Effect of a Sinorhizobium meliloti Strain with a Modified putA Gene on the Rhizosphere Microbial Community of Alfalfa

The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains. M403, a Sinorhizobium meliloti strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified putA (proline dehydrogenase) gene. This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at León, Spain. In this study, we determined the effects of these two strains on the indigenous microbial community. 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers. These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis. The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment. Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community.     

Plant-microbe association for rhizoremediation of chloronitroaromatic pollutants with Comamonas sp. strain CNB-1

Comamonas sp. strain CNB-1, isolated from activated sludge and having a strong ability to degrade 4-chloronitrobenzene (4CNB), was applied for rhizoremediation of 4CNB-polluted soil through association with alfalfa. Confocal laser scanning microscopy revealed that strain CNB-1 successfully colonized alfalfa roots. Determination of strain CNB-1 populations by cultivation method and by quantitative competitive PCR technique targeting the chloronitrobenzene nitroreductase gene showed that the population of strain CNB-1 in the rhizosphere was about 10-100 times higher than that in the bulk soil. Gnotobiotic and outdoor experiments showed that pollutant 4CNB was completely removed within 1 or 2 days after 4CNB application into soil, and that its phytotoxicity to alfalfa was eliminated by inoculation of strain CNB-1. Results from PCR-denaturing gradient gel electrophoresis and analysis of 16S rRNA gene libraries revealed that the indigenous soil microbial community mainly consisted of alphaproteobacteria, betaproteobacteria, gammaproteobacteria, the CFB bacteria (Cytophaga-Flavabacterium-Bacteriodes), and Acidobacteria. This microbial community was not significantly influenced by inoculation of strain CNB-1. Thus, this study has developed a Comamonas-alfalfa system for rhizoremediation of 4CNB.     

Effects of Pseudomonas putida modified to produce phenazine-1-carboxylic acid and 2,4-diacetylphloroglucinol on the microflora of field grown wheat

Pseudomonas putida WCS358r, genetically modified to have improved activity against soil-borne pathogens, was released into the rhizosphere of wheat. Two genetically modified derivatives carried the phzor the phl biosynthetic gene loci and constitutively produced either the antifungal compound phenazine-1-carboxylic acid (PCA) or the antifungal and antibacterial compound 2,4-diacetylphloroglucinol (DAPG). In 1997 and 1998, effects of single introductions of PCA producing derivatives on the indigenous microflora were studied. A transient shift in the composition of the total fungal microflora, determined by amplified ribosomal DNA restiction analysis (ARDRA), was detected. Starting in 1999, effects of repeated introduction of genetically modified microorganisms (GMMs) were studied. Wheat seeds coated with the PCA producer, the DAPG producer, a mixture of the PCA and DAPG producers, or WCS358r, were sown and the densities, composition and activities of the rhizosphere microbial populations were measured. All introduced strains decreased from 10⁷CFU per gram of rhizosphere sample to below the detection limit after harvest of the wheat plants. The phz genes were stably maintained in the PCA producers, and PCA was detected in rhizosphere extracts of plants treated with this strain or with the mixture of the PCA and DAPG producers. The phl genes were also stably maintained in the DAPG producing derivative of WCS358r. Effects of the genetically modified bacteria on the rhizosphere fungi and bacteria were analyzed by using amplified ribosomal DNA restriction analysis. Introduction of the genetically modified bacterial strains caused a transient change in the composition of the rhizosphere microflora. However, introduction of the GMMs did not affect the several soil microbial activities that were investigated in this study. This revised version was published online in August 2006 with corrections to the Cover Date.     

紫花苜蓿根际氢氧化细菌的分离及其对小麦种子促生作用的研究

从大豆植物根际分离的氢氧化细菌对植物的生长有促进作用,但是关于其他的豆科植物根际分离的氢氧化细菌是否也有促生作用的研究甚少。从紫花苜蓿根际土壤分离氢氧化细菌,并进行其对小麦种子促生实验的研究,判断氢氧化细菌是否有促生作用,从而丰富具有促生作用的根际微生物资源。采用MSA培养基,从铜川新区紫花苜蓿根际土壤中分离得到氢氧化细菌疑似菌株,对其进行TTC法检测菌株氢化酶活性和自养能力的特性,以获得氢氧化细菌;通过小麦种子的萌发进行促生实验验证。结果表明,16株菌株处理过的小麦根长分别增加25%～128%,芽长增长27%～73%,鲜重增加48%～103%。从苜蓿根际土壤分离出的氢氧化细菌均具有较明显的促生作用。     

Distribution of metabolic activity and phosphate starvation response of lux-tagged Pseudomonas fluorescens reporter bacteria in the barley rhizosphere.

