庐山亚热带森林生态系统六种不同类型土壤细菌多样性、青枯病生防细菌筛选及降解二氯苯胺机理研究
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摘要
本文采用传统培养和微生物分子生态学方法分析了庐山不同类型森林土壤的细菌多样性与土壤类型、海拔高度和季节因子的关系。并分析了该区域土壤细菌在解磷固氮,颉颃病原茵和降解有机农药污染方面的潜在能力。
我们结合PCR-DGGE分子指纹图谱方法和传统培养方法研究了庐山亚热带森林生态系统六种不同类型土壤(红壤、砖红性土壤、黄壤、黄棕壤、棕壤和山地草甸土)中细菌群落结构和颉颃细菌多样性变化规律。从微生物分子生态学角度分析不同土壤细菌群落结构变化与海拔高度、土壤类型和季节之间的关系。典型性对应分析(CCA)显示：土壤类型是细菌群落组成的主要驱动因子。DGGE图谱聚类分析与典型性对应分析(ccA)结果相一致,进一步证实了土壤类型影响细菌群落结构的组成。此外,我们还通过主成分分析(PCA)和细菌多样性指数揭示土壤类型影响土壤颉颃细菌丰富度、土壤颉颃细菌多样性与土壤总细菌多样性指数的对应关系。综合结果表明：土壤类型是决定庐山土壤总细菌结构和土壤颉颃细菌组成的重要影响因子。
利用分子生物学方法分析可培养的土壤颉颃细菌多样性与土壤类型之间关系。采用核糖体DNA扩增片段限制性酶切图谱分析(ARDRA)庐山土壤颉颃细菌多样性。从庐山土壤分离到125个土壤颉颃细菌可以分成13个ARDRA谱型,其中五个ARDRA谱型(G5、G13、G11、G9和G10)各分离到一个颉颃细菌；2个ARDRA谱型(G8和G12)分别拥有两个颉颃细菌；而G1、G2、G3、G4、G6和G7ARDRA谱型分别有32、18、36、14、5和11个颉颃细菌。每个ARDRA谱型中选取代表性菌株进行16S rRNA基因测序分析,显示庐山土壤颉颃细菌包括12个属,分布在五个不同的细菌门。其中红壤、砖红性土壤、棕壤和山地草甸土中分离的颉颃细菌比例较高,分别为22%、23%、25%和31%。选取24株土壤颉颃细菌进行番茄青枯病的温室防效实验,发现菌株M19具有最好的生防效果,达到59%。通过16S rRNA基因序列分析鉴定菌株M19属于拉恩氏菌属(Rahnella)细菌。本研究首次应用拉恩氏菌属(Rahnella)细菌作为番茄青枯病的生防细菌。
另外我们通过土壤富集培养、筛选到一株二氯苯胺降解菌株,编号为IMT21。经过对其形态特征、生理生化分析、16S rRNA基因序列分析,初步鉴定该菌株为Bacillus megaterium。Bacillus megaterium IMT21最适生长温度为30-40℃,最适生长pH为7.0。
接种菌株IMT21于含50ppmDCA的加富培养基中,通过LC-MS检测代谢产物,随着培养基中DCA含量的逐渐下降,发现在保留时间T=8.7/10.9分钟处有代谢底物峰的积累,此代谢物的保留时间和核质比(m/z 204)与3,4-二氯乙酰苯胺一致。由此推测IMT21主要通过乙酰化作用解毒二氯苯胺类化合物。IMT21能够通过乙酰化作用转化多种二氯苯胺类化合物为毒性较低的乙酰化产物,所以我们进一步克隆了芳基胺-N-乙酰转移酶基因(BmNat),并在大肠杆菌中进行异源表达和分析。同时还分析了菌株IMT21在土壤中降解DCA的过程对污染土壤细菌群落的影响。
本研究的主要发现与创新：第一、综合运用传统培养方法、PCR-DGGE分子指纹图谱技术和多元变量分析(PCA和CCA)方法解析庐山亚热带森林生态系统土壤细菌随海拔、土壤类型、季节的动态变化,发现土壤类型是影响细菌种群变化的主要因素；第二、首次应用拉恩氏菌属(Rahnella1细菌M19作为颉颃细菌用于番茄青枯病的生物防治；第三、分离鉴定了能同时降解五种二氯苯胺类化合物的Bacillus megateriumIMT21菌株,并首次克隆表达了巨大芽孢杆菌中的解毒基因芳基胺-N-乙酰转移酶基因(BmNat)。
In this study, the traditional culturable way and molecular microbial ecology methods was used to analysis the relationshinp of bacterial diversity and soil type, altitude and seasonal factors.We also analyze the soil bacteria's potential use in nitrogen-fixing, phosphorus dissolving, antagonistic pathogens and degradation of organic pesticide pollutions.
