甘草有益微生物的分离、鉴定及共接种效果初探
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摘要
本论文以内蒙占和青海省人工栽培的乌拉尔甘草为材料，研究甘草内生细菌分布的多样性。同时，还对内蒙古栽培甘草的根瘤菌和VA菌根进行了分析，并对内生细菌、根瘤菌、VA菌根的共接种效果进行了初步探讨，结果如下：
1、对内蒙古一年生、两年生和三年生乌拉尔甘草、青海两年生乌拉尔甘草健康植株根、茎、叶等组织中的内生细菌进行了分离和数量测定，共分离出细菌146株。结果表明，甘草根、茎、叶等组织内存在大量内生细菌，不同组织中内生细菌的分布密度不同。不同地域、不同年份甘草组织中内生细菌分布密度也存在较大差异。通过镜检观察芽孢杆菌是分离的优势菌，约占30％，实验中还分离到了黄单孢菌属(Xanthomonas)、假单孢菌属(Pseudomonas)、类芽孢杆菌属(paenibacillus)及短小杆菌属(Curtobacterium)。采用ERIC-PCR分析方法，发现甘草内生细菌存在特异的扩增条带，能够分辨出不同地区来源、不同组织中菌株的差异，说明甘草中内生细菌存在着丰富的多样性。
2、住PDA平板上测定了146株内生细菌凶株对西瓜枯萎病菌和烟草赤星病菌等15种病原菌的体外拮抗活性。共有23株内生细菌对一种或几种病原菌具有不同程度的拮抗活性，其中6株内生细菌对这些病原菌表现出较好的抑菌活性，对15种病原菌的抑制半径几乎都达到了7mm以上，特别是IS5对黄瓜枯萎病菌、棉花枯萎病菌的抑制半径都达到了13.0mm，抑菌率达剑65％，对病原菌的扩展具有明显的抑制作用。同时，IIL15菌株的培养滤液在DPA平板上抑制烟草赤星病菌和小麦纹枯病菌的生长，产生透明的抑菌圈，这说明细菌在生长代谢过程中产生了具有抑菌活性的抗生物质，抑制了真菌抱子的萌发。经常规的生理生化鉴定、Biolog微生物自动鉴定系统鉴定、16S rDNA序列分析，将6株拮抗菌分别鉴定为萎缩芽孢杆菌(Bacillus atrophaeus)、枯草芽孢杆菌(Bacillus subtils)、多粘类芽孢杆菌(Paenibacillus polymyxa)和Paenibacillus ehimensis。
3、从不同年份甘草根瘤中共得剑54个根瘤培养物，经菌落形态及镜检发现这些菌具有根瘤菌属的特征。采用ERIC-PCR分析方法，发现甘草根瘤菌存在特异的扩增条带，能够分辨出从不同年份植株上分离的根瘤菌的差异，说明甘草根瘤凶存在着丰富的多样性。在用根瘤菌同接甘草中，液体培养结瘤数多于半固体培养的，且根瘤较半固体培养的大。对结瘤效果好的菌株进行克隆测序，并分析序列比对结果，发现R31与Mesorhizobium temperatum(T)AF508208、R50与Mesorhizobium mediterraneum(T)L38825相似性都为99％，属于中慢生根瘤菌属。
4、分离甘草的VA菌根，并用三叶草进行单孢繁殖，将繁殖斤的菌根同接甘草。接种了菌根的甘草植株茎长、根粗、茎叶干重和根干重都有较大程度的提高，均比对照显著增加，并且不同处理对植株生长影响不同。可见接种VA菌根真菌显著促进了甘草的营养生长。将根瘤菌、内生细菌和菌根共接种甘草，实验结果表明，VA菌根真菌对甘草植株吸收氮、磷、钾有明显的促进作用，二种微生物接种后，复合接种比单一接种处理植株根茎叶中氮、磷、钾含量增加。这表明，甘草有益的根瘤菌和内生细凶能增强菌根对植株的促生长作用。因此，应用三种微生物共接种，对促进甘草生长，提高品质具有潜在的应用价值。
This paper was studied on isolation, identification and characteristics of endophytic bacteria、rhizobia and VA mycorrhizal fungi which were isolated from Glycyrrhiza uralensis plants, and the co-inoculated effect was also studied in the article. The results go as follows:
146 endophytic bacteria strains were isolated from different internal tissues of Glycyrrhiza uralensis plants collected from Innermongolia region and Qinghai province. There were many endophytic bacteria in tissue of the plant, and the densities of endophytic bacteria were varied depending on tissue of the plant. Xanthomonas、Bacillus、Pseudomonas、Paenibacillus、Curtobacterium were isolated, and Bacillus was the most prevalent endophytic bacteria among these strains. Of the 146 isolates, 6 strains exhibited extensive antagonistic activities against pathogenicbacteria. Characterization showed that these bacteria were Bacillus atrophaeus、B. subtilis、Paenibacillus polymyxa、P. ehimensis. This study indicated that selected 6 endophytic bacteria strains had potential for biological control of plant disease.