The purpose of this study was to determine the metabolic activity of Pseudomonas fluorescens DF57 in the barley rhizosphere and to assess whether sufficient phosphate was available to the bacterium. Hence, two DF57 reporter strains carrying chromosomal luxAB gene fusions were introduced into the rhizosphere. Strain DF57-40E7 expressed luxAB constitutively, making bioluminescence dependent upon the metabolic activity of the cells under defined assay conditions. The DF57-P2 reporter strain responded to phosphate limitation, and the luxAB gene fusion was controlled by a promoter containing regulatory sequences characteristic of members of the phosphate (Pho) regulon. DF57 generally had higher metabolic activity in a gnotobiotic rhizosphere than in the corresponding bulk soil. Within the rhizosphere the distribution of metabolic activity along the root differed between the rhizosphere soil and the rhizoplane, suggesting that growth conditions may differ between these two habitats. The DF57-P2 reporter strain encountered phosphate limitation in a gnotobiotic rhizosphere but not in a natural rhizosphere. This difference in phosphate availability seemed to be due to the indigenous microbial population, as DF57-P2 did not report phosphate limitation when established in the rhizosphere of plants in sterilized soil amended with indigenous microorganisms.     

Influence of intercropping and intercropping plus rhizobial inoculation on microbial activity and community composition in rhizosphere of alfalfa (Medicago sativa L.) and Siberian wild rye (Elymus sibiricus L.)

Alfalfa–Siberian wild rye intercropping is the predominant cropping system used to produce forage in China. In this study, the effects of intercropping and intercropping-rhizobial inoculation on soil enzyme activities, microbial biomass and bacterial community composition in the rhizosphere were examined. In both treatments, the yield of alfalfa, microbial biomass and activities of soil urease, invertase and alkaline phosphatase in the alfalfa rhizosphere were markedly increased, whereas there was a slight increase in the yield of Siberian wild rye, few impacts on soil microbial biomass, and decreased enzyme activities (except for urease) in the Siberian wild rye rhizosphere. Terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes indicated that Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were the major bacterial groups in the rhizosphere of both plants. However, intercropping and rhizobial inoculation induced some shifts in the relative abundance of them. Nitrosomonas and Nitrosospira groups were detected in all treatments by the T-RFLP patterns of ammonia monooxygenase subunit A ( amoA ) gene, but the relative abundance of Nitrosomonas increased and that of Nitrosospira decreased in the intercropping-rhizobial inoculation treatment. Both treatments tended to increase the diversity of amoA . Conclusively, the two treatments clearly affected soil microbial composition and soil enzyme activities, which might be reflected in changes in yield.     

Perturbation of maize rhizosphere microflora following seed bacterization with Burkholderia cepacia MCI 7

Introduction of a large quantity of exogenous microorganisms may disrupt a local ecosystem and affect the natural microflora. In this work we investigated the effects of the introduction of a plant growth promoting strain of Burkholderia cepacia into the rhizosphere of maize on both indigenous B. cepacia populations and microbial community structure of total culturable bacteria using the concept of r/K strategy. Moreover we studied the distribution of bacterial populations in the root system at various soil depths. Seed bacterization was used as application method. Root colonization of the introduced strain occurred mainly on roots close to the plant stem, whereas indigenous B. cepacia was recovered at higher amounts from the lower parts of root systems of mature plants. As far as total culturable bacteria are concerned, an almost uniform distribution in the root system of mature plants was observed. The release of the exogenous bacterial strain affected mainly the microbial populations of young growing plants rather than mature plants. Indeed it caused only short-term perturbations in the microbial community of maize rhizosphere. Colonization of maize roots by indigenous B. cepacia was not significantly affected by the presence of the exogenous strain.     