The composition of soil bacterial communities and antagonistic edaphic bacterias in six different soil type sites (Red soil、Lateritic soil、Yellow soil、Yellow brown soil、Brown soil、Meadow soil) from subtropical montane forest ecosystem in China were examined by using PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting 16S rRNA gene-and culturable approach.The linkage of soil type and temporal factors and bacterial community was analyzed by the ordination technique of canonical correspondence analysis (CCA). CCA Ordination of DGGE profiles showing that the primary forcing factor for the soil bacteria communities appeared to be soil type. Cluster analysis based on DGGE profiles agreed well with ordination analysis (CCA) confirming the soil type effect on bacteria communities. Moreover, we explored the soil type effect of the abundance of antagonistic edaphic bacteria by Principal component analysis (PCA). The proportion and diversity of antagonistic bacteria consistent with the phylotype diversity index from DGGE community. The results obtained in our study demonstrate clearly that soil type is important factor affecting the structure of total bacterial and antagonistic edaphic bacteria community.
ARDRA were used to profile the diversity of antagonistic edaphic bacteria, among the 125 antagonists,13 groups (G1 to G13) were formed according ARDRA fringerprint. There is 6 groups (G5, G13, G11, G9 and G10) contained only one isolate, respectively; 2 groups (G8 and G12) contained two isolates, respectively; G1, G2, G3, G4, G6 and G7 included 32, 18,36,14,5 and 11 isolates, respectively. A subset of representative isolates of each ARDRA group was identified by 16S rRNA gene sequence analysis. Results showed a wide variety of antagonists, comprising 12 different genera distributed in five bacterial divisions. High proportion of antagonistic edaphic bacteria were found in these types of soil (R= 22%, YR=23%, B=25%, and M=31%). In greenhouse, strain M19 had the best biocontrol efficiencies that about 59% and this is the first report of Rahnella sp. strain used as a BCA agaisnt R. solanacearum of tomato.
A dichloroanilines degrading strain named as IMT21 was isolated from enriched soil sample and identified as Bacillus megaterium according to its morphology、biochemical properties and 16S rRNA gene sequence. IMT21 can use 3,4-DCA as sole carbon source, biological properties of strain IMT21 were studied. The optimal temperature and pH were 30-40℃and 7.0, respectively.
Degradation of 3,4-DCA/3,5-DCA resulted in accumulation of a single metabolites with the retention time of 8.7/10.9 min, which had the same retention time and m/z (204) as 3,4-dichloroacetanilide, this suggests that the DCA were degraded via dichloroacetanilide metabolites. IMT21 can convert a wide variety of dichloroanilines to their corresponding acetanilides and dichloroaminophenol metabolites. In this study reported here, we cloned and expressed Arylamine N-acetyltransferases (NATs) in Escherichia coli. The expressed enzyme (BmNAT) was characterized.
我们结合PCR-DGGE分子指纹图谱方法和传统培养方法研究了庐山亚热带森林生态系统六种不同类型土壤(红壤、砖红性土壤、黄壤、黄棕壤、棕壤和山地草甸土)中细菌群落结构和颉颃细菌多样性变化规律。从微生物分子生态学角度分析不同土壤细菌群落结构变化与海拔高度、土壤类型和季节之间的关系。典型性对应分析(CCA)显示：土壤类型是细菌群落组成的主要驱动因子。DGGE图谱聚类分析与典型性对应分析(ccA)结果相一致,进一步证实了土壤类型影响细菌群落结构的组成。此外,我们还通过主成分分析(PCA)和细菌多样性指数揭示土壤类型影响土壤颉颃细菌丰富度、土壤颉颃细菌多样性与土壤总细菌多样性指数的对应关系。综合结果表明：土壤类型是决定庐山土壤总细菌结构和土壤颉颃细菌组成的重要影响因子。
利用分子生物学方法分析可培养的土壤颉颃细菌多样性与土壤类型之间关系。采用核糖体DNA扩增片段限制性酶切图谱分析(ARDRA)庐山土壤颉颃细菌多样性。从庐山土壤分离到125个土壤颉颃细菌可以分成13个ARDRA谱型,其中五个ARDRA谱型(G5、G13、G11、G9和G10)各分离到一个颉颃细菌；2个ARDRA谱型(G8和G12)分别拥有两个颉颃细菌；而G1、G2、G3、G4、G6和G7ARDRA谱型分别有32、18、36、14、5和11个颉颃细菌。每个ARDRA谱型中选取代表性菌株进行16S rRNA基因测序分析,显示庐山土壤颉颃细菌包括12个属,分布在五个不同的细菌门。其中红壤、砖红性土壤、棕壤和山地草甸土中分离的颉颃细菌比例较高,分别为22%、23%、25%和31%。选取24株土壤颉颃细菌进行番茄青枯病的温室防效实验,发现菌株M19具有最好的生防效果,达到59%。通过16S rRNA基因序列分析鉴定菌株M19属于拉恩氏菌属(Rahnella)细菌。本研究首次应用拉恩氏菌属(Rahnella)细菌作为番茄青枯病的生防细菌。
另外我们通过土壤富集培养、筛选到一株二氯苯胺降解菌株,编号为IMT21。经过对其形态特征、生理生化分析、16S rRNA基因序列分析,初步鉴定该菌株为Bacillus megaterium。Bacillus megaterium IMT21最适生长温度为30-40℃,最适生长pH为7.0。
