陕西黄土高原固氮植物沙棘根际微生物多样性研究
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摘要
为探索黄土高原地区非豆科固氮植物沙棘(Hippophae rhamnoides)根际促生菌在促进植被恢复和生态系统重建过程中的作用,以陕西关中地区结瘤沙棘根际土壤和根瘤为研究材料,通过对其微生物种群结构与土壤肥力之间关系的检测,以及根际氢氧化细菌、弗兰克氏菌(Frankia)和VA菌根(Vesicular-Avbuscular mycorrhiza)真菌多样性的研究,了解非豆科植物根际氢氧化细菌的种属分布情况,丰富现有关于氢氧化细菌分类的研究,并探索沙棘根际多种微生物联合促生的关系和机制。主要实验内容和结果如下：
本实验采用气体循环体系,电解水产生微量H2处理根际土壤,结果显示处理后的土壤中微生物种群结构发生改变,细菌和放线菌数量增加,其中氢氧化细菌数量显著增加,而真菌数量却有所减少；处理后土壤中微生物呼吸作用强度以及土壤酶的活性均有不同程度的提高,土壤有机质含量增加,植物激素类物质增多,说明H2处理后土壤的肥力有所提高。
采用持续通H2的气体循环培养体系和矿质盐(MSA)培养基分离出59株细菌。通过TTC试验、气相色谱法测定氧化H2能力以及自养生长检测,筛选出12株含氢化酶且氧化H2能力较强的菌株,初步确定为氢氧化细菌,其中菌株FS2的的氧化H2能力可达35.43μmol/L。根据菌株的形态特征、培养特征和生理生化特征,初步将这12株菌分属于芽孢杆菌属(Bacillus)、气单胞菌属(Aeromonas)、假单胞菌属(Pseudomonas)、微球菌属(Micrococcus)、黄单胞菌属(Xanthomonas)、短状杆菌属(Brachybacterium)、诺卡氏菌属(Nocardia)和脂肪杆菌属(Pimelobacter)。菌株FS2的16S rRNA基因序列(GenBank登录号为GU084156)与芽孢杆菌属同源性为99%,在系统发育树上位于同一分支,进一步将菌株FS2归为芽孢杆菌属(Bacillus)。
利用薄层层析的方法筛选出2株具有ACC脱氨酶的菌株FS2和WS4,并检测了对两株菌ACC脱氨酶活性的影响因素。结果表明两株菌在培养24h时可分解近90%的ACC,最适温度为30-35℃,最适pH为7-7.5。
从沙棘根瘤中分离出3株内生菌SY1,SY2,SY3,其属于慢生长型放线菌,在BAP液体培养基中形成颗粒状或絮状沉淀；在光学显微镜下,菌丝有分枝且粗细不一,无氮培养基诱导可产生泡囊,这些特征均符合弗兰克氏菌的一般性质,可将3株内生菌初步断定为弗兰克氏菌。通过对3株内生菌的生物学特性研究发现,菌株SY1和SY2为生理A型,菌株SY 3为生理AB型,利用较好的碳源是葡萄糖和Tween-80,利用较好的氮源是硫酸铵和硝酸钾。
利用酸性品红对沙棘根段染色,结果可见3批样品沙棘根系都有VA菌根(Vesicular-Avbuscular mycorrhiza)真菌侵染,侵染率分别为55.6%,72%,65%。采用湿筛倾析法从沙棘根际土壤鉴定出2属7种VA菌根真菌,包括球囊霉属(Glomus)6种：缩球囊霉(Glomus constrictum Trappe)、近明球囊霉(Glomus claroideum Schenck & Smith)、根内球囊霉(Glomus intraradices Schenck & Smith)、地球囊霉(Glomus geosporum Walker)、透光球囊霉(Glomus diaphanum Marton & walker)、网状球囊霉,(Glomus reticulatum Bhattacharjee & Mukerji);巨囊霉属(Gigaspora)1种：巨大巨孢囊霉(Gigaspora gigantean Gerd.Trappe)。
In order to explore the role of non-leguminous nitrogen fixing plant Hippophae rhamnoides'growth-promoting rhizobacteria in promoting vegetation restoration and ecosystem reconstruction in Loess Plateau, the soil and root nodule in Hippophae rhamnoides rhizosphere in Shaanxi province was used to detect the relationship between microbial population structure and soil fertility and to study the diversity of hydrogen-oxidizing bacteria, Frankia and Vesicular-Avbuscular mycorrhiza fungi. According to the analysis of the result of above experiments, we can realize the generic distribution of hydrogen-oxidizing bacteria, enrich the existing research about sort of hydrogen-oxidizing bacteria and explore the relationship and mechanism of polymicrobic growth-promoting in Hippophae rhamnoides rhizosphere. The main experimental content and results were as follows:
We used gas cycle incubation system to treated rhizosphere soils with H2 produced by water electrolysis. The result showed that the microbial population structure of the soil treated with H2 had changed:the number of bacteria and actinomyces had increased,and the number of hydrogen-oxidizing bacteria had increased significantly, but the number of fungi had declined. The microbes'respiration intensity and activity of soil enzyme in soil treated by H2 had enhanced to varying degrees; organic matter content of soil and planthormones in soil had grown in number, which explained that the soil fertility had enhanced with the treatment of H2.
