Lour.>Limonium bicolor(Bge.) Kuntz>Iris ensata Thunb>Lonicera japonica>Atriplex centralasiatica Iljin>CK, and the salt-tolerant micro-organisms’number and proportion has declined. The results indicated that the total number of physiological microorganism showed the following rule: Ammonifying>Aerobe azotobacter>Aerobe cellulolytic>Bacillus>Thiobacillus>Desulphurizing bacteria>Nitrosomonas>Nitrifiers>Dennitrifying bacteria. The amount of ammonfying and aerobe azotobacter took absolute dominance, the number of different plots followed as:Tamarix chinensis Lour.>Limonium bicolor(Bge.) Kuntz>Lonicera japonica>Iris ensata Thunb>Atriplex centralasiatica Iljin>CK. Various physiological microorganism groups of soil would not only help maintain soil fertility but also enhance the lives of other rhizosphere microbial activity. In this experiment, Tamarix chinensis Lour. had the greatest influence on soil microorganisms and could significantly increase their number of the soil, and the amounts of ammonification bacteria, aerobe azotobacter, aerobe cellulolytic and Nitrosomonas were the majority.
The results of RFLP and RAPD analysis indicated that after the cultivation of halophytes, the type and quantity of soil microbes were changed, RFLP analysis showed that the six plots’16S rDNA amplification products were digested by HinfI and Csp6I giving birth to the similar bands, even though they had different brightness. The RAPD fingerprints showed substantial differences among the different soil samples, and according to apparent changes in the number and size of amplified DNA fragments, the Shannon-weaver index of different plots was Tamarix chinensis Lour.>Lonicera japonica>CK>Limonium bicolor(Bge.) Kuntz>Atriplex centralasiatica Iljin>Iris ensata Thunb, and the Pielou index were CK>Lonicera japonica>Atriplex centralasiatica Iljin>Limonium bicolor(Bge.) Kuntz>Iris ensata Thunb>Tamarix chinensis Lour..
3.Ascertained the effect of long-term cultivation of halophytes on the microbial diversity.
Using the traditional means to determine the rhizosphere microbial changes in the types and quantities of different halophytes growing for long years, the result of the study indicated that the number of micro-organisms significantly increased after halophytes planted, and the Tamarix chinensis Lour. showed greater influence, which could significantly increase the number of micro-organisms, and at the same time the salt-tolerant bacteria’proportion was decreased significantly. After two-year cultivation, the number of microorganisms reached to the maximum, and the ratio of bacteria and fungi (B/F) significantly descended with the continuous cultivation. The total number of micro-organisms and nitrogen-transform bacteria were reduced, meanwhile, the number of fungi increased. The Suaeda heteroptera Kitagawa. had the similar effect, and it could increase the number of micro-organisms.
4. We primarily ascertained the seasonal dynamics of soil microorganism quantities.
There were significant changes of soil microorganism quantities in different seasons, and the microorganisms of different months showed a certain variation in the same halophytes’rhizosphere: the bacteria in every plot had an absolute advantage both in the number and proportion, and the number of micro-organisms were more in July, and less in September. In the same period, the number of soil microorganisms in the halophytes rhizosphere was: Tamarix chinensis Lour. >Suaeda heteroptera Kitagawa.>Limonium bicolor(Bge.) Kuntz. The dynamic changes of physiological microorganism groups had their own laws, but the number of each group still followed the rules: Ammonifying>Aerobe azotobacter > Aerobe cellulolytic > Bacillus > Thiobacillus > Desulphurizing bacteria>Nitrosomonas>Nitrifiers>Dennitrifying bacteria, no matter when it was in April, July or September.
引文
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