Greenhouse and Field Evaluations of an Autoselective System Based on an Essential Thymidylate Synthase Gene for Improved Maintenance of Plasmid Vectors in Modified Rhizobium meliloti

The stability of the thy autoselective system, based on an essential thymidylate synthase gene, for enhanced maintenance of plasmid vectors in Rhizobium meliloti was evaluated in the greenhouse and with field-grown alfalfa. The thy autoselective system consists of a free-replicating, broad-host-range plasmid vector containing a copy of the thyA gene from Lactococcus lactis subsp. lactis and a spontaneous mutant of R. meliloti deficient in thymidylate synthase (Thy(sup-)). Under greenhouse conditions, Thy(sup-) rhizobia did not persist in rooting solution alone unless supplemented with thymidine but survived in the presence of the host plant. Nodules formed on alfalfa plants grown in thymidine-free rooting solution and inoculated with Thy(sup-) rhizobia contained only Thy(sup+) revertants. In soil, Thy(sup-) rhizobia were compromised and failed to nodulate alfalfa. Thy(sup-) mutants containing a thy plasmid survived in the rhizosphere and nodulated alfalfa like the wild-type strain. The thy autoselective system was tested in the field with Thy(sup-) strain Rm24T and pPR602, a thy plasmid vector devoid of antibiotic resistance genes and marked with constitutively expressed lacZY. At 80 days after sowing, most rhizobia isolated from the nodules of field-grown alfalfa inoculated with Rm42T(pPR602) contained pPR602. The thy autoselective system proved useful to ensure maintenance of the plasmid vector under greenhouse and field conditions in R. meliloti.     

Relationship between the number of bacteria added to soil or seeds and their abundance and distribution in the rhizosphere of alfalfa

A study was conducted of the relationship between the density of several bacterial strains introduced into soil or onto seeds and their abundance in the rhizosphere of alfalfa. The abundance of six species in the rhizosphere was directly correlated with the density of bacteria initially added to soil. The density of six species in the rhizosphere of 15-day-old plants also was directly correlated with the density of each strain in nonrhizosphere soil. Tests of seven species added to soil at four inoculum densities showed that bacteria that survived well in the soil attained the highest densities in the rhizosphere and those that survived poorly in the soil were present at the lowest densities in the rhizosphere. Sixteen of 19 bacterial strains added to alfalfa seeds at 10⁷ or 10⁸ cells per g colonized the rhizosphere of 15-day-old plants, but nearly all of the cells were localized in the upper third of the rhizosphere. A study of 12 bacterial strains that failed to colonize the lower part of the rhizosphere if inoculated onto seeds showed that the bacteria colonized the entire rhizosphere of 15-day-old alfalfa plants if initially inoculated throughout the soil. The data suggest that the density of individual bacterial strains in the rhizosphere is dependent on their density in the soil and that seed inoculation only has an effect on the population in the proximal portion of the alfalfa root system.     

Effect of genetically modified Pseudomonas putida WCS358r on the fungal rhizosphere microflora of field-grown wheat

We released genetically modified Pseudomonas putida WCS358r into the rhizospheres of wheat plants. The two genetically modified derivatives, genetically modified microorganism (GMM) 2 and GMM 8, carried the phz biosynthetic gene locus of strain P. fluorescens 2-79 and constitutively produced the antifungal compound phenazine-1-carboxylic acid (PCA). In the springs of 1997 and 1998 we sowed wheat seeds treated with either GMM 2, GMM 8, or WCS358r (approximately 10(7) CFU per seed), and measured the numbers, composition, and activities of the rhizosphere microbial populations. During both growing seasons, all three bacterial strains decreased from 10(7) CFU per g of rhizosphere sample to below the limit of detection (10(2) CFU per g) 1 month after harvest of the wheat plants. The phz genes were stably maintained, and PCA was detected in rhizosphere extracts of GMM-treated plants. In 1997, but not in 1998, fungal numbers in the rhizosphere, quantified on 2% malt extract agar (total filamentous fungi) and on Komada's medium (mainly Fusarium spp.), were transiently suppressed in GMM 8-treated plants. We also analyzed the effects of the GMMs on the rhizosphere fungi by using amplified ribosomal DNA restriction analysis. Introduction of any of the three bacterial strains transiently changed the composition of the rhizosphere fungal microflora. However, in both 1997 and 1998, GMM-induced effects were distinct from those of WCS358r and lasted for 40 days in 1997 and for 89 days after sowing in 1998, whereas effects induced by WCS358r were detectable for 12 (1997) or 40 (1998) days. None of the strains affected the metabolic activity of the soil microbial population (substrate-induced respiration), soil nitrification potential, cellulose decomposition, plant height, or plant yield. The results indicate that application of GMMs engineered to have improved antifungal activity can exert nontarget effects on the natural fungal microflora.     

