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铅污染是当前土壤重金属污染亟待解决的难题之一，利用微生物与树木的互作来修复铅污染土壤是一个行之有效的方法。为了给利用植物内生细菌提升植物修复铅污染土壤效率提供支持，以毛竹的芽和叶为材料，分离耐铅内生细菌，采用生理分析和分子鉴定相结合的方法，对分离到的菌种进行鉴定。结果发现，在不同铅离子浓度的胁迫下，从毛竹芽和叶组织中分离出27株内生细菌，革兰氏染色和VP实验均为阳性。过氧化氢酶实验中，24株表现为阳性(+)，3株为阴性(-)；甲基红实验测定结果为阳性(++)4株、弱阳性(+)20株、阴性(-)3株。对细菌提取DNA，并PCR扩增16S rDNA序列，通过BLAST比对分析，并用MEGA软件建立发育树等分子鉴定手段，初步推断分离得到的内生细菌为假单胞菌属、蜡样芽孢杆菌和不动杆菌属。 相似文献
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种子被视为有益微生物的储存库和载体。与根际和叶际相比,种子相关细菌的研究还十分落后。本研究以长期绿肥植物与水稻轮作(稻-稻-紫云英,R-R-MV)和冬闲(稻-稻-冬闲,R-R-WF)两种种植模式的水稻种子为材料,分离种子内生细菌;在对菌株进行16S r RNA基因系统发育分析基础上,依据分离来源和分类地位选择代表菌株,进行溶磷、产IAA和铁载体等植物促生功能的检测。结果显示,绿肥轮作后水稻种子内生细菌的数量显著高于冬闲,群落组成也发生了明显变化。在属水平上,Pseudomonas和Xanthomonas是R-R-WF中的优势属,分别占28.57%和26.19%,而R-R-MV中Pantoea是优势属,占27.94%,其次是Pseudomonas(14.71%)和Paenibacillus(13.24%)。进行功能检测的31个菌株中,大多数分离株具有有益于植物的特性,表明种子内生细菌与植物的生长及健康密切相关。本研究不仅揭示了长期轮作条件可以改变种子的微生物组成,还获得了有益的微生物资源,将有助于种子内生菌作为生物接种剂在可持续农业中的研究和应用。 相似文献
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番茄内生拮抗细菌的分离鉴定及培养条件研究 总被引：1，自引：0，他引：1
针对番茄生产上灰霉病和叶霉病两大瘸害,为寻找安全、高效无污染的生防菌株及其最佳培养条件,本试验采用组织分离法从健康的番茄植株中分离出642个内生细菌菌株,并采用平板对峙法筛选出对番茄灰霉病菌和叶霉病菌拮抗作用强且稳定的两个菌株Thyy1和Jcxy8。通过形态学观察及生理生化特征测定,初步鉴定Jcxy8属环状芽孢杆菌（Bacillus circulans）,菌株Thhy1属枯草芽孢杆菌（Bacillus subtilis）。内生拮抗细菌在以豆饼粉为原料的6号培养基中生长速度快,发酵滤液对两种病原菌的抑制作用强。培养基初始pH值、培养时间、温度、通气量等对菌株生长及其抗菌物质的分泌有明显影响。以豆饼粉培养基、初始pH6．7、培养时问48h、温度30qc、并尽量增大培养通气量为菌株的最佳培养条件。 相似文献
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为揭示糯红高粱内生酵母菌多样性及筛选可利用菌株，本研究基于传统纯培养技术和Illumina MiSeq测序技术，研究杂交种金糯粱1号和地方种青壳洋根、茎和叶组织内生酵母菌的多样性，并对纯培养菌株产酶功能进行初筛。结果表明，分离纯化共获得36株内生酵母菌，其中金糯粱1号27株，青壳洋9株；根、茎、叶分别为6、7、23株。36株内生酵母隶属于1门3纲5目6科7属，担子菌门（Basidiomycota）为优势门，浅黄隐球酵母（Cryptococcus luteolus）为优势种，其中5株菌具有产4种酶的特性，隐球菌属（Cryptococcus）占比最多。基于酵母菌26S rDNA NL1F和NL2R区的高通量测序分析共检测到111个操作分类单元（OTU），隶属于2门83属，其中杂交种金糯粱1号和地方种青壳洋内生酵母菌群落多样性、群落结构不存在显著性差异，而不同组织间多样性、群落结构存在极显著性差异（P<0.001）。通过FUNGuild功能预测发现不同组织内生酵母菌的营养模式和生态功能群分为6种和16个，其中茎部独有功能群最多，叶部独有功能群最少。综上，糯红高粱不同品种和不同组织内生酵... 相似文献
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转几丁质酶基因水稻根系微生物群落分析 总被引：1，自引：1，他引：1
有用“直接观察法”和“分离培养法”对转基因水稻根系微生物群落的初步分析结果表明，导入外源水稻几丁质酶基因（RC24）的转基因水稻根内和根表的微生物群落发生显著变化，转基因水稻根部内生真菌总数显著减少，内生细菌总数显著增加，其内生细菌总数是未转基因亲本对照的10倍左右。采用“直接观察法”测得2个转基因水稻品种有内生真菌的根段率为55．2％和81．1％，而对照为100％，转基因水稻根系真菌和细菌种类与对照存在显著差异，有VA菌根泡囊的根段率显著降低。 相似文献
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不同石油污染程度土壤细菌群落多样性及优势菌群分析 总被引：1，自引：0，他引：1
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诸多研究发现毛竹入侵周围林分提高土壤pH，但未见统计性描述报道。为此，本研究将通过大数据分析证实此现象的普遍性以及探究伴随的土壤养分和微生物变化。本研究收录包含毛竹入侵有关土壤pH变化的42篇文献总计101组数据，采用整合分析方法（Meta-analysis）进行深入探讨。在研究土壤养分变化时，本文还结合了团队采集的12个毛竹入侵带的样地数据总计92个样品18组数据进行分析。结果表明，在所有的数据组中，84.9%的土壤在经过毛竹入侵后其pH有不同幅度的提升，说明毛竹入侵周围林分普遍提高土壤pH；土壤pH增加幅度随入侵阶段、毛竹纯林时间增加而增加，随土层深度增加而降低；入侵针叶林致土壤pH增加的幅度高于阔叶林。入侵的毛竹纯林与原生林相比，总体上显著降低（P<0.05）了土壤全氮（-15.9%）、硝态氮（-21.7%）、全碳（-2.0%），却增加（P<0.05）了土壤有效磷（+54.9%）、铵态氮（+14.7%）和碱解氮（+8.2%）。对27篇包含微生物数据的文献进行整合分析，结果表明毛竹入侵改变了微生物群落结构，增加（P<0.05）了放线菌门相对丰度（+25.86%），而降低（P<0.05）了酸杆菌门（-15.49%）、浮霉菌门（-26.66%）、拟杆菌门（-22.58%）的相对丰度。本研究通过Meta分析证明毛竹入侵周围林分提高土壤pH为普遍自然现象，结合土壤硝态氮和铵态氮指标的变化推测了导致土壤pH提升的可能机制，毛竹入侵提高了土壤细菌和真菌多样性，表明入侵对土壤微生物具有正反馈效应。 相似文献
