番茄根际微生物种群动态变化及多样性

采用盆栽试验的方法对番茄根际主要微生物种群在不同生育期的动态变化进行了跟踪研究.结果表明,在番茄整个生育期内,可培养细菌数量在初花期和初果期时最多;放线菌数量从苗期到末期逐渐减少;真菌数量逐渐增多.番茄对细菌根际效应明显.DGGE图谱显示不同生育期番茄根际均具有较高的细菌多样性.根际细菌种类和数量在初花期发生较为显著的变化,初果期根际群落多样性指数(H)和物种丰度(S)值都达到最高,微生物最丰富,是筛选拮抗菌的较好时期.     

不同培养基组合提高土壤细菌可培养性的研究

为选择性采用多培养基组合以提高土壤细菌可培养性,利用变性梯度凝胶电泳(DGGE)技术研究了贫营养、富营养和自然营养培养基在3种培养方式下获得细菌种群的差异。结果表明:平板培养条件下,细菌在贫营养培养基上生长较慢,菌落连续稳定形成。培养5d后,富营养的LB培养基和贫营养的R2A培养基获得菌落数最多,分别是贫营养的0.1×LB培养基获得菌落数的5.1倍和5.3倍。7种培养基中,LB培养基获得细菌种群数目最多,营养成分适当稀释后,培养物中有新的种群出现。贫营养培养基和富营养培养基培养物DGGE图谱相似性低,条带互补性强。三角瓶静置培养时,R2A和LB培养基获得细菌种群数目较多,其它几种培养基获得的细菌类群都能在这2种培养基中找到。试管静置培养条件下,LB培养基获得细菌种群数目最多,某些种群也只出现在R2A培养基和TSB培养基上,R2A及LB培养基与TSB培养基获得的细菌种群差异较为明显。研究结果为特殊培养基设计及选用合适培养基分离土壤细菌提供参考。     

AM真菌对青枯菌和根际细菌群落结构的影响

利用传统的平板培养与DGGE相结合的技术手段,研究了接种AM真菌对番茄根际土壤中的青枯菌和细菌群落结构的影响。结果表明,菌根根际土壤中的细菌总量和总DNA量都高于非菌根根际土壤,其中前者的青枯菌种群数量比后者低60倍;DGGE图谱也证实了AM真菌对青枯菌的抑制效应,还揭示出接种AM真菌对根际土壤中细菌群落结构所产生的复杂的影响。文章对AM真菌抑制青枯菌的机制进行了探讨。     

玉米根际与非根际解磷细菌的分布特点

植物光合作用产物约有 12 %～ 5 0 %通过根系进入根际土壤中 ,不同的植物 ,同一植物不同的生长发育时期 ,不仅根际分泌物的数量有差异 ,而且分泌物的种类也不同[4 ] 。这些分泌物不仅是微生物很好的培养基 ,而且一些分泌物可能抑制或有利于甚至刺激某些微生物的繁殖 ,从而导致根际微生物种群结构的变化。根际微生物的数量、活性和群落结构及其变化 ,直接影响到植物吸收水分、养分 ,也影响植物对恶劣环境的抵抗能力 ,尤其是与病菌的侵入和感染关系非常密切[6] 。Ｐ是植物最重要的营养元素之一 ,大多数土壤都具有很强的固定Ｐ的能力 ,Ｐ肥的利…     

番茄附生和内生细菌分离与群落相似性分析

为了溯清番茄生境以及各组织细菌群落间的相关性,以中杂302为试材,采用传统平板培养法结合16S r RNA拷贝数分析、变性梯度凝胶电泳(DGGE)分析,对番茄根区、根际土壤以及根、茎、叶片、果实、种子等组织附生和内生细菌的数量和群落相似性进行分析。结果表明,番茄根际的细菌数量最多,根区土壤次之;番茄组织附生细菌的数量显著低于根区和根际土壤中的细菌数量;组织附生细菌中,以根部最多,茎和叶片次之;组织内生细菌的数量较附生细菌少2-4个数量级,并以根内生细菌数量最多,种子表面胶状物中数量最少。DGGE细菌群落相似度分析表明,根际与组织内生样品间的相似度显著高于根区土壤,各组织内生细菌相互间的相似度最高,尤其是根、茎和叶片,以及种子、胎座、胶状物等相邻组织内生细菌间的相似度达到0.6以上。番茄组织尤其是相邻组织间的内生细菌群落结构相似,并与根际细菌密切相关。     