The persistence of bioluminescent Rhizobium meliloti strains L1 (RecA-) and L33 (RecA+) in non-sterile microcosms depends on the soil type,on the co-cultivation of the host legume alfalfa and on the presence of an indigenous R. meliloti population

In comparative analyses the influence of soil type, the rhizosphere of plants and the presence of an indigenous R. meliloti population on the population dynamics of bioluminescent R. meliloti strains L1 (RecA-) and L33 (RecA+) was assessed in microcosm studies. Both strains established better in a loamy and a clayey soil compared to a sandy soil. RecA- strain L1 showed a slightly but statistically significant reduced survival ability compared to RecA+ strain L33 (p 0.05). The presence of the host plant alfalfa stimulated the growth of both strains in non-sterile soil and no differences in the survival between both strains were observed. Co-cultivation of clover or wheat plants, respectively, neither positively nor negatively influenced the strains' survival. The most pronounced effect on the survival of both strains was exerted by the presence of an indigenous R. meliloti population. RecA- strain L1 showed a significantly impaired survival compared to RecA+ strain L33 (p 0.002). Moreover, no growth stimulation of strains L1 and L33 by the presence of the host plant alfalfa could be observed. These results indicate that the recA mutation affects the long-term rather than the short-term persistence of R. meliloti after environmental release.     

Variation of Microbial Rhizosphere Communities in Response to Crop Species, Soil Origin, and Inoculation with Sinorhizobium meliloti L33

Abstract A greenhouse study with soil–plant microcosms was conducted in order to compare the effect of crop species, soil origin, and a bacterial inoculant on the establishment of microbial communities colonizing plant roots. Two crop species, alfalfa (Medicago sativa) and rye (Secale cereale), were grown separately in two soils collected from agricultural fields at different locations and with differing histories of leguminous crop rotation. A subset of microcosms was inoculated at 10⁶ cfu g^-1 soil with the luciferase marker gene-tagged Sinorhizobium meliloti strain L33, a symbiotic partner of M. sativa. Microbial consortia were collected from the rhizospheres of alfalfa after 10 weeks of incubation and from rye after 11 weeks. S. meliloti L33 populations were one to two orders of magnitude higher in the rhizospheres of alfalfa than of rye. In soil with previous alfalfa cultivation, 80% of the alfalfa nodules were colonized by indigenous bacteria, while in the other soil alfalfa was colonized almost exclusively (>90%) with S. meliloti L33. Three community-level targeting approaches were used to characterize the variation of the extracted microbial rhizosphere consortia: (1) Community level physiological profiles (CLPP), (2) fatty acid methyl ester analysis (FAME), and (3) diversity of PCR amplified 16S rRNA target sequences from directly extracted ribosomes, determined by temperature gradient gel electrophoresis (TGGE). All approaches identified the crop species as the major determinant of microbial community characteristics. Consistently, the influence of soil was of minor importance, while a modification of the alfalfa-associated microbial community structure after inoculation with S. meliloti L33 was only consistently observed by using TGGE. Received: 20 October 1999; Accepted: 15 January 2000; Online Publication: 18 July 2000     

Rhizosphere microflora of plants used for the phytoremediation of bitumen-contaminated soil

The microbial communities and their degradative potential in rhizospheres of alfalfa (Medicago sativa) and reed (Phragmites australis) and in unplanted soil in response to bitumen contamination of soil were studied in pot experiments. According to the results of fluorescence microscopy, over a period of 27 months, bitumen contamination of soil reduced the total number of microorganisms more significantly (by 75%) in unplanted than in rhizosphere soil (by 42% and 7% for reed and alfalfa, respectively) and had various effects on some important physiological groups of microorganisms such as actinomycetes as well as nitrogen-fixing, nitrifying, denitrifying, ammonifying, phosphate-solubilizing, sulphur-oxidizing, cellulolytic and hydrocarbon-degrading microorganisms. The changes in the physiological structure of the microbial community under bitumen contamination were found to hinge on not merely the presence of plants but also their type. It was noted that the rhizosphere microflora of alfalfa was less inhibited by hydrocarbon pollution and had a higher degradative potential than the rhizosphere microflora of reed.     

Characterization of diverse 2,4-dichlorophenoxyacetic acid-degradative plasmids isolated from soil by complementation.

The diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative plasmids in the microbial community of an agricultural soil was examined by complementation. This technique involved mixing a suitable Alcaligenes eutrophus (Rifr) recipient strain with the indigenous microbial populations extracted from soil. After incubation of this mixture, Rifr recipient strains which grow with 2,4-D as the only C source were selected. Two A. eutrophus strains were used as recipients: JMP228 (2,4-D-), which was previously derived from A. eutrophus JMP134 by curing of the 2,4-D-degradative plasmid pJP4, and JMP228 carrying pBH501aE (a plasmid derived from pJP4 by deletion of a large part of the tfdA gene which encodes the first step in the mineralization of 2,4-D). By using agricultural soil that had been treated with 2,4-D for several years, transconjugants were obtained with both recipients. However, when untreated control soil was used, no transconjugants were isolated. The various transconjugants had plasmids with seven different EcoRI restriction patterns. The corresponding plasmids are designated pEMT1 to pEMT7. Unlike pJP4, pEMT1 appeared not to be an IncP1 plasmid, but all the others (pEMT2 to pEMT7) belong to the IncP1 group. Hybridization with individual probes for the tfdA to tfdF genes of pJP4 demonstrated that all plasmids showed high degrees of homology to the tfdA gene. Only pEMT1 showed a high degree of homology to tfdB, tfdC, tfdD, tfdE, and tfdF, while the others showed only moderate degrees of homology to tfdB and low degrees of homology to tfdC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction and environmental release of a Sinorhizobium meliloti strain genetically modified to be more competitive for alfalfa nodulation

Highly efficient nitrogen-fixing strains selected in the laboratory often fail to increase legume production in agricultural soils containing indigenous rhizobial populations because they cannot compete against these populations for nodule formation. We have previously demonstrated, with a Sinorhizobium meliloti PutA- mutant strain, that proline dehydrogenase activity is required for colonization and therefore for the nodulation efficiency and competitiveness of S. meliloti on alfalfa roots (J. I. Jiménez-Zurdo, P. van Dillewijn, M. J. Soto, M. R. de Felipe, J. Olivares, and N. Toro, Mol. Plant-Microbe Interact. 8:492-498, 1995). In this work, we investigated whether the putA gene could be used as a means of increasing the competitiveness of S. meliloti strains. We produced a construct in which a constitutive promoter was placed 190 nucleotides upstream from the start codon of the putA gene. This resulted in an increase in the basal expression of this gene, with this increase being even greater in the presence of the substrate proline. We found that the presence of multicopy plasmids containing this putA gene construct increased the competitiveness of S. meliloti in microcosm experiments in nonsterile soil planted with alfalfa plants subjected to drought stress only during the first month. We investigated whether this construct also increased the competitiveness of S. meliloti strains under agricultural conditions by using it as the inoculum in a contained field experiment at León, Spain. We found that the frequency of nodule occupancy was higher with inoculum containing the modified putA gene for samples that were analyzed after 34 days but not for samples that were analyzed later.     

Impacts of gene bioaugmentation with pJP4-harboring bacteria of 2,4-D-contaminated soil slurry on the indigenous microbial community

Gene bioaugmentation is a bioremediation strategy that enhances biodegradative potential via dissemination of degradative genes from introduced microorganisms to indigenous microorganisms. Bioremediation experiments using 2,4-dichlorophenoxyacetic acid (2,4-D)-contaminated soil slurry and strains of Pseudomonas putida or Escherichia coli harboring a self-transmissible 2,4-D degradative plasmid pJP4 were conducted in microcosms to assess possible effects of gene bioaugmentation on the overall microbial community structure and ecological functions (carbon source utilization and nitrogen transformation potentials). Although exogenous bacteria decreased rapidly, 2,4-D degradation was stimulated in bioaugmented microcosms, possibly because of the occurrence of transconjugants by the transfer of pJP4. Terminal restriction fragment length polymorphism analysis revealed that, although the bacterial community structure was disturbed immediately after introducing exogenous bacteria to the inoculated microcosms, it gradually approached that of the uninoculated microcosms. Biolog assay, nitrate reduction assay, and monitoring of the amoA gene of ammonia-oxidizing bacteria and nirK and nirS genes of denitrifying bacteria showed no irretrievable depressive effects of gene bioaugmentation on the carbon source utilization and nitrogen transformation potentials. These results may suggest that gene bioaugmentation with P. putida and E. coli strains harboring pJP4 is effective for the degradation of 2,4-D in soil without large impacts on the indigenous microbial community.     