Fifty nine bacterial strains were isolated by gas-cycle incubation system and MSA medium. Twelve strains among them which contained hydrogenase and had strong ability to oxidize hydrogen was selected by TTC test, gas chromatograph and autotrophic gowth detection, suggesting that they might be hydrogen-oxidizing bacteria initially. Hydrogen-oxidizing ability of strain FS2 was about 35.430μmol/L.Based on their morphological, cultural,and physiological and biochemical characteristics, these 12 strains were identified as members of genera Bacillus, Aeromonas, Pseudomonas, Micrococcus, Xanthomonas, Brachybacterium, Nocardia and Pimelobacter,respectively. The 16S rRNA gene sequence analysis suggested that strain FS2 (GenBank accession number GU084156) was clustered together with Bacillus in phylogenetic tree, with sequence similarity more than 99%.
We used thin layer chromatography to find out that strain FS2 and WS4 could consum-ed ACC and contained ACC deaminase.Then we detectd the influence factors about the activity of the ACC deaminase. The result showed that the two strains could decompose nearly 90% ACC in 24h and the optimal temperature was 30-35℃and the optimal pH was7.0-7.5.
Three endophyte strains SY1, SY2 and SY3 were isolated from root nodule of Hippophae rhamnoides,.They belonged to slow-growing actinomyces and formed flocculent or granular precipitate in the BAP liquid medium; their hyphae was filamentous with different diameters under microscope and could produce vesicle when growing in the nitrogen-free medium. Above characteristics was in line with general characteristics of Frankia, therefore the 3 strains could be identified as a member of Frankia preliminarily. The study of 3 Endophyte's biological property showed that strain SY1 and SY2 were divided into physiological group A and strain SY3 was divided into physiological group AB.Their optimal carbon sources were tween-80 and glucose, and optimal nitrogen sources were ammonium sulfate and nitre.
Acid fuchsin was used to dye root segment of Hippophae rhamnoides and the result showed that the Hippophae rhamnoides root system was infected by Vesicular-Avbuscular mycorrhiza fungi and the infecting rate were 55.6%,72% and 65%, respectively. SevenVA fungi species belonging to 2 genus were isolated and identified by sieving and deeanting proeedure from Hippophae rhamnoides rhizosphere soil.Six species were belonged to genus Glomus, including Glomus claroideum, Glomus consrictum, Glomus intraradices, Glomus geosporum, Glomus diaphanum and Glomus reticulatum,and Gigaspora gigantean was belonged to genus Gigaspora.
本实验采用气体循环体系,电解水产生微量H2处理根际土壤,结果显示处理后的土壤中微生物种群结构发生改变,细菌和放线菌数量增加,其中氢氧化细菌数量显著增加,而真菌数量却有所减少；处理后土壤中微生物呼吸作用强度以及土壤酶的活性均有不同程度的提高,土壤有机质含量增加,植物激素类物质增多,说明H2处理后土壤的肥力有所提高。
采用持续通H2的气体循环培养体系和矿质盐(MSA)培养基分离出59株细菌。通过TTC试验、气相色谱法测定氧化H2能力以及自养生长检测,筛选出12株含氢化酶且氧化H2能力较强的菌株,初步确定为氢氧化细菌,其中菌株FS2的的氧化H2能力可达35.43μmol/L。根据菌株的形态特征、培养特征和生理生化特征,初步将这12株菌分属于芽孢杆菌属(Bacillus)、气单胞菌属(Aeromonas)、假单胞菌属(Pseudomonas)、微球菌属(Micrococcus)、黄单胞菌属(Xanthomonas)、短状杆菌属(Brachybacterium)、诺卡氏菌属(Nocardia)和脂肪杆菌属(Pimelobacter)。菌株FS2的16S rRNA基因序列(GenBank登录号为GU084156)与芽孢杆菌属同源性为99%,在系统发育树上位于同一分支,进一步将菌株FS2归为芽孢杆菌属(Bacillus)。
利用薄层层析的方法筛选出2株具有ACC脱氨酶的菌株FS2和WS4,并检测了对两株菌ACC脱氨酶活性的影响因素。结果表明两株菌在培养24h时可分解近90%的ACC,最适温度为30-35℃,最适pH为7-7.5。
从沙棘根瘤中分离出3株内生菌SY1,SY2,SY3,其属于慢生长型放线菌,在BAP液体培养基中形成颗粒状或絮状沉淀；在光学显微镜下,菌丝有分枝且粗细不一,无氮培养基诱导可产生泡囊,这些特征均符合弗兰克氏菌的一般性质,可将3株内生菌初步断定为弗兰克氏菌。通过对3株内生菌的生物学特性研究发现,菌株SY1和SY2为生理A型,菌株SY 3为生理AB型,利用较好的碳源是葡萄糖和Tween-80,利用较好的氮源是硫酸铵和硝酸钾。
利用酸性品红对沙棘根段染色,结果可见3批样品沙棘根系都有VA菌根(Vesicular-Avbuscular mycorrhiza)真菌侵染,侵染率分别为55.6%,72%,65%。采用湿筛倾析法从沙棘根际土壤鉴定出2属7种VA菌根真菌,包括球囊霉属(Glomus)6种：缩球囊霉(Glomus constrictum Trappe)、近明球囊霉(Glomus claroideum Schenck & Smith)、根内球囊霉(Glomus intraradices Schenck & Smith)、地球囊霉(Glomus geosporum Walker)、透光球囊霉(Glomus diaphanum Marton & walker)、网状球囊霉,(Glomus reticulatum Bhattacharjee & Mukerji);巨囊霉属(Gigaspora)1种：巨大巨孢囊霉(Gigaspora gigantean Gerd.Trappe)。