本溪山樱根际与非根际解磷细菌群落结构及动态变化

利用选择性培养基,对不同基质中的本溪山樱(Cerasus sachalinensis) 根际与非根际解磷细菌进行了分离、鉴定和分类,分析了3种不同基质中根际与非根际解磷细菌数量和类群的变化.结果表明,从3种不同配比的基质中分离纯化获得的解磷细菌分别属于13个属,以芽孢杆菌属(Bacillus)、假单胞菌属(Pseudomonas)和沙雷铁氏菌属(Serratia)为主.其中添加炉渣的基质最适于解磷细菌的生长与繁殖,其种群数量最高,但类群的多样性指数低于另外两种土壤.本溪山樱不同生育期根际与非根际解磷细菌种群数量不同,新梢停长期根际中定殖的解磷细菌种群最多（共分离到6个菌属）,新梢迅速生长期和落叶期较少,萌芽期最少.根际土壤解磷细菌多样性亦随生育期发生变化,新梢迅速生长期最高,落叶期次之,新梢停长期最低.非根际土壤则有随生育期逐渐减小的趋势.解磷细菌的根际效应较明显     

一株嗜麦芽寡养单胞菌的分离及其生物学特性

近年来,基于高通量测序技术对植物根际土壤微生物菌群结构的测定结果表明,土壤中绝大多数微生物难以通过传统的富营养培养基实现培养和分离。为进一步探索分离土壤中寡营养微生物的方法,结合多种培养因素,以土壤浸出液为基础培养基设计寡营养培养基分离土壤细菌菌株。并通过菌株的细胞形态、生理生化特征、16S rRNA和gyrB基因序列等对其中一株菌进行鉴定,同时对其蛋白酶与铁载体的产生能力进行检测以评价其在植物根际促生方面的应用潜力。利用土壤浸出液做培养基是有效分离土壤寡营养细菌的方法 ;编号为S35的菌株鉴定为嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia),具有蛋白酶和铁载体产生能力。采用的寡营养培养基可以分离获得在富营养培养基上不易分离的细菌,得到S. maltophilia S35菌株在植物促生方面具有一定的应用潜力。     

一株柠条内生解磷菌的分离鉴定及实时荧光定量PCR检测

结合形态观察与16S rDNA序列测定对柠条根系内分离筛选得到的解磷细菌C9进行鉴定,Blast比对结果表明C9为泛菌(Pantoea vagans).在无机磷液体培养基培养条件下,用钼锑抗比色法研究它的解磷能力.结果表明,随着时间的延长,培养液中速效磷含量逐渐增加到4.45mg/L,溶液pH可降至4.2.进一步利用实时荧光定量PCR检测柠条根系中、柠条根际土壤与柠条根围土壤中该细菌存在的相对基因拷贝数,结果发现该基因在3种样品中的数量为:柠条根系＞柠条根际土壤＞柠条根围土壤,表明泛菌解磷细菌能聚集生长在柠条根系内,随着与根系接触距离的增加而呈逐级递减趋势.     

福建省稻田土壤细菌群落的16S rDNA-PCR-DGGE分析

用不依赖细菌培养的16S rDNA-PCR-DGGE方法对福建省6个不同地区12个取样点的稻田土壤进行细菌群落结构分析.对12份样品直接提取其总DNA,用F341GC/R534引物扩增16SrDNA基因的V3可变区,结合DGGE(denaturing gradient gel electrophoresis)技术分析样品细菌群落组成.结果表明,福建省不同地区的稻田土壤之间细菌群落结构存在较大差异.犬体上可分为闽东、闽南、闽北、闽西4个大类.同一地区的根际土和表土样品之间也存在差异,但差异相对较低,其中龙岩根际土和表土细菌群落结构相似性最大,永泰差异性最大.回收了DGGE图谱中11个条带,测序结果经过Blast比对表明其中10个条带代表的细菌是不可培养的,显示了DGGE技术的优越性.     

