人参连作对土壤微生物群落的影响研究
摘要
人参(Panax ginseng C. A. Mey.)具有很高的药用价值,但强忌连作特性严重影响了人参的规范化栽培,老参地重茬栽参会导致人参长势变弱、病害高发等问题。本课题从不同年限人参栽培土壤的微生物群落结构及碳源代谢能力着手,以期从人参栽培土壤微生物角度解析人参连作障碍机理,为人参生产提供理论依据。本课题从人参主产区吉林省抚松县大方村、黄泥村和五里地村分别采集1～6年生人参根际土壤(紧贴根表皮的土壤)和根围土壤(根周围的土壤)。
利用随机扩增多态性DNA标记法(RAPD)分析了土壤微生物的遗传多样性,结果表明,直播参根际土壤微生物的遗传多样性高于根围土壤,而移栽人参的根围土壤微生物的遗传多样性高于根际土壤。在根际土壤中,大方村和黄泥村三年生人参根际土壤的遗传多样性最高。
利用BiologTM EcoPlate开展了人参栽培土壤微生物碳源利用能力的研究。结果表明,随人参生长年限增加,根际土壤微生物的碳源利用能力表现为先升高,后降低。与根围土壤相比,根际土壤微生物的碳源利用能力相对较高；部分人参根际土壤的山农多样性指数以及碳源利用率高于与之对应的人参根围土壤。主成分分析结果表明,大方村和五里地村的大部分根际土壤聚在一起,而大部分根围土壤聚在一起；黄泥村的主成分分析图中,生长年限相同的人参栽培土壤聚在一起。
利用扩增核糖体DNA限制性内切酶分析方法分析发现,大方村人参栽培土壤主要山α-变形菌纲Alphaproteobacteria、β-变形菌纲Betaproteobacteria、γ-变形菌纲Gammaproteobacteria、拟杆菌门Bacteroidetes、酸杆菌门Acidobacteria、厚壁菌门Firmicutes、疣微菌门Verrucomicrobia七个门或纲的细菌以及Unknown bacteria组成,随着人参栽培年限的增长,细菌种群结构会发生变化。在一年生、二年生三年生以及五年生(3+2)人参根际土以及三年生人参根围土中均有发现假单胞菌Pseudomonas、伯克霍尔德菌Burkholderia、芽孢杆菌Bacillus等,他们中部分是对病原真菌生长具有抑制作用的细菌,其中一年生和二年生人参根际土中所占比例较高。由饱和度曲线可知,在所有土壤样品中,只有六年生(3+3)人参根围土细菌多样性大于六年生(3+3)人参根际土,其余土壤样品均是根际土壤细菌多样性高于其对应的围土壤细菌多样性。
用自毒物质处理新林土,经Biolog分析发现,未经自毒物质处理的对照组土壤微生物碳源代谢能力明显高于处理过的土壤；主成分分析结果表明,对照组与其余土壤样品被划分成两组,说明二者的碳源利用特征存在明显差别。经邻苯二甲酸二异丁酯、丁二酸二异丁酯以及混合自毒物质处理的土壤微生物对碳源的利用能力较低；RAPD结果聚类分析表明,三份土壤样品的相似系数比较高,但微生物遗传多样性较低。
Ginseng(Panax ginseng C. A. Meyer), is usually used as adaptogenic, anti-aging etc. health tonic. It is mainly cultivated in China, Korea, and Japan etc. Asian countries. It has been regarded as one of the most important remedies in oriental medicine for more than1,000years. As herbaceous perennial plant, ginseng grows at least six years from sow to harvest. During the long growing process, soilborne diseases made severe threaten on the health of P. ginseng root. Rhizosphere and nonrhizosphere soils of one to six years ginseng were sampled from Dafang, Huangni and Wulidi in Jilin province.