In order to explore the role of non-leguminous nitrogen fixing plant Hippophae rhamnoides'growth-promoting rhizobacteria in promoting vegetation restoration and ecosystem reconstruction in Loess Plateau, the soil and root nodule in Hippophae rhamnoides rhizosphere in Shaanxi province was used to detect the relationship between microbial population structure and soil fertility and to study the diversity of hydrogen-oxidizing bacteria, Frankia and Vesicular-Avbuscular mycorrhiza fungi. According to the analysis of the result of above experiments, we can realize the generic distribution of hydrogen-oxidizing bacteria, enrich the existing research about sort of hydrogen-oxidizing bacteria and explore the relationship and mechanism of polymicrobic growth-promoting in Hippophae rhamnoides rhizosphere. The main experimental content and results were as follows:
We used gas cycle incubation system to treated rhizosphere soils with H2 produced by water electrolysis. The result showed that the microbial population structure of the soil treated with H2 had changed:the number of bacteria and actinomyces had increased,and the number of hydrogen-oxidizing bacteria had increased significantly, but the number of fungi had declined. The microbes'respiration intensity and activity of soil enzyme in soil treated by H2 had enhanced to varying degrees; organic matter content of soil and planthormones in soil had grown in number, which explained that the soil fertility had enhanced with the treatment of H2.
Fifty nine bacterial strains were isolated by gas-cycle incubation system and MSA medium. Twelve strains among them which contained hydrogenase and had strong ability to oxidize hydrogen was selected by TTC test, gas chromatograph and autotrophic gowth detection, suggesting that they might be hydrogen-oxidizing bacteria initially. Hydrogen-oxidizing ability of strain FS2 was about 35.430μmol/L.Based on their morphological, cultural,and physiological and biochemical characteristics, these 12 strains were identified as members of genera Bacillus, Aeromonas, Pseudomonas, Micrococcus, Xanthomonas, Brachybacterium, Nocardia and Pimelobacter,respectively. The 16S rRNA gene sequence analysis suggested that strain FS2 (GenBank accession number GU084156) was clustered together with Bacillus in phylogenetic tree, with sequence similarity more than 99%.
We used thin layer chromatography to find out that strain FS2 and WS4 could consum-ed ACC and contained ACC deaminase.Then we detectd the influence factors about the activity of the ACC deaminase. The result showed that the two strains could decompose nearly 90% ACC in 24h and the optimal temperature was 30-35℃and the optimal pH was7.0-7.5.
Three endophyte strains SY1, SY2 and SY3 were isolated from root nodule of Hippophae rhamnoides,.They belonged to slow-growing actinomyces and formed flocculent or granular precipitate in the BAP liquid medium; their hyphae was filamentous with different diameters under microscope and could produce vesicle when growing in the nitrogen-free medium. Above characteristics was in line with general characteristics of Frankia, therefore the 3 strains could be identified as a member of Frankia preliminarily. The study of 3 Endophyte's biological property showed that strain SY1 and SY2 were divided into physiological group A and strain SY3 was divided into physiological group AB.Their optimal carbon sources were tween-80 and glucose, and optimal nitrogen sources were ammonium sulfate and nitre.
Acid fuchsin was used to dye root segment of Hippophae rhamnoides and the result showed that the Hippophae rhamnoides root system was infected by Vesicular-Avbuscular mycorrhiza fungi and the infecting rate were 55.6%,72% and 65%, respectively. SevenVA fungi species belonging to 2 genus were isolated and identified by sieving and deeanting proeedure from Hippophae rhamnoides rhizosphere soil.Six species were belonged to genus Glomus, including Glomus claroideum, Glomus consrictum, Glomus intraradices, Glomus geosporum, Glomus diaphanum and Glomus reticulatum,and Gigaspora gigantean was belonged to genus Gigaspora.
引文
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