辣椒青枯病罹病与健康植株根际土壤微生物群落多样性研究

系统研究和分析辣椒青枯病常发地发病与健康植株土壤微生物群落结构特征,为辣椒青枯病的绿色防治提供理论依据.基于16SrDNA基因高通量测序技术,对辣椒青枯病发病和健康植株根际土壤微生物群落结构和组成进行分析,同时采用biologyeco平板培养技术研究其土壤微生物群落代谢多样性和功能多样性的特征.结果表明,辣椒青枯病发病和健康植株根际土壤微生物群落组成之间存在显著差异,辣椒青枯病发病土壤的OTU为4566个,辣椒青枯病健康土壤的OTU为4167个.依据OTU所属细菌物种信息对土壤细菌群落结构进行分析,变形菌门在发病和健康土壤中均为优势细菌类群,其次为放线菌门类群.其中健康植株根际土壤中芽单胞菌门(Gemmatimonadetes)、装甲菌门(Armatimonadetes)的相对丰度比发病植株的分别高出了4.37,3.87倍,而发病植株根际土壤中厚壁菌门(Firmicutes)的相对丰度比健康植株的高出了3.87倍.辣椒青枯病发病土壤和健康土壤的土壤微生物代谢多样性也存在显著差异,同时,健康土壤中其微生物群落代谢得到显著增强,特别是对酚类化合物的利用显著增多,对辣椒抗病性存在显著的影响.研究表明,辣椒青枯病发病和健康植株根际土壤微生物群落组成和结构之间存在显著差异,并且健康土壤的微生物群落对酚类化合物的利用显著增强.     

Influence of different Sinorhizobium meliloti inocula on abundance of genes involved in nitrogen transformations in the rhizosphere of alfalfa (Medicago sativa L.)

Inoculation of leguminous seeds with selected rhizobial strains is practised in agriculture to ameliorate the plant yield by enhanced root nodulation and nitrogen uptake of the plant. However, effective symbiosis between legumes and rhizobia does not only depend on the capacity of nitrogen fixation but also on the entire nitrogen turnover in the rhizosphere. We investigated the influence of seed inoculation with two indigenous Sinorhizobium meliloti strains exhibiting different efficiency concerning plant growth promotion on nitrogen turnover processes in the rhizosphere during the growth of alfalfa. Quantification of six target genes (bacterial amoA, nirK, nirS, nosZ, nifH and archaeal amoA) within the nitrogen cycle was performed in rhizosphere samples before nodule formation, at bud development and at the late flowering stage. The results clearly demonstrated that effectiveness of rhizobial inocula is related to abundance of nifH genes in the late flowering phase of alfalfa. Moreover, other genes involved in nitrogen turnover had been affected by the inocula, e.g. higher numbers of amoA copies were observed during flowering when the more effective strain had been inoculated. However, the respective gene abundances differed overall to a greater extent between the three plant development stages than between the inoculation variants.     

Effect of Genetically Modified Pseudomonas putida WCS358r on the Fungal Rhizosphere Microflora of Field-Grown Wheat

We released genetically modified Pseudomonas putida WCS358r into the rhizospheres of wheat plants. The two genetically modified derivatives, genetically modified microorganism (GMM) 2 and GMM 8, carried the phz biosynthetic gene locus of strain P. fluorescens 2-79 and constitutively produced the antifungal compound phenazine-1-carboxylic acid (PCA). In the springs of 1997 and 1998 we sowed wheat seeds treated with either GMM 2, GMM 8, or WCS358r (approximately 10⁷ CFU per seed), and measured the numbers, composition, and activities of the rhizosphere microbial populations. During both growing seasons, all three bacterial strains decreased from 10⁷ CFU per g of rhizosphere sample to below the limit of detection (10² CFU per g) 1 month after harvest of the wheat plants. The phz genes were stably maintained, and PCA was detected in rhizosphere extracts of GMM-treated plants. In 1997, but not in 1998, fungal numbers in the rhizosphere, quantified on 2% malt extract agar (total filamentous fungi) and on Komada's medium (mainly Fusarium spp.), were transiently suppressed in GMM 8-treated plants. We also analyzed the effects of the GMMs on the rhizosphere fungi by using amplified ribosomal DNA restriction analysis. Introduction of any of the three bacterial strains transiently changed the composition of the rhizosphere fungal microflora. However, in both 1997 and 1998, GMM-induced effects were distinct from those of WCS358r and lasted for 40 days in 1997 and for 89 days after sowing in 1998, whereas effects induced by WCS358r were detectable for 12 (1997) or 40 (1998) days. None of the strains affected the metabolic activity of the soil microbial population (substrate-induced respiration), soil nitrification potential, cellulose decomposition, plant height, or plant yield. The results indicate that application of GMMs engineered to have improved antifungal activity can exert nontarget effects on the natural fungal microflora.