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A total of 98 non-symbiotic endophytic bacterial strains isolated from soybean root nodules were classified into eight rDNA types in ARDRA analysis and 21 BOX types in BOX-PCR. The phylogenetic analysis of 16S rDNA identified these strains as Pantoea, Serratia, Acinetobacter, Bacillus, Agrobacterium, and Burkholderia. Limited genetic diversity was revealed among these bacteria since most of the strains (85.7%) were found in three very similar rDNA types corresponding to Pantoea agglomerans, and many strains shared the same BOX-PCR patterns. The inoculation of nodule endophytes had no significant effects on the growth and nodulation of soybean, but most of the strains produced indoleacetic acid (IAA), could solubilize mineral phosphate, and could fix nitrogen, implying that they are a valuable pool for discovering plant growth promoting bacteria. Our results demonstrated that the nodule endophytes were common in soybean and their diversity was affected by the plant's character and the soil conditions. The 99% similarities found in the nifH genes of Bradyrhizobium japonicum and of the endophytic Bacillus strains strongly indicated that horizontal transfer of symbiotic genes happened between the symbiotic bacteria and the endophytes. 相似文献
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Apiraya Thepsukhon Somporn Choonluchanon Shikeyuki Tajima Mika Nomura Soraya Ruamrungsri 《Journal of plant nutrition》2013,36(9):1424-1438
Endophytic bacteria carrying out dinitrogen (N2) fixation and indole acetic acid (IAA) synthesis were firstly identified in C. alismatifolia, a globally important flower crop. Their potential as growth promoters to stimulate the rapid growth of host plant was also examined. It will be beneficial to reduce the propagation period of tissue culture plantlets, and also utilize as a biofertilizer for rhizome production in the field. Seven endophytic bacteria were isolated from the leaf, four isolates from the leaf base, and two from the rhizome. ECS203, a gram-negative bacterium with a round shape, showed the highest N2 fixation at 4.2 nmol C2H4/106 cells/hr, and ECS202 showed the highest IAA synthesis at 296 μL μg ? 1 protein. Three selected isolates of N2-fixing and IAA synthesizing endophytic bacteria, i.e., ECS202, ECS203, and ECS204, isolated from the leaf base, were used to reinoculate Curcuma plantlets derived from tissue culture. Then, plants were grown in sterilized sand for 2 months and weekly supplied with N-free nutrient solution. Plant growth, colonization, nitrogen fixation, and IAA synthesis were measured at two months after planting. The inoculated plants clearly showed a better performance of plant growth and yield in terms of the plant height, plant weight, leaf area, and diameter of new rhizomes compared with uninoculated plants. The chlorophyll content and N concentration of leaves and roots also increased in inoculated plants. Endophytic bacteria from inoculated plants colonized the roots, rhizome, and leaf base. Partial sequence analysis using 16S rDNA indicated that the isolate ECS202 corresponded to Sphingomonas pseudosanguinis (99.2% similarity over 1,371 bp), ECS203 to Bacillus drentensis (99.4% similarity over 1,450 bp) and ECS204 to Bacillus methylotrophicus (99.9% similarity over 13,06 bp). 相似文献
