luxAB基因标记的K2116L菌株在棉花根际中的定殖

采用三亲本杂交法将发光酶luxAB基因转移进棉花根际促生菌芽胞杆菌K2116菌株中,获得标记菌株K2116L.标记菌株连续传代15次均未发生质粒丢失现象,表明标记菌株具有较好的遗传稳定性;K2116L菌株的生长及其释钾能力未受到标记质粒的影响.K2116L菌株在灭菌和非灭菌的黄褐土、黄潮土和红壤3种土壤中均能长期存活;在灭菌土壤中的数量稍高于非灭菌土壤;在3种土壤中的数量依次为:黄褐土＞黄潮土＞红壤;在不同土壤中,K2116L菌株具有与土著菌株进行空间和营养竞争的能力.采用根盒试验追踪标记菌株在棉花根部的定殖动态,棉花播种12d时标记菌株在0～2、2～4 cm深度根际土壤定殖密度达到最大;18 d时在4 cm以下的深度达到最高水平.棉花播种18 d时标记菌株在所有深度的根表均达到最高定殖水平,0～2 cm根段定殖密度为1.76×106cfu·g-1,8cm以下根段达到1.6×105cfu·g-1.补充营养后,根际和根表标记的菌株数量均有明显上升.试验数据显示,随着根的生长标记菌株不断向根尖方向扩散.     

巨大芽胞杆菌luxAB标记菌株的根际定殖研究

通过三亲本杂交方法成功地用发光酶基因luxAB标记巨大芽胞杆菌ATCC14581,所获得的标记菌株ATCC14581-L在不同的条件下能稳定发光.将该标记菌株制成微生物接种剂,并利用土壤微缩系统将其接种小麦进一步研究它在小麦根际的定殖动态和散布规律.结果发现,ATCC14581-L在灭菌土壤中的定殖水平高于不灭菌土壤,在垂直方向上主要的定殖在0～7cm根段间,且随深度增加而降低.ATCC14581-L在小麦种后第7d之前就已达到最高定殖水平,在初始接种量为3.40×107cfu/g根情况下,第7d时灭菌土壤处理的根际菌数为2.54×105cfu/g根,而不灭菌土壤的根际菌数为8.87×104cfu/g根;随着时间的增长,定殖数量明显降低.     

硅酸盐细菌NBT菌株在小麦根际定殖的初步研究

对硅酸盐细菌NBT菌株进行了耐药性标记,得到稳定的链霉素抗性标记菌NBT菌株,采用选择性培养基分离计数,通过琼脂平板和盆栽试验,研究了标记菌NBT在小麦根际的定殖动态及影响因素。结果表明,在灭菌土盆栽中,播种后9d左右NBT菌株在小麦根际的定殖水平达最高(1．4×10^8cfu·g^-1根土),播种后54d左右趋向稳定,NBT菌株细胞数量为2．4×10^3cfu·g^-1根土;未灭菌土盆栽中,播种后9d左右NBT菌株的定殖水平达最高(3．8×10^8cfu·g^-1根土),60d左右趋向稳定,菌数为3．1×10^3cfu·g^-1根土,牛物和非牛物因素对NBT菌株定殖小麦根系有影响。     

黄单胞菌P5310-luxAB菌株在水稻根际的吸附与土壤中的存活

目的研究标记菌株在水稻国丰1号幼根根表的吸附动态规律和不同类型土壤中存活情况,为禾本科植物凝集素介导作用、吸附的影响参数和联合固氮的研究及应用提供科学依据。方法采用三亲本杂交法将发光基因luxAB导入水稻根际促生菌黄单胞菌（Xanthom onassp）P5310菌株中,标记菌株P5310-luxAB能稳定遗传,luxAB基因的导入不影响标记菌株生长特性。结果 P5310-luxAB菌株存在稳定型和松散型两种吸附,随标记菌株浓度变化的吸附规律符合Langmu ir等温吸附方程,水稻根表最大吸附量qm=3.33×109CFU/g,吸附系数α=2.86×10-8。凝集素处理的水稻根表对标记菌株的亲和力增强,吸附量也随之增大。标记菌株在灭菌土壤和不灭菌土壤中都能很好的存活。在前25 d,不灭菌土壤中菌落数要低于灭菌土壤,随着营养的消耗,细菌明显减少;但补充营养后,在灭菌土壤和不灭菌土壤中标记菌株数量都快速增加,证明标记菌株与不灭菌土壤中的土著微生物的竞争占优势,很快与灭菌土壤中的菌数接近,证明菌株能在土壤中很好地存活与定植。结论以上研究结果为菌株P5310的开发应用提供了可靠的实验数据。     