分蘖洋葱根系分泌物对黄瓜幼苗生长及根际土壤微生物的影响

以不同化感潜力分蘖洋葱为供体,黄瓜为受体,研究了分蘖洋葱根系分泌物对黄瓜幼苗生长、根际土壤微生物数量及细菌群落结构的影响.结果表明:不同化感潜力分蘖洋葱根系分泌物对黄瓜幼苗生长均具有促进作用,且随着浓度的升高,促进作用增强,相同浓度下,化感潜力强、弱供体之间差异不显著;不同化感潜力分蘖洋葱根系分泌物均增加了黄瓜根际土壤细菌和放线菌数量,降低了真菌和尖镰孢菌数量,化感潜力强的品种(L-06)效果更显著;不同化感潜力分蘖洋葱根系分泌物均能提高黄瓜根际土壤细菌群落丰富度,差异条带的序列片段经比对推测为3大细菌类群:Actinobacteria(放线菌纲)、Proteobacteria(变形菌纲)和Anaerolineaceae(厌氧绳菌纲),其中厌氧绳菌只出现在化感潜力强(L-06)的处理中.化感潜力强(L-06)、浓度高(10 mL·株-1)的分蘖洋葱根系分泌物更有利于黄瓜根际土壤细菌群落丰富度的提高.     

Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed

The bacterial rhizosphere communities of three host plants of the pathogenic fungus Verticillium dahliae, field-grown strawberry (Fragaria ananassa Duch.), oilseed rape (Brassica napus L.), and potato (Solanum tuberosum L.), were analyzed. We aimed to determine the degree to which the rhizosphere effect is plant dependent and whether this effect would be increased by growing the same crops in two consecutive years. Rhizosphere or soil samples were taken five times over the vegetation periods. To allow a cultivation-independent analysis, total community DNA was extracted from the microbial pellet recovered from root or soil samples. 16S rDNA fragments amplified by PCR from soil or rhizosphere bacterium DNA were analyzed by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere which became more pronounced in the second year. DGGE patterns of oilseed rape and potato rhizosphere communities were more similar to each other than to the strawberry patterns. In both years seasonal shifts in the abundance and composition of the bacterial rhizosphere populations were observed. Independent of the plant species, the patterns of the first sampling times for both years were characterized by the absence of some of the bands which became dominant at the following sampling times. Bacillus megaterium and Arthrobacter sp. were found as predominant populations in bulk soils. Sequencing of dominant bands excised from the rhizosphere patterns revealed that 6 out of 10 bands resembled gram-positive bacteria. Nocardia populations were identified as strawberry-specific bands.     

Bulk and Rhizosphere Soil Bacterial Communities Studied by Denaturing Gradient Gel Electrophoresis: Plant-Dependent Enrichment and Seasonal Shifts Revealed

The bacterial rhizosphere communities of three host plants of the pathogenic fungus Verticillium dahliae, field-grown strawberry (Fragaria ananassa Duch.), oilseed rape (Brassica napus L.), and potato (Solanum tuberosum L.), were analyzed. We aimed to determine the degree to which the rhizosphere effect is plant dependent and whether this effect would be increased by growing the same crops in two consecutive years. Rhizosphere or soil samples were taken five times over the vegetation periods. To allow a cultivation-independent analysis, total community DNA was extracted from the microbial pellet recovered from root or soil samples. 16S rDNA fragments amplified by PCR from soil or rhizosphere bacterium DNA were analyzed by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere which became more pronounced in the second year. DGGE patterns of oilseed rape and potato rhizosphere communities were more similar to each other than to the strawberry patterns. In both years seasonal shifts in the abundance and composition of the bacterial rhizosphere populations were observed. Independent of the plant species, the patterns of the first sampling times for both years were characterized by the absence of some of the bands which became dominant at the following sampling times. Bacillus megaterium and Arthrobacter sp. were found as predominant populations in bulk soils. Sequencing of dominant bands excised from the rhizosphere patterns revealed that 6 out of 10 bands resembled gram-positive bacteria. Nocardia populations were identified as strawberry-specific bands.     

Analysis of the Dynamics of Bacterial Communities in the Rhizosphere of the Chrysanthemum via Denaturing Gradient Gel Electrophoresis and Substrate Utilization Patterns

In order to gain a better understanding of the spatial and temporal dynamics of bacterial communities of the rhizosphere of the chrysanthemum, two complementary methods were used: a molecular bacterial community profiling method, i.e., 16S rRNA gene-based PCR followed by denaturing gradient gel electrophoresis (DGGE), and an agar plate method in which 11 sole-carbon-source utilization tests were used. The DGGE patterns showed that the bacterial communities as determined from direct rhizosphere DNA extracts were largely stable along developing roots of the chrysanthemum, with very little change over time or between root parts of different ages. The patterns were also similar to those produced with DNA extracts obtained from bulk soil samples. The DGGE patterns obtained by using microbial colonies from dilution plates as the source of target DNA were different from those found with the direct DNA extracts. Moreover, these patterns showed differences among plant replicates but also among replicate plates. Results obtained with the sole-carbon-source utilization tests indicated that the metabolic profile of the bacterial communities in the rhizosphere of the root tip did not change substantially during plant growth. This suggests selective development of specific bacterial populations by the presence of a root tip. On the other hand, the metabolic profile of bacterial communities in the rhizosphere of the root base changed during plant growth. With eight sole-carbon-source utilization tests, a significant effect of the development stage of the plant on the number of bacteria which were able to grow on these carbon sources was observed.     