54 rhizobia were isolated from Glycyrrhiza uralensis plants collected from Innermongolia region. Genomic DNA of the rhizobia were amplified and analyzed by PCR with primers specific for ERIC sequence. Each strain showed different profile of band patterns when PCR products were analyzed in agarose gel electrophoresis. The strains of the same species were resolved by unique bands specific for each strain. The representative strains were chosen to inoculate into Glycyrrhiza uralensis. Whole 16SrDNA sequences of the effective strain R50 and R31 were determined and then analyzed in phylogenesis, which indicated that R50 and R31 were 99% homology to the tested strains of Mesorhizobium in 16S rDNA, respectively, and thus both of them belonged to Mesorhizobium.
VA mycorrhizal fungi on Glycyrrhiza uralensis plants were isolated by wet sieving and decanting method and single spore inoculation. On the basis of the micromorphological characteries of spores, most stains were Glomus. The biomass of the plants is significantly increased by inoculating VAM. To investigate the effect of VAM、Rhizobium and endophytic bacteria on N、K and P utilization and the growth of Glycyrrhiza uralensis, four treatments were applied. Both the single and the dual inoculation could help the plant growing quickly and healthily ,and could increase the absorption of N、K and P. The effect of dual inoculation was superior to single inoculation, and the treble inoculation was superior to dual inoculation. The present data indicated that the inoculation of VAM、Rhizobia and endophytic bacteria improved N、K and P utilization , and consequently increased the yield of Glycyrrhiza uralensis together.
1、对内蒙古一年生、两年生和三年生乌拉尔甘草、青海两年生乌拉尔甘草健康植株根、茎、叶等组织中的内生细菌进行了分离和数量测定，共分离出细菌146株。结果表明，甘草根、茎、叶等组织内存在大量内生细菌，不同组织中内生细菌的分布密度不同。不同地域、不同年份甘草组织中内生细菌分布密度也存在较大差异。通过镜检观察芽孢杆菌是分离的优势菌，约占30％，实验中还分离到了黄单孢菌属(Xanthomonas)、假单孢菌属(Pseudomonas)、类芽孢杆菌属(paenibacillus)及短小杆菌属(Curtobacterium)。采用ERIC-PCR分析方法，发现甘草内生细菌存在特异的扩增条带，能够分辨出不同地区来源、不同组织中菌株的差异，说明甘草中内生细菌存在着丰富的多样性。
2、住PDA平板上测定了146株内生细菌凶株对西瓜枯萎病菌和烟草赤星病菌等15种病原菌的体外拮抗活性。共有23株内生细菌对一种或几种病原菌具有不同程度的拮抗活性，其中6株内生细菌对这些病原菌表现出较好的抑菌活性，对15种病原菌的抑制半径几乎都达到了7mm以上，特别是IS5对黄瓜枯萎病菌、棉花枯萎病菌的抑制半径都达到了13.0mm，抑菌率达剑65％，对病原菌的扩展具有明显的抑制作用。同时，IIL15菌株的培养滤液在DPA平板上抑制烟草赤星病菌和小麦纹枯病菌的生长，产生透明的抑菌圈，这说明细菌在生长代谢过程中产生了具有抑菌活性的抗生物质，抑制了真菌抱子的萌发。经常规的生理生化鉴定、Biolog微生物自动鉴定系统鉴定、16S rDNA序列分析，将6株拮抗菌分别鉴定为萎缩芽孢杆菌(Bacillus atrophaeus)、枯草芽孢杆菌(Bacillus subtils)、多粘类芽孢杆菌(Paenibacillus polymyxa)和Paenibacillus ehimensis。
3、从不同年份甘草根瘤中共得剑54个根瘤培养物，经菌落形态及镜检发现这些菌具有根瘤菌属的特征。采用ERIC-PCR分析方法，发现甘草根瘤菌存在特异的扩增条带，能够分辨出从不同年份植株上分离的根瘤菌的差异，说明甘草根瘤凶存在着丰富的多样性。在用根瘤菌同接甘草中，液体培养结瘤数多于半固体培养的，且根瘤较半固体培养的大。对结瘤效果好的菌株进行克隆测序，并分析序列比对结果，发现R31与Mesorhizobium temperatum(T)AF508208、R50与Mesorhizobium mediterraneum(T)L38825相似性都为99％，属于中慢生根瘤菌属。
4、分离甘草的VA菌根，并用三叶草进行单孢繁殖，将繁殖斤的菌根同接甘草。接种了菌根的甘草植株茎长、根粗、茎叶干重和根干重都有较大程度的提高，均比对照显著增加，并且不同处理对植株生长影响不同。可见接种VA菌根真菌显著促进了甘草的营养生长。将根瘤菌、内生细菌和菌根共接种甘草，实验结果表明，VA菌根真菌对甘草植株吸收氮、磷、钾有明显的促进作用，二种微生物接种后，复合接种比单一接种处理植株根茎叶中氮、磷、钾含量增加。这表明，甘草有益的根瘤菌和内生细凶能增强菌根对植株的促生长作用。因此，应用三种微生物共接种，对促进甘草生长，提高品质具有潜在的应用价值。