巨大芽胞杆菌luxAB标记菌株的根际定殖研究

通过三亲本杂交方法成功地用发光酶基因luxAB标记巨大芽胞杆菌ATCC1 4581 ,所获得的标记菌株ATCC1 45 81 L在不同的条件下能稳定发光。将该标记菌株制成微生物接种剂 ,并利用土壤微缩系统将其接种小麦进一步研究它在小麦根际的定殖动态和散布规律。结果发现 ,ATCC1 45 81 L在灭菌土壤中的定殖水平高于不灭菌土壤 ,在垂直方向上主要的定殖在 0～ 7cm根段间 ,且随深度增加而降低。ATCC1 45 81 L在小麦种后第 7d之前就已达到最高定殖水平 ,在初始接种量为 3     

运用生色基因标记黄瓜根围促生菌(PGPR)筛选菌株

采用三亲交配方法 ,通过Tn7转座系统将lacZY标记基因导入黄瓜根围促生菌 (PG PR)筛选菌株PseudomonasfluorescensCN1 1 6和PseudomonascorrugataCN31的利福平抗性突变株中 ;标记假单胞菌菌株则被赋予了利用乳糖作为唯一碳源的能力 ,在只有乳糖的M9培养基上生长能分解X Gal,菌落显出特有的蓝色 ;经Southern杂交分析 ,证明标记基因lacZY存在于转化菌株的染色体上 ;经验证标记菌株标记性状稳定 ,与对应的野生菌株比较其它性状如培养性状、形态特征、生防效果等基本不变 ;PGPR菌株利福平抗性和生色基因标记的结合 (双标记 )能最大限度地将土壤中引入的PGPR菌株与土著细菌分开 ,检测下限可达 1 0CFU mL ,为PGPR在根围的分子生态学研究提供了一个较好的工具。     

两株PGPR菌株的花生定殖及对根际细菌群落结构的影响北大核心CSCD

为探讨2株根际促生菌耐酪氨酸束村氏菌P9和吡咯伯克霍尔德氏菌P10对花生的促生机制。利用GFP及利福平对2个菌株进行标记、结合扫描电镜观察,追踪了2株PGPR菌株在花生组织中的定殖动态;并通过16S rRNA全长测序对菌株接种花生根际土壤的细菌多样性进行分析。结果表明,利福平标记的P9和P10菌株具有良好的遗传稳定性,其生长和促生特性与原始菌株基本一致。GFP标记菌株可在花生的根尖及其分生区定殖;利福平标记菌株可稳定定殖在土壤及花生的根、茎部,且接菌30 d后定殖数仍保持在10CFU/g数量级。与未接菌植株根际土壤相比,P9、P10及混合菌株接种组的细菌群落相似性更高;接菌组的Flavihumibacter、unidentified_Rhizobiaceae的相对丰度显著增加,芽孢杆菌属、链霉菌属等的丰度较CK有不同程度增加,溶杆菌属、无色杆菌属及假黄单胞菌属等的丰度降低。2株PGPR菌株均可通过直接定殖在植株组织中、间接影响土壤细菌群落结构而发挥对花生的促生作用,混合菌株接种效果更优。研究结果明析了2株促生菌的促生机制,并为菌株的应用提供了科学依据。     