Rhizosphere Microbial Community Structure in Relation to Root Location and Plant Iron Nutritional Status

Root exudate composition and quantity vary in relation to plant nutritional status, but the impact of the differences on rhizosphere microbial communities is not known. To examine this question, we performed an experiment with barley (Hordeum vulgare) plants under iron-limiting and iron-sufficient growth conditions. Plants were grown in an iron-limiting soil in root box microcosms. One-half of the plants were treated with foliar iron every day to inhibit phytosiderophore production and to alter root exudate composition. After 30 days, the bacterial communities associated with different root zones, including the primary root tips, nonelongating secondary root tips, sites of lateral root emergence, and older roots distal from the tip, were characterized by using 16S ribosomal DNA (rDNA) fingerprints generated by PCR-denaturing gradient gel electrophoresis (DGGE). Our results showed that the microbial communities associated with the different root locations produced many common 16S rDNA bands but that the communities could be distinguished by using correspondence analysis. Approximately 40% of the variation between communities could be attributed to plant iron nutritional status. A sequence analysis of clones generated from a single 16S rDNA band obtained at all of the root locations revealed that there were taxonomically different species in the same band, suggesting that the resolving power of DGGE for characterization of community structure at the species level is limited. Our results suggest that the bacterial communities in the rhizosphere are substantially different in different root zones and that a rhizosphere community may be altered by changes in root exudate composition caused by changes in plant iron nutritional status.     

Rhizosphere microbial community structure in relation to root location and plant iron nutritional status

Root exudate composition and quantity vary in relation to plant nutritional status, but the impact of the differences on rhizosphere microbial communities is not known. To examine this question, we performed an experiment with barley (Hordeum vulgare) plants under iron-limiting and iron-sufficient growth conditions. Plants were grown in an iron-limiting soil in root box microcosms. One-half of the plants were treated with foliar iron every day to inhibit phytosiderophore production and to alter root exudate composition. After 30 days, the bacterial communities associated with different root zones, including the primary root tips, nonelongating secondary root tips, sites of lateral root emergence, and older roots distal from the tip, were characterized by using 16S ribosomal DNA (rDNA) fingerprints generated by PCR-denaturing gradient gel electrophoresis (DGGE). Our results showed that the microbial communities associated with the different root locations produced many common 16S rDNA bands but that the communities could be distinguished by using correspondence analysis. Approximately 40% of the variation between communities could be attributed to plant iron nutritional status. A sequence analysis of clones generated from a single 16S rDNA band obtained at all of the root locations revealed that there were taxonomically different species in the same band, suggesting that the resolving power of DGGE for characterization of community structure at the species level is limited. Our results suggest that the bacterial communities in the rhizosphere are substantially different in different root zones and that a rhizosphere community may be altered by changes in root exudate composition caused by changes in plant iron nutritional status.     

Comparative analysis of bacterial diversity in the rhizosphere of tomato by culture-dependent and -independent approaches

The microbiome in the rhizosphere–the region surrounding plant roots–plays a key role in plant growth and health, enhancing nutrient availability and protecting plants from biotic and abiotic stresses. To assess bacterial diversity in the tomato rhizosphere, we performed two contrasting approaches: culture-dependent and -independent. In the culture-dependent approach, two culture media (Reasoner’s 2A agar and soil extract agar) were supplemented with 12 antibiotics for isolating diverse bacteria from the tomato rhizosphere by inhibiting predominant bacteria. A total of 689 bacterial isolates were clustered into 164 operational taxonomic units (OTUs) at 97% sequence similarity, and these were found to belong to five bacterial phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, and Firmicutes). Of these, 122 OTUs were retrieved from the antibiotic-containing media, and 80 OTUs were recovered by one specific antibiotic-containing medium. In the culture-independent approach, we conducted Illumina MiSeq amplicon sequencing of the 16S rRNA gene and obtained 19,215 high-quality sequences, which clustered into 478 OTUs belonging to 16 phyla. Among the total OTUs from the MiSeq dataset, 22% were recovered in the culture collection, whereas 41% of OTUs in the culture collection were not captured by MiSeq sequencing. These results showed that antibiotics were effective in isolating various taxa that were not readily isolated on antibiotic-free media, and that both contrasting approaches provided complementary information to characterize bacterial diversity in the tomato rhizosphere.     