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Yuichi Saeki Ai Kaneko Toshiaki Hara Koutaro Suzuki Takeo Yamakawa Minh Thi Nguyen Yoshitaka Nagatomo Shoichiro Akao 《Soil Science and Plant Nutrition》2005,51(7):1043-1052
In order to analyze the phylogeny of soybean-nodulating bacteria in alkaline soils in Vietnam, indigenous soybean-nodulating bacteria were isolated from root nodules by cultivating three kinds of Rj -soybean cultivars on two alkaline soils in Vietnam. The 120 isolates were classified into two major genera of soybean-nodulating rhizobia, namely Bradyrhizobium and Sinorhizobium genera, based on a growth analysis on medium and PCR-RFLP analyses of 16S rDNA and of the 16S–23S rDNA internal transcribed spacer (ITS) region. Most of the isolates of B. japonicum were extra-slow-growing and their ITS types were similar to that of B. japonicum USDA 135. They were not isolated from the soybean cultivar CNS used as Rj2 Rj3 genotype. Isolates of Sinorhizobium were divided into two groups, S. fredii and S. sp., based on a PCR-RFLP analysis of 16S rDNA. Furthermore, PCR-RFLP analysis of the 16S–23S rDNA ITS region enabled to separate them into five types, three ITS types associated with S. fredii and two with S. sp. Sinorhizobium was frequently isolated from the three soybean cultivars on two soils. From the isolate ratio, it was suggested that B. japonicum strains similar to B. japonicum USDA 135 and S. fredii predominated in the alkaline soils of Vietnam. Additionally, our findings indicated that the Rj -genotypes affected not only the compatibility, but also the preference for nodulation between the host soybean and rhizobia. 相似文献
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Identification and characterization of rhizobacteria isolated by enrichment culture on spinach roots
Rhizobacteria can be used for biological control and environmental restoration. In this study, we performed enrichment culture of rhizobacteria, identified isolates, and investigated the physiological properties of the bacterial isolates. Five bacteria differing in their colony morphology were isolated from spinach roots as enriched rhizobacteria. Four isolates were identified by sequencing of 16S rDNA as β and γ-Proteobacteria; 16S rDNA sequencing was not completed on one isolate. Based on microscopic observation, we determined that at least two types of bacteria differing in their morphology co-existed in this isolate, and that it may not be possible to culture the two types separately. Based on tests of substrate utilization, we could not find the characteristics that were common to the isolates. One of the five isolates was inoculated into non-sterile soil, and we examined its root-colonizing ability. The test strain which was not detected in the non-rhizosphere soil, accounted for about 20% of the total bacteria on the roots. These results suggested that enrichment culture might be useful for isolating bacteria with a high root-colonizing ability 相似文献
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采集四川省汉源县富泉乡万顺铅锌矿区5个不同重金属浓度的土壤样品,进行了微生物数量及放线菌多样性的研究。经分离、纯化得到43株不同的放线菌,然后对其进行BOXAIR-PCR和16SrDNAPCR-RFLP分析。结果表明,铅锌矿区重金属复合污染对土壤微生物数量有较大的影响,随着铅锌矿区重金属污染程度的加剧,土壤微生物的总数下降。相关性分析表明,重金属含量与细菌数量呈极显著负相关（P〈0.01）,与放线菌数量、真菌数量呈显著负相关（P〈0.05）。供试菌株的16SrDNA用HaeⅢ、HinfⅠ和TaqⅠ酶切后具有32种遗传图谱类型。BOXAIR-PCR的聚类结果表明在86%的水平上,所有菌株分为10个遗传类型,结果基本与16SrDNAPCR-RFLP聚类差异不大。来源于高重金属的含量样品的菌株基本聚在一起,可能是重金属含量影响了放线菌的分布。同时,16SrDNA序列聚类分析结合系统发育树分析表明链霉菌属是汉源铅锌矿区主要的放线菌属并且具有遗传多样性。 相似文献