This paper was studied on isolation, identification and characteristics of endophytic bacteria、rhizobia and VA mycorrhizal fungi which were isolated from Glycyrrhiza uralensis plants, and the co-inoculated effect was also studied in the article. The results go as follows:
146 endophytic bacteria strains were isolated from different internal tissues of Glycyrrhiza uralensis plants collected from Innermongolia region and Qinghai province. There were many endophytic bacteria in tissue of the plant, and the densities of endophytic bacteria were varied depending on tissue of the plant. Xanthomonas、Bacillus、Pseudomonas、Paenibacillus、Curtobacterium were isolated, and Bacillus was the most prevalent endophytic bacteria among these strains. Of the 146 isolates, 6 strains exhibited extensive antagonistic activities against pathogenicbacteria. Characterization showed that these bacteria were Bacillus atrophaeus、B. subtilis、Paenibacillus polymyxa、P. ehimensis. This study indicated that selected 6 endophytic bacteria strains had potential for biological control of plant disease.
54 rhizobia were isolated from Glycyrrhiza uralensis plants collected from Innermongolia region. Genomic DNA of the rhizobia were amplified and analyzed by PCR with primers specific for ERIC sequence. Each strain showed different profile of band patterns when PCR products were analyzed in agarose gel electrophoresis. The strains of the same species were resolved by unique bands specific for each strain. The representative strains were chosen to inoculate into Glycyrrhiza uralensis. Whole 16SrDNA sequences of the effective strain R50 and R31 were determined and then analyzed in phylogenesis, which indicated that R50 and R31 were 99% homology to the tested strains of Mesorhizobium in 16S rDNA, respectively, and thus both of them belonged to Mesorhizobium.
VA mycorrhizal fungi on Glycyrrhiza uralensis plants were isolated by wet sieving and decanting method and single spore inoculation. On the basis of the micromorphological characteries of spores, most stains were Glomus. The biomass of the plants is significantly increased by inoculating VAM. To investigate the effect of VAM、Rhizobium and endophytic bacteria on N、K and P utilization and the growth of Glycyrrhiza uralensis, four treatments were applied. Both the single and the dual inoculation could help the plant growing quickly and healthily ,and could increase the absorption of N、K and P. The effect of dual inoculation was superior to single inoculation, and the treble inoculation was superior to dual inoculation. The present data indicated that the inoculation of VAM、Rhizobia and endophytic bacteria improved N、K and P utilization , and consequently increased the yield of Glycyrrhiza uralensis together.
引文
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