LuxAB标记荧光假单胞菌的菜心根际定殖研究

目的:利用三亲本杂交方法将luxAB发光酶基因标记至荧光假单胞菌PF20001上,所获得的标记菌株PF20001-Lux能稳定发光。将该标记菌株制成微生物制剂,接种菜心进一步研究它在菜心根际的定殖动态和散布规律。方法:利用三亲本杂交方法将luxAB发光酶基因标记至荧光假单胞菌PF20001上,将该菌施与菜心生长土壤中,通过接合子发光检测及发光菌落的平板计数来分析荧光假单胞菌在菜心根际的定殖分布情况。结果:PF20001-Lux在根系周围的土壤中的有一定的定殖率,在根内主要定殖在3～4cm根内。PF20001-Lux在菜心种植后7d前就达到最高定殖水平达3.8×105CFU/g,随后逐渐下降。结论:PF20001-lux在菜心根际具备良好的适应能力。     

海洋细菌L_1-9双抗菌株的定殖能力及其对黄瓜枯萎病的防治作用

采用抗生素标记法,对海洋多黏类芽孢杆菌L1-9菌株进行标记,抗性菌株L1-9Str,rif对链霉素和利福平的抗性浓度分别为160μg/m L和20μg/m L。双抗菌株L1-9Str,rif的抑菌特性及其对链霉素和利福平的抗性经多次传代仍比较稳定。盆栽试验表明,该双抗菌株能在黄瓜根部土壤及根组织、茎基部、子叶和真叶组织中定殖。菌株L1-9Str,rif在黄瓜外根际、根际和根表土壤及黄瓜组织中的定殖动态基本一致,初期黄瓜组织中L1-9Str,rif菌量较少,随着时间的延长,菌量逐渐增加,达到高峰后逐渐减少。菌株L1-9Str,rif在根表土壤中菌量最多(1.76×109 CFU/g),其次是根际土壤,外根际土壤中菌量较少;在黄瓜组织中,菌株L1-9Str,rif其在子叶中的定殖能力最强(5.63×104 CFU/g),其次是根和茎基部(0-2 cm);调查至第26 d时在根部土壤中的含菌量仍保持在稳定的水平,其中根表土壤中含菌量最高(2.41×107 CFU/g),在黄瓜组织样品中,子叶中的含菌量最高(4.15×104 CFU/g);温室防病实验结果表明,菌株L1-9和L1-9Str,rif菌株对黄瓜枯萎病具有良好的防治效果,不同时期防效均达70%以上。上述结果表明来自海洋的多黏类芽孢杆菌L1-9菌株能在黄瓜根部土壤及幼苗组织中定殖,是一株有潜力的黄瓜枯萎病生防菌株。     

利用发光酶基因标记技术跟踪棉花根圈中的绿针假单胞菌PL9L

采用细菌转化和杂交的方法,成功地将全套发光酶基因标记系统Tn7luxCDABE引入绿针假单胞菌(Pseudomonas chlororaphis)PL9,得到稳定的发光标记菌PL9L。采用发光菌落平板计数法和X射线胶片自显影法,通过盆栽试验和盒裁试验,研究了发光标记菌PL9L在棉花根圈的定殖动态和分布规律。盆栽试验结果表明,在灭菌土盆栽中,播种后6d左右PL9L在棉花根圈的定殖水平达最高(31×109cfu/g根土),播种后56d左右趋向稳定,PL9L数量为17×102cfu/g根土;未灭菌土盆载中,播种后8d左右PL9L的定殖水平达最高(11×109cfu/g根土),46d左右趋向稳定,菌数为14×102cfu/g根土。盒栽试验结果表明,PL9L可从种子向根尖方向扩散,但并不与根的伸长生长同步,播种后36d,灭菌土盒栽中PL9L可扩散至种子下方120cm以内,而未灭菌土盒栽中PL9L扩散至110cm以内。在棉花根尖区域均未检测到PL9L。     

Induced Reporter Gene Activity, Enhanced Stress Resistance, and Competitive Ability of a Genetically Modified Pseudomonas fluorescens Strain Released into a Field Plot Planted with Wheat