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Tomoyoshi HASHIMOTO Miyo KOGA Yoshikuni MASAOKA 《Soil Science and Plant Nutrition》2009,55(5):647-659
This research aimed to determine whether a diluted nutrient broth (DNB) medium was different from a conventional nutrient broth (NB) medium when counting and isolating denitrifying bacteria in surface and subsurface upland soils. To this end, we investigated populations of denitrifying bacteria isolated from the surface to a depth of 4 m of subsurface upland soil that had received slurry. The DNB medium gave higher viable counts of denitrifying bacteria than the NB medium and a higher isolation ratio of denitrifying bacterial isolates. In total, 74 isolates from the DNB medium (D-isolates) and 26 isolates from the NB medium (N-isolates) were collected. We characterized their denitrifying activity and analyzed the diversity of 16S rDNA and denitrifying-related genes. Seventy-three percent of the D-isolates were oligotrophic denitrifying bacteria. The N2 -producing, oligotrophic denitrifying bacteria, largely of α-Proteobacteria, increased in the D-isolates. The D-isolates and the N-isolates had some taxonomic overlapping on a phylogenetic tree based on 16S rDNA. It was not possible to identify the denitrification phenotype (N2 -producing or N2 O-producing) on the phylogenetic tree. Phylogenetic groups of isolates corresponded to nirK groups, except in some isolates in which horizontal gene transfer might have occurred. The terminal gas emission of the isolates was consistent with the existence of the nosZ gene. The DNB medium may be very useful in isolating N2 -producing denitrifying α-Proteobacteria. Its use highlights the ecological significance of oligotrophic isolates and the different viable counts resulting from the selectivity of conventional and diluted media. 相似文献
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Plant growth promoting (PGP) bacteria associated with sugarcane are a promising alternative for the expansion of this crop in Southern Brazil. In this study bacterial strains from different sugarcane fields were isolated to estimate their diversity, to evaluate some of their PGP activities and to use them as inoculant strains in field experiment. Samples of rhizospheric soil, roots, and stems of sugarcane were collected in six Rio Grande do Sul localities. The isolation of bacteria was made in three different N-free media. DNA from each isolate was subjected to nifH or 16S rDNA PCR-RFLP, and to the 16S rDNA partial sequencing. Five hundred and sixteen strains were isolated and several PGP characteristics were analyzed. Shannon index was used to evaluate the bacterial diversity. Indexes varying from 0.94 to 2.46 were obtained. Soil pH and clay were the characteristics most closely related to bacterial diversity. Achromobacter, Agrobacterium, Burkholderia, Gluconacetobacter, and Stenotrophomonas were the most abundant genera. Concerning the PGP activities, indolic compounds production was detected in 368 isolates; 138 isolates were able to solubilize phosphate; and 