接种菌株IMT21于含50ppmDCA的加富培养基中,通过LC-MS检测代谢产物,随着培养基中DCA含量的逐渐下降,发现在保留时间T=8.7/10.9分钟处有代谢底物峰的积累,此代谢物的保留时间和核质比(m/z 204)与3,4-二氯乙酰苯胺一致。由此推测IMT21主要通过乙酰化作用解毒二氯苯胺类化合物。IMT21能够通过乙酰化作用转化多种二氯苯胺类化合物为毒性较低的乙酰化产物,所以我们进一步克隆了芳基胺-N-乙酰转移酶基因(BmNat),并在大肠杆菌中进行异源表达和分析。同时还分析了菌株IMT21在土壤中降解DCA的过程对污染土壤细菌群落的影响。
本研究的主要发现与创新：第一、综合运用传统培养方法、PCR-DGGE分子指纹图谱技术和多元变量分析(PCA和CCA)方法解析庐山亚热带森林生态系统土壤细菌随海拔、土壤类型、季节的动态变化,发现土壤类型是影响细菌种群变化的主要因素；第二、首次应用拉恩氏菌属(Rahnella1细菌M19作为颉颃细菌用于番茄青枯病的生物防治；第三、分离鉴定了能同时降解五种二氯苯胺类化合物的Bacillus megateriumIMT21菌株,并首次克隆表达了巨大芽孢杆菌中的解毒基因芳基胺-N-乙酰转移酶基因(BmNat)。
In this study, the traditional culturable way and molecular microbial ecology methods was used to analysis the relationshinp of bacterial diversity and soil type, altitude and seasonal factors.We also analyze the soil bacteria's potential use in nitrogen-fixing, phosphorus dissolving, antagonistic pathogens and degradation of organic pesticide pollutions.
The composition of soil bacterial communities and antagonistic edaphic bacterias in six different soil type sites (Red soil、Lateritic soil、Yellow soil、Yellow brown soil、Brown soil、Meadow soil) from subtropical montane forest ecosystem in China were examined by using PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting 16S rRNA gene-and culturable approach.The linkage of soil type and temporal factors and bacterial community was analyzed by the ordination technique of canonical correspondence analysis (CCA). CCA Ordination of DGGE profiles showing that the primary forcing factor for the soil bacteria communities appeared to be soil type. Cluster analysis based on DGGE profiles agreed well with ordination analysis (CCA) confirming the soil type effect on bacteria communities. Moreover, we explored the soil type effect of the abundance of antagonistic edaphic bacteria by Principal component analysis (PCA). The proportion and diversity of antagonistic bacteria consistent with the phylotype diversity index from DGGE community. The results obtained in our study demonstrate clearly that soil type is important factor affecting the structure of total bacterial and antagonistic edaphic bacteria community.
ARDRA were used to profile the diversity of antagonistic edaphic bacteria, among the 125 antagonists,13 groups (G1 to G13) were formed according ARDRA fringerprint. There is 6 groups (G5, G13, G11, G9 and G10) contained only one isolate, respectively; 2 groups (G8 and G12) contained two isolates, respectively; G1, G2, G3, G4, G6 and G7 included 32, 18,36,14,5 and 11 isolates, respectively. A subset of representative isolates of each ARDRA group was identified by 16S rRNA gene sequence analysis. Results showed a wide variety of antagonists, comprising 12 different genera distributed in five bacterial divisions. High proportion of antagonistic edaphic bacteria were found in these types of soil (R= 22%, YR=23%, B=25%, and M=31%). In greenhouse, strain M19 had the best biocontrol efficiencies that about 59% and this is the first report of Rahnella sp. strain used as a BCA agaisnt R. solanacearum of tomato.
A dichloroanilines degrading strain named as IMT21 was isolated from enriched soil sample and identified as Bacillus megaterium according to its morphology、biochemical properties and 16S rRNA gene sequence. IMT21 can use 3,4-DCA as sole carbon source, biological properties of strain IMT21 were studied. The optimal temperature and pH were 30-40℃and 7.0, respectively.
Degradation of 3,4-DCA/3,5-DCA resulted in accumulation of a single metabolites with the retention time of 8.7/10.9 min, which had the same retention time and m/z (204) as 3,4-dichloroacetanilide, this suggests that the DCA were degraded via dichloroacetanilide metabolites. IMT21 can convert a wide variety of dichloroanilines to their corresponding acetanilides and dichloroaminophenol metabolites. In this study reported here, we cloned and expressed Arylamine N-acetyltransferases (NATs) in Escherichia coli. The expressed enzyme (BmNAT) was characterized.
引文
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