The fates of Pseudomonas fluorescens R2fR and its mutant derivative RIWE8, which contains a lacZ reporter gene responsive to wheat root exudate, were compared in a field microplot. Inoculant survival, root colonization, translocation, resistance to stress factors, and reporter gene activity were assessed in bulk and wheat rhizosphere soils. Populations of both strains declined gradually in bulk and wheat rhizosphere soils and on the wheat rhizoplane as determined by specific CFU and immunofluorescence (IF). In samples from both bulk soil and wheat rhizosphere, IF cell counts were up to 3 orders of magnitude greater than the corresponding numbers of CFU after 120 days, indicating the presence of nonculturable inoculant cells. Estimates of RIWE8-specific target DNA molecule numbers in bulk soil samples 3 and 120 days after inoculation by most-probable-number PCR coincided with the corresponding CFU values. Transport of both strains to deeper soil layers was observed by 3 days after introduction into the microplot. Both strains colonized wheat roots similarly, and cells were seen scattered on the surface of 1-month-old wheat seedling roots by immunogold labelling-scanning electron microscopy. On average, reporter gene activity was significantly higher in wheat rhizosphere soil containing RIWE8 cells than in bulk soil or in soils containing R2fR cells. For both strains, resistance to the four stress factors ethanol, high temperature, high osmotic tension, and oxidative stress increased progressively with residence in soil. Cells from the rhizosphere of 11-day-old seedlings showed similar levels of resistance to osmotic and oxidative stresses and enhanced resistance to ethanol and heat as compared to cells from bulk soil. By 37 days, populations of R2fR and RIWE8 in the rhizosphere were significantly more sensitive to osmotic stress than were populations in bulk soil, whereas differences in response to the other stress factors were less evident. Hence, except for the induction of reporter gene expression in strain RIWE8 in the wheat rhizosphere, the data indicated that there were no great differences in the ecological properties in soil between the lacZ-modified and parental strains.     

Identification and Characterization of Rhizosphere-Competent Bacteria of Wheat

To obtain rhizosphere-competent bacteria which could subsequently be modified for the development of biological control agents, bacteria were isolated from the rhizosphere and rhizoplane of wheat and barley plants by standard techniques. Of these isolates, 60 were selected for field testing as spring wheat seed inoculants in 1985. Isolates were marked genetically for resistance to antibiotics via selection of spontaneous mutants to detect and monitor isolates in the field. Forty-three days after planting, the average log₁₀ CFU/mg (dry weight) of roots and rhizosphere soil for the mutant isolates sampled ranged from 0 to 3.4. Twenty mutant isolates were retested in 1986. A total of 4 isolates were not detected, but the other 16 had an average root colonization value of log₁₀ 2.1 CFU and a range of log₁₀ 0.9 CFU to log₁₀ 3.2 CFU when sampled 32 days after planting. The average colonization value dropped to log₁₀ 1.1 CFU 51 days later. Some isolates detected previously were not detected in the second sampling; others had root colonization values similar to those obtained in the first sampling. Mutant isolates of rhizosphere bacteria included Bacillus pumilus, Bacillus subtilis, Pseudomonas fluorescens, Streptomyces spp., Xanthomonas maltophilia, and a saprophytic coryneform. Mixtures of isolates from different genera and species were compatible on seeds and roots.     

Survival of genetically modified Pseudomonas fluorescens introduced into subtropical soil microcosms