390 were siderophores producers. The inoculation of sugarcane with Gluconacetobacter diazotrophicus VI27 strain showed a significant increase in the number of sets germinated, in the amount of soluble solids, and in the yield of sugarcane juice compared with the control. As a conclusion, a diverse population of PGP bacteria was found in the sugarcane samples. These bacteria, especially G. diazotrophicus strain VI27, could be used as biofertilizers of sugarcane as well as other cereal crops under controlled conditions to avoid or reduce the use of standard N fertilizers. 相似文献
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Pham Q. Hung Senthil M. Kumar V. Govindsamy K. Annapurna 《Biology and Fertility of Soils》2007,44(1):155-162
Endophytic bacteria were isolated from surface sterilized stems, root, and nodules of wild and cultivated soybean varieties.
Various phenotypic traits that are expected to be involved in the persistence and functions of the bacteria were analyzed.
Most of the isolates from soybean were motile and indoleacetic acid producers, and 70 and 33% of isolates excreted cellulase
and pectinase, respectively. These traits may be involved in endophytic characteristics in soybean. Some isolates were resistant
to Str100, formed capsules, and produced fluorescent pigments. Molecular characterization of selected 35 endophytic bacteria by 16S
rDNA–polymerase chain reaction–restriction fragment length polymorphism showed two main clusters at 48 and 43% similarity
coefficients in which most of the endophytes belonged. The genetic variation was more among endophytes isolated from Glycine max tissues than from G. soja. 相似文献
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Genetic diversity and relationship between Bradyrhizobium strains isolated from blackgram and cowpea
Lilly M. Saleena P. Loganathan Sunita Rangarajan Sudha Nair 《Biology and Fertility of Soils》2001,34(4):276-281
The genetic diversity of bradyrhizobial strains associated with blackgram and cowpea grown in two different agricultural soils (non-saline and saline) along the coastline of Tamil Nadu has been analysed. Phenotypically indistinguishable isolates were analysed for DNA polymorphism using random amplification of polymorphic DNA (RAPD) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of 16S rDNA and nifD. Although these bacteria belong to a group with a broad host range, RAPD analysis showed a considerable level of genetic diversity among the strains isolated from different host plants. Soil pH and salinity seem to have an effect on the selection of natural populations as revealed by PCR-RFLP of 16S rDNA. A combination of PCR-RFLP genotyping with nodulation studies indicates that monocropping of blackgram and the salinity of the soil have made ineffective rhizobia the dominant genotype, thereby creating an ecological burden on their other compatible hosts. A group of strains and a type strain sharing three different 16S PCR-RFLP types were shown to have the same set of symbiotic genes as inferred from the PCR-RFLP pattern of nifD. Another group of cowpea rhizobia that were found to be effective nitrogen fixers and sharing distinct 16S profiles were found to have a different set of symbiotic genes. 相似文献