转基因生防菌308R(pCPP430)对番茄根围菌群的影响

研究目的在于了解转基因生防菌308R(pCPP430)对番茄根围菌群代谢能力和群落结构的影响。实验中使用了两种互为补充的方法,即单一碳源利用测试(SCSU)和ERIC-PCR,对分别以308R(pCPP430)悬液、308R悬液和无菌水蘸根处理的番茄植株根围菌群进行比较。SCSU菌落计数的聚类分析表明,308R(pCPP430)和308R处理的根围菌重复之间相似性好,水处理的相似性差。主成分分析也得到了相同的结果。ERIC-PCR聚类结果表明,10种碳源,其中8种水处理和308R处理聚为一类。实验为生防菌与植物的互作提供一些依据,为根围菌群结构研究提供一些新的思路。     

Increased Complexity of the Species Composition of Lactic Acid Bacteria in Human Feces Revealed by Alternative Incubation Condition

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with primers specific for lactic acid bacteria (LAB) was applied to investigate various media and incubation conditions to recover LAB from human feces. Samples were plated on selective and nonselective media and incubated under standard condition (37 degrees C, anaerobiosis) for fecal LAB as well as alternative condition (30 degrees C, 2% O2). PCR-DGGE analyses of resuspended bacterial biomass (RBB) obtained from agar plates revealed that the species composition of the recovered LAB was affected more strongly by the incubation condition than by the used medium. It was observed that food-associated LAB, such as Lactobacillus sakei and Leuconostoc mesenteroides, hitherto not described as intestinal inhabitants, are more easily selected when the alternative incubation condition is used. Identification of randomly picked colonies grown under the alternative condition showed that L. sakei is one of the predominant food-associated LAB species, reaching counts of up to 106 CFU/g feces. Comparison of the results of bacteriological culture with those obtained by PCR-DGGE analysis of the RBB showed that investigation of RBB is a fast and reliable method to gain insight into the species composition of culturable LAB in feces.     

一株番茄青枯病生防菌的鉴定与防病、定殖能力初探

摘要：【目的】采用根系分泌物培养基筛选到一株番茄根际优势细菌YPP-9。本文分析测定该菌株对植物青枯病菌茄科雷尔氏菌的拮抗作用和控病能力,及其在番茄根际的定殖能力,并系统分析该菌株的分类学地位。【方法】以平板双重培养法和温室盆栽试验分别测定菌株对病原菌的拮抗能力和对番茄青枯病的控病能力;利用变性梯度凝胶电泳技术分析菌株在番茄根际的定殖能力;以形态学和生理生化特性以及16S rRNA基因序列分析确定菌株的分类地位。【结果】菌株YPP-9对茄科雷尔氏菌SSF-4的平板抑菌带宽为5 mm,其盆栽控制番茄青枯病的效果达63.7%。菌株YPP-9在番茄根际具有较好的定殖能力。该菌株培养24 h后菌落呈奶酪色,革兰氏染色阳性,菌体杆状、大小1.8-4.1 μm×0.9-1.1 μm,形成芽孢,芽孢中生或偏端生且为近似柱形,孢囊不膨大,无伴孢晶体,侧生鞭毛。菌株生长pH范围为pH 5.5-8.5且最适生长pH为6.0,生长温度范围为20℃-45℃且最适生长温度为30℃。The BIOLOG GP2结果显示该菌为芽孢杆菌属。16S rRNA基因序列分析显示该菌株与Bacillus fumarioli的亲缘关系最近且序列相似性为97%,且其序列号为FJ231500。该菌株的G+C含量为41.9%,甲基萘醌主要类型为MK-7,细胞壁脂肪酸的主要种类为C14:0 iso、C15:0 iso 和C16:0 iso以及C16 : 1ω7c alcohol且含量分别为28.27%、19.59%、12.93%和10.88%。【结论】菌株YPP-9对茄科雷尔氏菌具有良好的拮抗作用和盆栽控病能力,且能良好的定殖于番茄根际。分类学上,该菌株归入芽胞杆菌属（Bacillus）,并可能是一个新的种。