Abstract A genetically modified strain of Pseudomonas fluorescens and its parent showed grossly similar decline rates following introduction into subtropical clay and sandy soils. In unplanted clay soit at pH 6.9 and 25°C, population densities declined progressively from about 10⁸ to 10³ colony forming units (cfu) g⁻¹ dry soil over 75 days, but in unplanted sandy soil the introduced populations could not be detected after 25 days. In clay soil at pH 8.7 or 4.7, or at environmental temperature, decay rates were enhanced as compared to those at pH 6.9 and 25°C. Counts of introduced strains in clay bulk soil and in rhizosphere and rhizoplane of maize suggested that the introduced bacteria competed well with the native bacteria, and colonized the roots at about 10⁶ cfu g⁻¹ dry root at 25°C, over 20 days. However, rhizoplane colonization was lower at environmental temperature. The decay rate of both strains was slower in planted than in unplanted sandy soil. The population densities in the rhizosphere and rhizoplane in the sandy soil were significantly lower than those in the clay soil. Both introduced strains colonized the maize roots in both soils, using seeds coated with bacteria in 1% carboxymethyl cellulose. Introduced cells were localized at different sites along the roots of plants developing in clay soil, with higher densities in the original (near the seeds) and root hair zones as compared to the intermediate zones. No significant difference was observed between the extent of root colonization of the genetically modified strain and its parent.     

Colonization of Wheat Roots by an Exopolysaccharide-Producing Pantoea agglomerans Strain and Its Effect on Rhizosphere Soil Aggregation

The effect of bacterial secretion of an exopolysaccharide (EPS) on rhizosphere soil physical properties was investigated by inoculating strain NAS206, which was isolated from the rhizosphere of wheat (Triticum durum L.) growing in a Moroccan vertisol and was identified as Pantoea aglomerans. Phenotypic identification of this strain with the Biotype-100 system was confirmed by amplified ribosomal DNA restriction analysis. After inoculation of wheat seedlings with strain NAS206, colonization increased at the rhizoplane and in root-adhering soil (RAS) but not in bulk soil. Colonization further increased under relatively dry conditions (20% soil water content; matric potential, −0.55 MPa). By means of genetic fingerprinting using enterobacterial repetitive intergenic consensus PCR, we were able to verify that colonies counted as strain NAS206 on agar plates descended from inoculated strain NAS206. The intense colonization of the wheat rhizosphere by these EPS-producing bacteria was associated with significant soil aggregation, as shown by increased ratios of RAS dry mass to root tissue (RT) dry mass (RAS/RT) and the improved water stability of adhering soil aggregates. The maximum effect of strain NAS206 on both the RAS/RT ratio and aggregate stability was measured at 24% average soil water content (matric potential, −0.20 MPa). Inoculated strain NAS206 improved RAS macroporosity (pore diameter, 10 to 30 μm) compared to the noninoculated control, particularly when the soil was nearly water saturated (matric potential, −0.05 MPa). Our results suggest that P. agglomerans NAS206 can play an important role in the regulation of the water content (excess or deficit) of the rhizosphere of wheat by improving soil aggregation.     

Beneficial bacteria of agricultural importance

The rhizosphere is the soil–plant root interphase and in practice consists of the soil adhering to the root besides the loose soil surrounding it. Plant growth-promoting rhizobacteria (PGPR) are potential agents for the biological control of plant pathogens. A biocontrol strain should be able to protect the host plant from pathogens and fulfill the requirement for strong colonization. Numerous compounds that are toxic to pathogens, such as HCN, phenazines, pyrrolnitrin, and pyoluteorin as well as, other enzymes, antibiotics, metabolites and phytohormones are the means by which PGPR act, just as quorum sensing and chemotaxis which are vital for rhizosphere competence and colonization. The presence of root exudates has a pronounced effect on the rhizosphere where they serve as an energy source, promoting growth and influencing the root system for the rhizobacteria. In certain instances they have products that inhibit the growth of soil-borne pathogens to the advantage of the plant root. A major source of concern is reproducibility in the field due to the complex interaction between the plant (plant species), microbe and the environment (soil fertility and moisture, day length, light intensity, length of growing season, and temperature). This review listed most of the documented PGPR genera and discussed their exploitation.     