The genetic diversity of microbial community of the selected soils were characterized by culture-independent approaches, random amplified polymorphic DNA (RAPD). Results showed that, genetic diversity index of rhizosphere soil of ginseng that had not been transplanted was higher than the nonrhizosphere soil, while, after been transplanted, the diversity index of rhizosphere soil was lower than the nonrhizosphere soil. Genetic diversity index of the rhizosphere soil of3yr-ginseng was the highest one in both Dafang and Huangni.
Metabolic function variance of microbe in the soil samples were characterized by community level physiological profiles (CLPP) using BiologTM EcoPlate. With the increasing of cultivating years, the carbon substrates metabolic activities of soil microbes-at three sites were increased and then dtscreased finally. AW CD value and utilization for six types of carbon source of the rihzosphere soil were higher than the corresponding nonrihzosphere soil. Except for the5yr-rhizosphere soil WLD2+3R sampled from Wulidi, the Shannon diversity indexes of the rihzosphere soils were higher than the nonrihzosphere soils. In principal component analysis of Dafang and Huangni, most of the rihzosphere soils gathered together, while most of the nonrihzosphere soils gathered. In Wulidi, soils with the same cultivating years gathered.
We also analysed the microbial community structure of the rihzosphere soils and nonrihzosphere soils sampled from Dafang. We found that, Verrucomicrobia, Acidobacteria, Proteobacteria, Bacteroidetes, Acidobacteria, Firmicutes, Verruco-microbia were the dominants in the soils. With the increasing of cultivating years, plant disease preventive or plant growth promoting bacteria, such as Pseudomonas, Burkholderia, Bacillus, etc., tended to be rare.
We made autotoxins solution, and sprayed on the new forest soil sampled from Fusong of Jilin Province. After three months, we examined the varience of microbial community using RAPD, results showed that the soils with the treatment of diisobutyl phthalate, diisobutyl succinate and the mixed autotoxins have lower genetic diversity index than the the CK and the soils treated with the other autotoxins. Cluster analysis showed that similarity coefficient of these soils is relatively high. In the metabolic function variance analysis of microbe in the soil samples characterized by community level physiological profiles (CLPP) using Bio logTM EcoPlate, we found that, the carbon substrates metabolic activity of control is higher than those treated with autotoxins. The AWCD value of soils that treated by diisobutyl phthalate, diisobutyl succinate and the mixed autotoxins were lower than those treated by the other autotoxins. In the principal component analysis, control was seperated with the soils treated by autoxins.
利用随机扩增多态性DNA标记法(RAPD)分析了土壤微生物的遗传多样性,结果表明,直播参根际土壤微生物的遗传多样性高于根围土壤,而移栽人参的根围土壤微生物的遗传多样性高于根际土壤。在根际土壤中,大方村和黄泥村三年生人参根际土壤的遗传多样性最高。
利用BiologTM EcoPlate开展了人参栽培土壤微生物碳源利用能力的研究。结果表明,随人参生长年限增加,根际土壤微生物的碳源利用能力表现为先升高,后降低。与根围土壤相比,根际土壤微生物的碳源利用能力相对较高；部分人参根际土壤的山农多样性指数以及碳源利用率高于与之对应的人参根围土壤。主成分分析结果表明,大方村和五里地村的大部分根际土壤聚在一起,而大部分根围土壤聚在一起；黄泥村的主成分分析图中,生长年限相同的人参栽培土壤聚在一起。
利用扩增核糖体DNA限制性内切酶分析方法分析发现,大方村人参栽培土壤主要山α-变形菌纲Alphaproteobacteria、β-变形菌纲Betaproteobacteria、γ-变形菌纲Gammaproteobacteria、拟杆菌门Bacteroidetes、酸杆菌门Acidobacteria、厚壁菌门Firmicutes、疣微菌门Verrucomicrobia七个门或纲的细菌以及Unknown bacteria组成,随着人参栽培年限的增长,细菌种群结构会发生变化。在一年生、二年生三年生以及五年生(3+2)人参根际土以及三年生人参根围土中均有发现假单胞菌Pseudomonas、伯克霍尔德菌Burkholderia、芽孢杆菌Bacillus等,他们中部分是对病原真菌生长具有抑制作用的细菌,其中一年生和二年生人参根际土中所占比例较高。由饱和度曲线可知,在所有土壤样品中,只有六年生(3+3)人参根围土细菌多样性大于六年生(3+3)人参根际土,其余土壤样品均是根际土壤细菌多样性高于其对应的围土壤细菌多样性。
用自毒物质处理新林土,经Biolog分析发现,未经自毒物质处理的对照组土壤微生物碳源代谢能力明显高于处理过的土壤；主成分分析结果表明,对照组与其余土壤样品被划分成两组,说明二者的碳源利用特征存在明显差别。经邻苯二甲酸二异丁酯、丁二酸二异丁酯以及混合自毒物质处理的土壤微生物对碳源的利用能力较低；RAPD结果聚类分析表明,三份土壤样品的相似系数比较高,但微生物遗传多样性较低。
Ginseng(Panax ginseng C. A. Meyer), is usually used as adaptogenic, anti-aging etc. health tonic. It is mainly cultivated in China, Korea, and Japan etc. Asian countries. It has been regarded as one of the most important remedies in oriental medicine for more than1,000years. As herbaceous perennial plant, ginseng grows at least six years from sow to harvest. During the long growing process, soilborne diseases made severe threaten on the health of P. ginseng root. Rhizosphere and nonrhizosphere soils of one to six years ginseng were sampled from Dafang, Huangni and Wulidi in Jilin province.
The genetic diversity of microbial community of the selected soils were characterized by culture-independent approaches, random amplified polymorphic DNA (RAPD). Results showed that, genetic diversity index of rhizosphere soil of ginseng that had not been transplanted was higher than the nonrhizosphere soil, while, after been transplanted, the diversity index of rhizosphere soil was lower than the nonrhizosphere soil. Genetic diversity index of the rhizosphere soil of3yr-ginseng was the highest one in both Dafang and Huangni.
Metabolic function variance of microbe in the soil samples were characterized by community level physiological profiles (CLPP) using BiologTM EcoPlate. With the increasing of cultivating years, the carbon substrates metabolic activities of soil microbes-at three sites were increased and then dtscreased finally. AW CD value and utilization for six types of carbon source of the rihzosphere soil were higher than the corresponding nonrihzosphere soil. Except for the5yr-rhizosphere soil WLD2+3R sampled from Wulidi, the Shannon diversity indexes of the rihzosphere soils were higher than the nonrihzosphere soils. In principal component analysis of Dafang and Huangni, most of the rihzosphere soils gathered together, while most of the nonrihzosphere soils gathered. In Wulidi, soils with the same cultivating years gathered.
We also analysed the microbial community structure of the rihzosphere soils and nonrihzosphere soils sampled from Dafang. We found that, Verrucomicrobia, Acidobacteria, Proteobacteria, Bacteroidetes, Acidobacteria, Firmicutes, Verruco-microbia were the dominants in the soils. With the increasing of cultivating years, plant disease preventive or plant growth promoting bacteria, such as Pseudomonas, Burkholderia, Bacillus, etc., tended to be rare.
We made autotoxins solution, and sprayed on the new forest soil sampled from Fusong of Jilin Province. After three months, we examined the varience of microbial community using RAPD, results showed that the soils with the treatment of diisobutyl phthalate, diisobutyl succinate and the mixed autotoxins have lower genetic diversity index than the the CK and the soils treated with the other autotoxins. Cluster analysis showed that similarity coefficient of these soils is relatively high. In the metabolic function variance analysis of microbe in the soil samples characterized by community level physiological profiles (CLPP) using Bio logTM EcoPlate, we found that, the carbon substrates metabolic activity of control is higher than those treated with autotoxins. The AWCD value of soils that treated by diisobutyl phthalate, diisobutyl succinate and the mixed autotoxins were lower than those treated by the other autotoxins. In the principal component analysis, control was seperated with the soils treated by autoxins.
引文
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