External and Internal Root Colonization of Maize by Two Pseudomonas Strains: Enumeration by Enzyme-Linked Immunosorbent Assay (ELISA)

Maize root colonization by two fluorescent Pseudomonas strains M.3.1. and TR335, isolated respectively from maize and tomato roots, were studied in hydroponic conditions. Each bacterium was inoculated separately, and three different colonization areas were studied: nutrient solution, rhizoplane, and endorhizosphere. The two Pseudomonas strains established large rhizosphere populations, and rhizoplane colonization of the entire root system was similar for both strains. However, strain M.3.1. colonized the endorhizosphere more efficiently than strain TR335. Seminal root cuttings from the tip to the seed allowed the assessment of colonization of three different root areas (i.e., root cap and elongation area, root-hair zone, and mature zone). Rhizoplane colonizations of all these three areas by M.3.1. were significantly the same, whereas strain TR335 colonized the rhizoplane of the root cap and elongation area more actively than the root-hair zone and mature zone. Population size of the strain M.3.1. in the internal tissue of these areas was greater than that of strain TR335. Co-inoculations of the two strains indicated a stimulation of the population size of strain M.3.1. regardless of root area studied, whereas population size of strain TR335 remained unchanged. These results demonstrated that external and internal maize root tissues were colonized to a greater extent by a strain isolated from a maize rhizosphere than by one isolated from another rhizosphere. Received: 26 September 1996 / Accepted: 1 November 1996     

Disease suppression and crop improvement through fluorescent pseudomonads isolated from cultivated soils

Three fluorescent pseudomonads isolated from rhizosphere/rhizoplane of crop plants showed in vitro antibiosis against seven fungal and two bacterial plant pathogens on iron-deficient KB medium. Seed bacterization of chick- pea (Cicer arientinum L.), egg plant (Solanum melongena L.), soybean (Glycine max Merr.) and tomato (Lycopersicon esculentum Mill.) with these organisms showed an increased seed germination, shoot height, root length, fresh weight, dry weight and yield. Seed bacterization with one of these strains, RB 8, reduced the number of chick-pea wilted plants in wilt-sick (Fusarium oxysporum f.sp. ciceris) soil. Addition of iron into the soil eliminated the disease suppression. The disease suppression and/or growth enhancement along with the positive root colonization by these organisms indicate their possible use as plant growth-promoting rhizobacteria (PGPR)/biocontrol agents against chick-pea wilt.     

Growth of bacteria in the rhizoplane and the rhizosphere of rape seedlings

Abstract The growth of 10 isolates of rhizosphere bacteria was compared in the rhizoplane (RP), rhizosphere (RS) and non-rhizosphere soil of a model system with rape seedlings growing in sterile sand. The colonization of the RP differed little among isolates. However, the bacterial isolates differed according to their degree of dependence on the root for growth, as judged by RS:RP and plant:non-plant ratios for CFU. These two ratios were correlated with changes in viability and 'physiological status' (as judged by γ values, Hattori, T. (1983) J. Gen. Appl. Microbiol. 29, 9–16).     

Detection of recombinant Pseudomonas putida in the wheat rhizosphere by fluorescence in situ hybridization targeting mRNA and rRNA

A method was developed to detect a specific strain of bacteria in wheat root rhizoplane using fluorescence in situ hybridization and confocal microscopy. Probes targeting both 23S rRNA and messenger RNA were used simultaneously to achieve detection of recombinant Pseudomonas putida (TOM20) expressing toluene o-monooxygenase (tom) genes and synthetic phytochelatin (EC20). The probe specific to P. putida 23S rRNA sequences was labeled with Cy3 fluor, and the probe specific to the tom genes was labeled with Alexa647 fluor. Probe specificity was first determined, and hybridization temperature was optimized using three rhizosphere bacteria pure cultures as controls, along with the P. putida TOM20 strain. The probes were highly specific to the respective targets, with minimal non-specific binding. The recombinant strain was inoculated into wheat seedling rhizosphere. Colonization of P. putida TOM20 was confirmed by extraction of root biofilm and growth of colonies on selective agar medium. Confocal microscopy of hybridized root biofilm detected P. putida TOM20 cells emitting both Cy3 and Alexa647 fluorescence signals.     

Bacterial origin and community composition in the barley phytosphere as a function of habitat and presowing conditions

An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.